USOO86826 19B2
(12) United States Patent (10) Patent No.: US 8,682,619 B2 Amodei et al. (45) Date of Patent: Mar. 25, 2014
(54) DEVICE INCLUDING ALTERED filed on Oct. 2, 2007, and a continuation-in-part of MICROORGANISMS, AND METHODS AND application No. 1 1/900,870, filed on Sep. 12, 2007, SYSTEMIS OF USE and a continuation-in-part of application No. (75) Inventors: Dario G. Amodei, Princeton, NJ (US); 11/900,776, filed on Sep. 12, 2007, and a Mahalaxmi Gita Bangera, Renton, WA continuation-in-part of application No. 1 1/900,773, (US); Xiaoyan Robert Bao, Cambridge, filed on Sep. 12, 2007, now Pat. No. 8,252,570, and a MA (US); Anna Bershteyn, Somerville, continuation-in-part of application No. 1 1/701,163, MA (US); Brett Bethke, Arlington, MA filed on Jan. 31, 2007, now Pat. No. 8,354.258, and a (US); Philip A. Eckhoff, Bellevue, WA continuation-in-part of application No. 1 1/452.019, (US); Kevin Michael Esvelt, filed on Jun. 12, 2006, and a continuation-in-part of Cambridge, MA (US); Kyle B. application No. 1 1/451,994, filed on Jun. 12, 2006, and Gustafson, Lausanne (CH); Edward K. a continuation-in-part of application No. 1 1/451,986, Y. Jung, Bellevue, WA (US); William filed on Jun. 12, 2006, now Pat. No. 8,053,220, and a Michael Kaminsky, Alexandria, VA continuation-in-part of application No. 1 1/389.268, (US); Jordin T. Kare, Seattle, WA (US); filed on Mar. 24, 2006, and a continuation-in-part of Lily Yvonne Kim, Brookline, MA (US); application No. 1 1/304,499, filed on Dec. 14, 2005, Eric C. Leuthardt, St. Louis, MO (US); now Pat. No. 8.278,094, and a continuation-in-part of Erez Lieberman, Cambridge, MA (US); application No. 1 1/304,492, filed on Dec. 14, 2005, Ankur Moitra, Cambridge, MA (US); now Pat. No. 7,855,062, and a continuation-in-part of Christopher Somogyi, Woodinville, WA application No. 1 1/304,486, filed on Dec. 14, 2005, (US); Clarence T. Tegreene, Bellevue, now Pat. No. 8,198,080. WA (US); Lowell L. Wood, Jr., Bellevue, WA (US); Jeremiah James (51) Int. C. Zartman, Montgomery, AL (US) GOIN 33/48 (2006.01) (73) Assignee: The Invention Science Fund I, LLC, GOIN3L/00 (2006.01) Bellevue, WA (US) G06F 17/50 (2006.01) (*) Notice: Subject to any disclaimer, the term of this G06F 9/00 (2011.01) patent is extended or adjusted under 35 (52) U.S. C. U.S.C. 154(b) by 402 days. CPC ...... G06F 19/3468 (2013.01) (21) Appl. No.: 12/802,149 USPC ...... 703/1702/19, 702/27 (22) Filed: May 28, 2010 (58) Field of Classification Search None (65) Prior Publication Data See application file for complete search history. US 2011 FOO28945A1 Feb. 3, 2011 (56) References Cited Related U.S. Application Data U.S. PATENT DOCUMENTS (63) Continuation-in-part of application No. 12/802,148, 4,352.581 A 10, 1982 Leuthold et al. filed on May 28, 2010, and a continuation-in-part of 4,627,853. A 12/1986 Campbell et al. application No. 12/806,637, filed on May 28, 2010, and a continuation-in-part of application No. (Continued) 127657,604, filed on Jan. 22, 2010, and a FOREIGN PATENT DOCUMENTS continuation-in-part of application No. 127657,605, filed on Jan. 22, 2010, and a continuation-in-part of WO WO95/24929 9, 1995 application No. 127657,606, filed on Jan. 22, 2010, and WO WO96,40947 12/1996 a continuation-in-part of application No. 127657,607, (Continued) filed on Jan. 22, 2010, and a continuation-in-part of application No. 127657,608, filed on Jan. 22, 2010, and OTHER PUBLICATIONS a continuation-in-part of application No. 127657,609, Auxotrophic, 2007, one page. 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No. 127657,606, entitled COM APPLICATIONS POSITIONS AND METHODS FOR THERAPEUTIC DELIVERY WITH MICROORGANISMS, naming Roder The present application is related to and claims the benefit ick A. Hyde, Edward K.Y. Jung, Royce A. Levien, Robert W. of the earliest available effective filing date(s) from the fol Lord, Mark A. Malamud, John D. Rinaldo, Jr. and Lowell L. lowing listed application(s) (the “Related Applications') 10 Wood, Jr. as inventors, filed 22 Jan. 2010, which is currently (e.g., claims earliest available priority dates for other than co-pending, or is an application of which a currently co provisional patent applications or claims benefits under 35 pending application is entitled to the benefit of the filing date. USC S 119(e) for provisional patent applications, for any and For purposes of the USPTO extra-statutory requirements, all parent, grandparent, great-grandparent, etc. applications the present application constitutes a continuation-in-part of 15 U.S. patent application Ser. No. 127657,607, entitled COM of the Related Application(s)). POSITIONS AND METHODS FOR THERAPEUTIC DELIVERY WITH MICROORGANISMS, naming Roder RELATED APPLICATIONS ick A. Hyde, Edward K.Y. Jung, Royce A. Levien, Robert W. Lord, Mark A. Malamud, John D. Rinaldo, Jr. and Lowell L. For purposes of the USPTO extra-statutory requirements, Wood, Jr. as inventors, filed 22 Jan. 2010, which is currently the present application constitutes a continuation-in-part of co-pending, or is an application of which a currently co U.S. patent application Ser. No. 12/802,148, entitled pending application is entitled to the benefit of the filing date. DEVICE INCLUDING ALTERED MICROORGANISMS, For purposes of the USPTO extra-statutory requirements, AND METHODS AND SYSTEMS OF USE, naming Dario the present application constitutes a continuation-in-part of G. Amodei, Mahalaxmi Gita Bangera, Xiaoyan Robert Bao, 25 U.S. patent application Ser. No. 127657,608, entitled COM Anna Bershteyn, Brett Bethke, Philip A. Eckhoff, Kevin POSITIONS AND METHODS FOR THERAPEUTIC Michael Esvelt, Kyle Gustafson, Edward K.Y. Jung, William DELIVERY WITH MICROORGANISMS, naming Roder Michael Kaminsky, Jordin T. Kare, Lily Yvonne Kim, Eric C. ick A. Hyde, Edward K.Y. Jung, Royce A. Levien, Robert W. Leuthardt, Erez Lieberman, Ankur Moitra, Christopher Lord, Mark A. Malamud, John D. Rinaldo, Jr. and Lowell L. Somogyi, Clarence T. Tegreene, Lowell L. Wood, Jr. and 30 Wood, Jr. as inventors, filed 22 Jan. 2010, which is currently Jeremiah James Zartman as inventors, filed 28 May 2010, co-pending, or is an application of which a currently co which is currently co-pending, or is an application of which a pending application is entitled to the benefit of the filing date. currently co-pending application is entitled to the benefit of For purposes of the USPTO extra-statutory requirements, the filing date. the present application constitutes a continuation-in-part of For purposes of the USPTO extra-statutory requirements, 35 U.S. patent application Ser. No. 127657,609, entitled COM the present application constitutes a continuation-in-part of POSITIONS AND METHODS FOR THERAPEUTIC U.S. patent application Ser. No. 12/806,637, entitled DELIVERY WITH MICROORGANISMS, naming Roder DEVICE INCLUDING ALTERED MICROORGANISMS, ick A. Hyde, Edward K.Y. Jung, Royce A. Levien, Robert W. AND METHODS AND SYSTEMS OF USE, naming Dario Lord, Mark A. Malamud, John D. Rinaldo, Jr. and Lowell L. G. Amodei, Mahalaxmi Gita Bangera, Xiaoyan Robert Bao, 40 Wood, Jr. as inventors, filed 22 Jan. 2010, which is currently Anna Bershteyn, Brett Bethke, Philip A. Eckhoff, Kevin co-pending, or is an application of which a currently co Michael Esvelt, Kyle Gustafson, Edward K.Y. Jung, William pending application is entitled to the benefit of the filing date. Michael Kaminsky, Jordin T. Kare, Lily Yvonne Kim, Eric C. For purposes of the USPTO extra-statutory requirements, Leuthardt, Erez Lieberman, Ankur Moitra, Christopher the present application constitutes a continuation-in-part of Somogyi, Clarence T. Tegreene, Lowell L. Wood, Jr. and 45 U.S. patent application Ser. No. 12/462,114, entitled Jeremiah James Zartman as inventors, filed 28 May 2010, DEVICE INCLUDING BONE CAGE AND METHOD FOR which is currently co-pending, or is an application of which a TREATMENT OF DISEASE IN A SUBJECT, naming Ed currently co-pending application is entitled to the benefit of Harlow, Edward K.Y. Jung, Robert Langer, Eric C. Leuthardt, the filing date. Elizabeth A. Sweeney and Lowell L. Wood, Jr. as inventors, For purposes of the USPTO extra-statutory requirements, 50 filed 28 Jul. 2009, which is currently co-pending, or is an the present application constitutes a continuation-in-part of application of which a currently co-pending application is U.S. patent application Ser. No. 127657,604, entitled COM entitled to the benefit of the filing date. POSITIONS AND METHODS FOR THERAPEUTIC For purposes of the USPTO extra-statutory requirements, DELIVERY WITH MICROORGANISMS, naming Roder the present application constitutes a continuation-in-part of ick A. Hyde, Edward K.Y. Jung, Royce A. Levien, Robert W. 55 U.S. patent application Ser. No. 1 1/975,605, entitled BLOOD Lord, Mark A. Malamud, John D. Rinaldo, Jr. and Lowell L. BRAIN BARRIER DEVICE, naming Ed Harlow, Roderick Wood, Jr. as inventors, filed 22 Jan. 2010, which is currently A. Hyde, Edward K. Y. Jung, Robert Langer, Eric C. co-pending, or is an application of which a currently co Leuthardt, and Lowell L. Wood, Jr. as inventors, filed 18 Oct. pending application is entitled to the benefit of the filing date. 2007, which is currently co-pending, or is an application of For purposes of the USPTO extra-statutory requirements, 60 which a currently co-pending application is entitled to the the present application constitutes a continuation-in-part of benefit of the filing date. U.S. patent application Ser. No. 127657,605, entitled COM For purposes of the USPTO extra-statutory requirements, POSITIONS AND METHODS FOR THERAPEUTIC the present application constitutes a continuation-in-part of DELIVERY WITH MICROORGANISMS, naming Roder U.S. patent application Ser. No. 11/974,750, entitled BONE ick A. Hyde, Edward K.Y. Jung, Royce A. Levien, Robert W. 65 SEMI-PERMEABLE DEVICE, naming Ed Harlow, Edward Lord, Mark A. Malamud, John D. Rinaldo, Jr. and Lowell L. K. Y. Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. Wood, Jr. as inventors, filed 22 Jan. 2010, which is currently Wood, Jr. as inventors, filed 15 Oct. 2007, which is currently US 8,682,619 B2 3 4 co-pending, or is an application of which a currently co For purposes of the USPTO extra-statutory requirements, pending application is entitled to the benefit of the filing date. the present application constitutes a continuation-in-part of For purposes of the USPTO extra-statutory requirements, U.S. patent application Ser. No. 1 1/701,163, entitled DIA the present application constitutes a continuation-in-part of TOM DEVICE, naming Edward K. Y. Jung, Robert Langer, U.S. patent application Ser. No. 11/974,852, entitled BONE and Eric C. Leuthardt as inventors, filed 31 Jan. 2007, which SEMI-PERMEABLE DEVICE, naming Ed Harlow, Edward is currently co-pending, or is an application of which a cur K. Y. Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. rently co-pending application is entitled to the benefit of the Wood, Jr. as inventors, filed 15 Oct. 2007, which is currently filing date. co-pending, or is an application of which a currently co For purposes of the USPTO extra-statutory requirements, pending application is entitled to the benefit of the filing date. 10 the present application constitutes a continuation-in-part of For purposes of the USPTO extra-statutory requirements, U.S. patent application Ser. No. 1 1/452,019, entitled BLOOD the present application constitutes a continuation-in-part of BRAIN BARRIER DEVICE, naming Ed Harlow, Roderick U.S. patent application Ser. No. 11/974,798, entitled BONE A. Hyde, Edward K. Y. Jung, Robert Langer, Eric C. SEMI-PERMEABLE DEVICE, naming Ed Harlow, Edward 15 Leuthardt, and Lowell L. Wood, Jr. as inventors, filed 12 Jun. K. Y. Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. 2006, which is currently co-pending, or is an application of Wood, Jr. as inventors, filed 15 Oct. 2007, which is currently which a currently co-pending application is entitled to the co-pending, or is an application of which a currently co benefit of the filing date. pending application is entitled to the benefit of the filing date. For purposes of the USPTO extra-statutory requirements, For purposes of the USPTO extra-statutory requirements, the present application constitutes a continuation-in-part of the present application constitutes a continuation-in-part of U.S. patent application Ser. No. 1 1/451,994, entitled CHOR U.S. patent application Ser. No. 11/906,664, entitled BONE OID PLEXUS DEVICE, naming Ed Harlow, Roderick A. CELL DELIVERY DEVICE, naming Ed Harlow, Edward K. Hyde, Edward K. Y. Jung, Robert Langer, Eric C. Leuthardt, Y. Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. and Lowell L. Wood, Jr. as inventors, filed 12 Jun. 2006, Wood, Jr. as inventors, filed 2 Oct. 2007, which is currently 25 which is currently co-pending, or is an application of which a co-pending, or is an application of which a currently co currently co-pending application is entitled to the benefit of pending application is entitled to the benefit of the filing date. the filing date. For purposes of the USPTO extra-statutory requirements, For purposes of the USPTO extra-statutory requirements, the present application constitutes a continuation-in-part of the present application constitutes a continuation-in-part of U.S. patent application Ser. No. 11/906,581, entitled BONE 30 U.S. patent application Ser. No. 1 1/451,986, entitled CHOR CELL DELIVERY DEVICE, naming Ed Harlow, Edward K. OID PLEXUS DEVICE, naming Ed Harlow, Roderick A. Y. Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. Hyde, Edward K.Y. Jung, Robert Langer, Eric C. Leuthardt, Wood, Jr. as inventors, filed 2 Oct. 2007, which is currently and Lowell L. Wood, Jr. as inventors, filed 12 Jun. 2006, co-pending, or is an application of which a currently co which is currently co-pending, or is an application of which a pending application is entitled to the benefit of the filing date. 35 currently co-pending application is entitled to the benefit of For purposes of the USPTO extra-statutory requirements, the filing date. the present application constitutes a continuation-in-part of For purposes of the USPTO extra-statutory requirements, U.S. patent application Ser. No. 11/906,580, entitled BONE the present application constitutes a continuation-in-part of CELL DELIVERY DEVICE, naming Ed Harlow, Edward K. U.S. patent application Ser. No. 1 1/389.268, entitled BLOOD Y. Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. 40 BRAIN BARRIER DEVICE, naming Ed Harlow, Roderick Wood, Jr. as inventors, filed 2 Oct. 2007, which is currently A. Hyde, Edward K. Y. Jung, Robert Langer, Eric C. co-pending, or is an application of which a currently co Leuthardt, and Lowell L. Wood, Jr. as inventors, filed 24 Mar. pending application is entitled to the benefit of the filing date. 2006, which is currently co-pending, or is an application of For purposes of the USPTO extra-statutory requirements, which a currently co-pending application is entitled to the the present application constitutes a continuation-in-part of 45 benefit of the filing date. U.S. patent application Ser. No. 11/900,870, entitled BONE For purposes of the USPTO extra-statutory requirements, DELIVERY DEVICE, naming Ed Harlow, Edward K. Y. the present application constitutes a continuation-in-part of Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. Wood, U.S. patent application Ser. No. 11/304,499, entitled BONE Jr. as inventors, filed 12 Sep. 2007, which is currently co SEMI-PERMEABLE DEVICE, naming Ed Harlow, Edward pending, or is an application of which a currently co-pending 50 K. Y. Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. application is entitled to the benefit of the filing date. Wood, Jr. as inventors, filed 14 Dec. 2005, which is currently For purposes of the USPTO extra-statutory requirements, co-pending, or is an application of which a currently co the present application constitutes a continuation-in-part of pending application is entitled to the benefit of the filing date. U.S. patent application Ser. No. 11/900,776, entitled BONE For purposes of the USPTO extra-statutory requirements, DELIVERY DEVICE, naming Ed Harlow, Edward K. Y. 55 the present application constitutes a continuation-in-part of Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. Wood, U.S. patent application Ser. No. 11/304,492, entitled BONE Jr. as inventors, filed 12 Sep. 2007, which is currently co CELL DELIVERY DEVICE, naming Ed Harlow, Edward K. pending, or is an application of which a currently co-pending Y. Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. application is entitled to the benefit of the filing date. Wood, Jr. as inventors, filed 14 Dec. 2005, which is currently For purposes of the USPTO extra-statutory requirements, 60 co-pending, or is an application of which a currently co the present application constitutes a continuation-in-part of pending application is entitled to the benefit of the filing date. U.S. patent application Ser. No. 11/900,773, entitled BONE For purposes of the USPTO extra-statutory requirements, DELIVERY DEVICE, naming Ed Harlow, Edward K. Y. the present application constitutes a continuation-in-part of Jung, Robert Langer, Eric C. Leuthardt, and Lowell L. Wood, U.S. patent application Ser. No. 11/304,486, entitled BONE Jr. as inventors, filed 12 Sep. 2007, which is currently co 65 DELIVERY DEVICE, naming Ed Harlow, Roderick A. pending, or is an application of which a currently co-pending Hyde, Edward K. Y. Jung, Robert Langer, Eric C. Leuthardt, application is entitled to the benefit of the filing date. and Lowell L. Wood, Jr. as inventors, filed 14 Dec. 2005, US 8,682,619 B2 5 6 which is currently co-pending, or is an application of which a In an embodiment, a system comprises at least one com currently co-pending application is entitled to the benefit of puting device; one or more instructions on a recordable the filing date. medium that when executed on the at least one computing device cause the at least one computing device to receive a SUMMARY first input associated with a first possible dataset, the first possible dataset including data representative of at least one The present disclosure relates to various devices, systems, parameter for making or administering at least one treatment methods (including computer-implemented methods), and device to at least one biological tissue, wherein the at least one computer program products relating to administering altered treatment device includes at least one semi-permeable barrier microorganisms to a biological tissue. 10 structured to Substantially enclose at least one auxotrophic In an embodiment, a device, comprises at least one semi microorganism; wherein the at least one auxotrophic micro permeable barrier Substantially enclosing at least one aux organism includes at least one nucleic acid construct encod otrophic microorganism, the at least one auxotrophic micro ing at least one therapeutic agent; and one or more instruc tions on a recordable medium that when executed on the at organism including at least one nucleic acid construct least one computing device cause the computing device to encoding at least one therapeutic agent, the barrier defining an generate an output to a user readable display. interior region and exterior region; and wherein the device In an embodiment, a computer-implemented method com includes at least one metabolite required by the at least one prises executing one or more instructions located on a record auxotrophic microorganism. able medium for regulating dispensing of at least one treat In an embodiment, an implantable device, comprises an ment device including at least one semi-permeable barrier semi-permeable barrier Substantially enclosing at least one Substantially enclosing at least one auxotrophic microorgan auxotrophic microorganism, the barrier defining an interior ism, the at least one auxotrophic microorganism including at region and exterior region of the device; the interior region least one nucleic acid construct encoding at least one thera including at least one reservoir configured for containing the peutic agent, the barrier defining an interior region and exte at least one auxotrophic microorganism, wherein the at least 25 rior region; wherein the device includes at least one metabo one auxotrophic microorganism includes at least one nucleic lite required by the at least one auxotrophic microorganism. acid construct encoding at least one therapeutic agent; at least In an embodiment, a computer program product, com one port configured to allow transport of the at least one prises a recordable medium bearing one or more instructions therapeutic agent to the exterior region of the device; and at for regulating dispensing of at least one treatment device least one osmotic pump configured to facilitate transport of 30 including an semi-permeable barrier structured to Substan the at least one therapeutic agent through the at least one port. tially enclose at least one auxotrophic microorganism, and In an embodiment, a method of treating a disease or con wherein the at least one auxotrophic microorganism includes dition in a Subject, comprises administering at least one at least one nucleic acid construct encoding at least one thera implantable device including an semi-permeable barrier peutic agent. structured to Substantially enclose at least one auxotrophic 35 microorganism, the structure defining an interior region and BRIEF DESCRIPTION OF THE FIGURES an exterior region of the device; wherein the at least one auxotrophic microorganism includes at least one nucleic acid FIGS. 1A and 1B depict a diagrammatic view of an aspect construct encoding at least one therapeutic agent; and of an embodiment of a device that substantially encloses at wherein the at least one implantable device dispenses to the 40 least one altered microorganism. subject an effective amount of the at least one therapeutic FIGS. 2A, 2B, and 2C depict a diagrammatic view of an agent. aspect of an embodiment of a device that substantially In an embodiment, a composition, comprises at least one encloses at least one altered microorganism. auxotrophic microorganism including at least one pH induc FIGS. 3A, 3B, and 3C depict a diagrammatic view of an ible promoter operably coupled to at least one heterologous 45 aspect of an embodiment of a device, with closable openings. genetic element encoding at least one therapeutic agent for at FIGS. 4A and 4B depict a diagrammatic view of an aspect least one biological tissue, and including at least one genetic of an embodiment of a device. FIG. 4A shows a cross-sec element inducible to initiate death of the at least one aux tional view with the endothelial cell ablumenal surface ori otrophic microorganism; and wherein the at least one com ented toward the exterior of the device. FIG. 4B shows a position includes at least one metabolite required by the at 50 cross-sectional view with the endothelial cell ablumenal Sur least one auxotrophic microorganism. face oriented toward the internal cavity. In an embodiment, a system comprises at least one com FIGS.5A and 5B depict a diagrammatic view of an aspect puting device; at least one treatment device including at least of an embodiment of a device. FIG. 5A shows a cross-sec one semi-permeable barrier Substantially enclosing at least tional view with the epithelial cell apical surface oriented one auxotrophic microorganism, the at least one auxotrophic 55 toward the exterior of the device. FIG. 5B shows a cross microorganism including at least one nucleic acid construct sectional view with the epithelial cell apical surface oriented encoding at least one therapeutic agent, the barrier defining an toward the internal cavity. interior region and exterior region; wherein the device FIGS. 6A, 6B, 6C, and 6D show scanning electron micro includes at least one metabolite required by the at least one graphs of centric and pinnate diatoms. FIG. 6A shows frus auxotrophic microorganism; the treatment device further 60 tules and girdle bands of a centric diatom, Biddulphia reticu comprising at least one pump for dispensing at least one lata (size bar-10 um). FIG. 6B shows frustules, raphes, and therapeutic agent, the pump including electronic circuitry girdle bands of two Diplomeis sp. pennate diatoms (size configured to send or receive signals from the computing bar=10 um). FIG. 6C shows frustules of a centric diatom, device; and one or more instructions on a recordable medium Eupodiscus radiatus (size bar-20 Lum). FIG. 6D shows frus that when executed on the computing device cause the com 65 tules and girdle bands of a centric diatom, Melosira varians puting device to regulate dispensing of the at least one thera (size bar=10 um). (PLoS Biol. (2004) 2010):e306: 1512 peutic agent from the at least one treatment device. 1515). US 8,682,619 B2 7 8 FIGS. 7A and 7B show scanning electron micrographs of ods, and systems for preventing or treating an infectious diatoms with interlocking fingerlike protuberances at the clo disease or a pathological condition in the Subject are dis sure of the frustules. FIG. 7A shows Cymatoseira belgica. closed. The pathological condition includes, but is not limited FIG. 7B shows Aulacoseira italica (left) and Aulacoseira to, infectious disease, neoplastic disease, atherosclerosis, valida (right). 5 hypertension, autoimmune disease, diabetes, or Substance FIG. 8 depicts a partial view of particular genetic elements addiction. utilized in various embodiments disclosed herein. In an embodiment, the device includes a semi-permeable FIG.9 depicts a partial view of particular genetic elements barrier Substantially enclosing at least one altered microor utilized in various embodiments disclosed herein. ganism, the semi-permeable barrier defining an interior FIGS. 10A, 10B, and 10C depict diagrammatic views of 10 region and an exterior region of the barrier. In an embodiment, various aspects of a device. FIG.10C is a cross-sectional view the at least one altered microorganism includes at least one of FIG. 10A. auxotrophic microorganism. In an embodiment, the device FIG. 11 depicts diagrammatic views of various aspects of a includes at least one metabolite (e.g., an essential nutrient) device. required by the at least one altered microorganism. In an FIG. 12 depicts a partial view of a method relating to 15 embodiment, the device includes at least one semi-permeable various aspects of embodiments disclosed herein. barrier Substantially enclosing at least one auxotrophic FIG. 13 depicts a partial view of a system relating to vari microorganism, the at least one auxotrophic microorganism ous aspects of the embodiments disclosed herein. including at least one nucleic acid construct encoding at least FIG. 14 depicts a partial view of FIG. 13. one therapeutic agent, the barrier defining an interior region FIG. 15 depicts a partial view of FIG. 13. and exterior region; and wherein the device includes at least FIG. 16 depicts a partial view of FIG. 13. one metabolite required by the at least one auxotrophic FIG. 17 depicts a partial view of FIG. 13. microorganism. FIG. 18 depicts a partial view of FIG. 13. In an embodiment, the semi-permeable barrier provides FIG. 19 depicts a partial view of a system relating to vari selectivity for egress from the interior region to the exterior ous aspects of the embodiments disclosed herein. 25 region of the barrier. In an embodiment, the semi-permeable FIG. 20 depicts a partial view of FIG. 19. barrier provides selectivity for ingress from the exterior FIG. 21 depicts a partial view of FIG. 19. region to the interior region of the barrier. In an embodiment, FIG.22 depicts a partial view of FIG. 19. the device includes one or more bone barriers, diatom barri FIG. 23 depicts a partial view of FIG. 19. ers, polymer or copolymer barriers, protein barriers, element FIG. 24 depicts a partial view of FIG. 19. 30 barriers, vitamin barriers, mineral barriers, chloroplast barri FIG. 25 depicts a partial view of FIG. 19. ers, or carbohydrate barriers. In an embodiment, the bone FIG. 26 depicts a partial view of a computer-implemented barrier includes a bone cage. In an embodiment, the diatom method relating to various aspects of the embodiments dis barrier includes a diatom cage. In an embodiment, the poly closed herein. mer or copolymer barrier includes a polymer or copolymer FIG. 27 depicts a partial view of FIG. 26. 35 Cage. FIG. 28 depicts a partial view of FIG. 26. In an embodiment, the device is useful in a method for FIG. 29 depicts a computer program product relating to vaccinating a subject. In an embodiment, the device is useful various aspects of the embodiments disclosed herein. in a method for treating any disease or condition where an FIG. 30 depicts a partial view of FIG. 29. increase in the immune response in the Subject is desired. The FIG. 31 depicts a partial view of FIG. 29. 40 device is useful in a method for treating an infectious disease or neoplastic disease in the Subject. In an aspect, stem cells or DETAILED DESCRIPTION tissues or other cell or tissue types can be configured to restructure the one or more semi-permeable barriers. In the following detailed description, reference is made to The device including one or more semi-permeable barriers the accompanying drawings, which form a parthereof. In the 45 is configured for implantation in a soft tissue of the Subject. In drawings, similar symbols typically identify similar compo an embodiment, the device is configured for injection into the nents, unless context dictates otherwise. The illustrative Soft tissue of a subject using a syringe. In an embodiment, the embodiments described in the detailed description, drawings, device is configured to be at least one of biocompatible or and claims are not meant to be limiting. Other embodiments biodegradable. In an embodiment, the device including one or may be utilized, and other changes may be made, without 50 more semi-permeable barriers can remain in the Soft tissue departing from the spirit or scope of the Subject matter pre over an extended period of time to deliver at least one thera sented here. peutic agent to the Subject to treat a pathological condition. The present application uses formal outline headings for The device including one or more cages can deliver a dosage clarity of presentation. However, it is to be understood that the of the immunogen and the adjuvant over a long term. Follow outline headings are for presentation purposes, and that dif 55 ing injection into a soft tissue of the Subject, the device includ ferent types of Subject matter may be discussed throughout ing one or more bone cages can deliver a temporal series of the application (e.g., method(s) may be described under com vaccinations or booster vaccinations as may be required for a position heading(s) and/or kit headings; and/or descriptions specific vaccination schedule. The device can be configured of single topics may span two or more topic headings). Hence, to provide one or more chambers and/or multiple pore sizes the use of the formal outline headings is not intended to be in 60 for controlled release of the one or more immunogens and one any way limiting. or more adjuvants. Devices, methods, and systems are disclosed herein that In an embodiment, the device including one or more semi relate to delivering at least one therapeutic agent to a biologi permeable barriers refers to a rigid, semi-rigid, or otherwise cal tissue or organ. In an embodiment, the biological tissue or structurally Supportive structure with one or more external organ is part of a subject, as described herein. 65 walls, and at least one internal cavity. In an embodiment, the Devices, methods, and systems are disclosed herein for device including one or more semi-permeable barriers refers stimulating an immune response in a Subject. Devices, meth to a pliable structure. In an embodiment, the device includes US 8,682,619 B2 10 at least one of a rigid, semi-rigid, or pliable structure. In an The trigger can stimulate immediate release of the at least one embodiment, the semi-permeable barrier includes at least one therapeutic agent from the device. Alternatively, the trigger interior region and at least one exterior region. can stimulate the synthesis of the at least one therapeutic In an embodiment, one or more semi-permeable barriers agent by cells incorporated in the device. The trigger can be a can be structurally supportive to include, or contain biologi biomolecule. Examples of trigger biomolecules include, but cally active molecules, e.g., one or more immunogens and are not limited to, pathogen associated biomolecules (e.g., one or more adjuvants, or one or more cells or tissues config toxins, polysaccharides, double stranded RNA, CpG poly ured to produce at least one therapeutic agent, Such as one or nucleotides), tumor associated biomolecules (e.g., tumor more immunogens and/or one or more adjuvants. In an antigens, tumor markers), other disease associated biomol embodiment, the device including one or more cages can be 10 ecules (e.g., B-amyloid), allergens (e.g., food allergen), or configured to deliver the at least one therapeutic agent, Such other biomolecules (e.g., drugs of abuse). Alternatively, the as one or more immunogens and the one or more adjuvants to trigger can be a physiological change induced by the patho the soft tissue of a Subject and to raise an immune response to gen, tumor, disease, or allergic response. Examples of a the immunogen providing a method for treating a pathologi 15 physiological change include, but are not limited to, changes cal condition in the Subject. In an embodiment, the device in pH, temperature, osmolarity, hypoxia, and ion concentra including one or more cages can be configured to be non tions. Examples of a physiological change further include, but weight-bearing to the skeletal structure when implanted into are not limited to, increases in concentrations of endogenous a soft tissue of a Subject. In an embodiment, the device includ compounds in the Subject such as radical oxygen species, ing one or more cages is not configured to provide intrinsic cytokines, nitric oxide, anti-microbial peptides, or pro-in support to the bone or skeletal structure of the subject. flammatory molecules. In an embodiment, one or more immunogens and one or The device can be implantable indicating it is able to be more adjuvants, optionally in combination with a semi-per placed within a subject. The device including one or more meable membrane and/or one or more cells or tissues, can be semi-permeable barriers can be implanted by methods placed within the internal cavity of the semi-permeable bar 25 including, but not limited to, Surgery, injection, Suppository, rier. In an embodiment, one or more immunogens and the one and inhalation. The device including one or more semi-per or more adjuvants in combination with the one or more cells meable barriers can be placed, for example, Subcutaneously, or tissues may not include bone tissue. In an aspect, the cells intramuscularly, intraperitoneally, intravenously, intravitre or tissue may include stem cells or progenitor cells, e.g., ally, intra-arterially, intraarteriolarly, in capillary beds, Sub mesodermal cells, osteoblasts or osteoclasts, configured to 30 dermally, intradermally, orally, rectally, or nasally. The synthesize or degrade bone tissue. In an aspect, the one or device including one or more semi-permeable barriers can be more cells or tissues are modified to produce one or more implanted during a surgical procedure, or can be injected immunogens and/or one or more adjuvants. The external wall using, for example, a hollow bore needle, such as those used of the device can be any shape, including, but not limited to, for biopsies. Alternatively, injection can be by a gun, Such as spherical, oval, rectangular, Square, trapezoidal or modified 35 those used for anesthetic darts. The device including one or versions of these shapes. The internal cavity, or interior of the more semi-permeable barriers can be implanted in any loca device, can also be any shape, including, but not limited to, tion in a Subject appropriate for the desired treatment, Such spherical, oval, rectangular, Square, trapezoidal or modified locations are well-known to health care workers including, versions of these shapes. Moreover, the internal cavity of the but not limited to, physicians and nurses, as well as Veterinary, device can be configured to include or define one or more 40 animal husbandry, fish, game, Zoo, bird, reptile, and exotic portions that are in fluid communication or are isolated por animal officials. tions from each other. The device including one or more The device including one or more semi-permeable barriers semi-permeable barriers can be configured to release the at can be implanted in well-vascularized soft tissue, including, least one therapeutic agent (e.g., one or more immunogens but not limited to, liver, kidney, muscle, lung, cardiac and/or and the one or more adjuvants) in a timed release manner over 45 brain tissue. In an aspect, the device including one or more an extended time frame according to desired or recommended semi-permeable barriers is implanted in less well-vascular dosage and timing of a vaccination schedule. ized tissue including, but not limited to, joints, cartilage, and In an embodiment, the release of the at least one therapeu fat. The device including one or more semi-permeable barri tic agent from the semi-permeable barrier can be temporally ers can be implanted behind the blood brain barrier. The controlled. Temporal release can be controlled by the prop 50 device including a semi-permeable barrier can be implanted erties of the semi-permeable barrier, e.g., compartments, wall in the bladder, uterus, or vagina. thickness, or pores in the cage, the formulation of the at least Biocompatible refers to a material the body generally one therapeutic agent, or a combination thereof. Temporally accepts without a significant immune response/rejection or controlled release of the at least one therapeutic agent from excessive fibrosis. In an aspect, Some immune response and/ the device is useful for vaccines or other therapeutic agents 55 or fibrosis is desired. In an aspect, vascularization is desired. requiring multiple doses (e.g., primary immunization and In an aspect, vascularization is not desired. secondary immunization to establish memory cells respon The device including one or more semi-permeable barriers sive to the pathogenic organism or pathogenic condition). For can be implanted in a Subject including mammal, reptile, bird, example, Vaccines recommended for multiple doses can amphibian, or fish. In an aspect, the Subject includes domes include providing one dosage of the device including the one 60 ticated, wild, research, Zoo, sports, pet, primate, marine, and or more bone cages to the Subject maintaining the temporally farm animals. The animal can be a mammal. The mammal can controlled release of the one or more immunogen and the one be a primate. In a further aspect, the primate can be a human. or more adjuvant for up to 24 months. Animals include, but are not limited to, human, bovine, por The release of the at least one therapeutic agent from the cine, Swine, Ovine, murine, canine, avian, feline, equine, or device can be controlled, for example, by a trigger, a pump, a 65 rodent. Domesticated and/or farm animals include, but are not biomolecule, a specific analyte, a pathogen or tumor cell, limited to, chickens, horses, cattle, pigs, sheep, donkeys, pulse of radiation, or an externally-administered compound. mules, rabbits, goats, ducks, geese, chickens, and turkeys. US 8,682,619 B2 11 12 Wild animals can include, but are not limited to, non-human 107 uL., up to 10°uL, up to 10u L, up to 10 uL., up to 10 primates, bear, deer, elk, raccoons, squirrels, wolves, coyotes, uL to 0.01 uL., up to 0.1 uL., up to 1 uL., up to 10 uL., up to 100 opossums, foxes, skunks, and cougars. Research animals uL or up to 1000 uL. include, but are not limited to, rats, mice, hamsters, guinea The internal cavity of the semi-permeable barrier can hold pigs, rabbits, pigs, dogs, cats, and non-human primates. Pets one or more cells. Inanaspect, the cell can be the immunogen, include, but are not limited to, dogs, cats, gerbils, hamsters, e.g., a live, attenuated or inactivated pathogen or tumor cell. guinea pigs and rabbits. Reptiles include, but are not limited In an aspect, the cell can be engineered to generate one or to, Snakes, lizards, alligators, crocodiles, iguanas, and turtles. more immunogen and/or one or more adjuvant. Examples of Avian animals include, but are not limited to, chickens, ducks, cell types that can be included in the cavity of the semi 10 permeable barrier include, but are not limited to, a virus, a geese, owls, sea gulls, eagles, hawks, and falcons. Fish bacterium, a fungus, aparasite, or a mammaliancell. The type include, but are not limited to, farm-raised, wild, pelagic, of cell depends upon the nature of the vaccine and the immu coastal, sport, commercial, fresh water, salt water, and tropi nogen. In an aspect, the type of cell and the number of cells cal. Marine animals include, but are not limited to, whales, held in the cavity may be dependent upon the size of the cells sharks, seals, sea lions, walruses, penguins, dolphins, and 15 relative to the size of the cavity. Viruses can range in diameter fish. from about 20 nm to about 400 nm. The human immunode As discussed herein, in an embodiment the semi-perme ficiency virus (HIV) is approximately 90 to 160 nm in diam able barrier includes at least one external wall. In an embodi eter. Bacteria can range in size from about 0.1 um to about 600 ment, the external wall of the one or more semi-permeable um over a single dimension. Escherichia coli represent a barriers can be any dimension, preferably an integerum from bacillus of about average size measuring 1 to 1.5um in width about 1 um to about 1,000 um including approximately, but and 2 to 6 um in length. In an aspect, the cell can be a fungus not limited to, 2 um, 3 um, 4 Jum, 5um, 8 um, 10um, 12 um, that is a genetically engineered yeast strain. Saccharomyces 15um, 20 Lum, 25um, 50 um, 100 um, 200 um, 300 um, 500 cerevisiae, a common yeast strain, ranges in diameter from 3 um, 600 um, 800 um and 1,000 um. In an aspect, the external to 6 Jum. In an aspect, the cell can be a blood borne parasite, wall can be approximately 1 um to 1,000 um, 2 um to 500 um, 25 e.g., Plasmodium. In another example, blood borne parasites 3 um to 250 um, 4 um to 100 um, 5um to 50 um, 5um to 10 range in size from about 1 to 30 um by about 1 to 5 um um, 2 um to 20 Jum, 1 um to 50 um, 5um to 25um, or 2 um to depending upon the type of parasite and the stage of devel 8 um in width. In an aspect, the width is not uniform through opment. Plasmodium sporozoites are about 1 um in width and out the structure. about 5um in length. Mammalian cells can range in diameter In an embodiment, the semi-permeable barrier includes an 30 from about 3 um to about 100 um. For example, circulating internal cavity. In an embodiment, the diameter of the internal human tumor cells of epithelial origin are about 15 to 20 um cavity of the one or more semi-permeable barriers can be any in diameter. Chinese hamster ovary (CHO) cells, a cell type integer um from approximately 1 to approximately 1,000 commonly used for genetic engineering, are about 14.5um in including, but not limited to approximately, 2 um, 3 um, 4 um, diameter. The Volume of an average human cell ranges from 5um, 8 um, 10um, 12 Jum, 15um, 20um, 25um, 50 um, 100 35 about 500 to about 4000 cubicum. As an example, a spherical um, 200 um, 300 um, 500 um, 600 um, 800 um or 1,000 um. mammalian cell with a diameter of 10 um has a calculated In an aspect, the diameter of the internal cavity can be volume of approximately 523 cubic um while a spherical approximately 1 um to 1,000um, 2 um to 800 um, 5um to 750 bacterium with a diameter of 1 um has a calculated volume of um, 10um to 500 um, 20 um to 250 um, 10um to 100 um, 5 approximately 0.523 um, Suggesting that 1000 bacteria may um to 50 um, 1 um to 10 um, 2 um to 20 Jum, 1 um to 50 um, 40 be loaded into a space that accommodates one mammalian 50 Lum to 500 um, or 250 um to 1,000 um in width. In an cell. aspect, the diameter of the internal cavity can be approxi Additionally, implantable devices are disclosed herein that mately up to 1 mm to 1 cm in width, including, but not limited include a semi-permeable barrier Substantially enclosing at to, up to 1 mm, up to 10 mm, up to 100 mm, or up to 1 cm or least one altered microorganism, the barrier defining an inte more in width. In an aspect, the internal diameter is not 45 rior region and an exterior region, as described herein. In an uniform throughout the structure. For example, the internal embodiment, the at least one altered microorganism includes diameter can be up to 1 mm in one dimension and up to 3 cm at least one nucleic acid construct encoding at least one thera in a second dimension. peutic agent. In an embodiment, the altered microorganism The volume of the internal cavity can be any integer cubic includes an auxotrophic microorganism. In an embodiment, um from about 1 um to about 10" um including, but not 50 the device includes at least one metabolite (e.g., an essential limited to, 1 cubicum, 8 cubicum, 27 cubicum, 64 cubicum, nutrient) required by the at least one altered microorganism 125 cubic 512 cubicum, 1000 cubicum, 1700 cubicum,3400 (e.g., auxotrophic microorganism). cubicum, 8000 cubicum, 1.5x10 cubicum, 1.25x10 cubic As described herein, in an embodiment, the semi-perme um, 10 cubicum, 8x10 cubicum,3x107 cubicum, 10 cubic able barrier includes one or more of a bone barrier, diatom um, 2x10 cubic um, 5x10 cubic um, 10 cubic um, 10" 55 barrier, polymer or co-polymer barrier, protein barrier, ele cubicum 10' cubicum, and 10' cubicum. In an aspect, the ment barrier, vitamin barrier, mineral barrier, chloroplast bar volume of the internal cavity can be approximately up to 100 rier, or carbohydrate barrier. In an embodiment, the semi cubicum, up to 1000 cubicum, up to 10 cubicum, 10 cubic permeable barrier includes one or more of collagen, elastin, um, up to 10 cubicum, up to 107 cubic um, up to 10 cubic albumin, gelatin, extracellular matrix, acrylate, methacrylate, um, up to 10 cubicum, up to 10' cubicum, up to 10' cubic 60 fibrinogen, thrombin, fibrin, cellulose, poly(hydroxyal um, or up to 10' cubicum. kanoate), poly(e-caprolactone), polysaccharide, poly The liquid Volume capacity of the internal cavity can be any thioester, starch, amylase, polylactone, polyphosphaZene, integer microliter (uL) from about 10 uL to about 1000 uL polycyanaoacrylate, poly(lactic acid), poly(glycolic acid), including approximately, but not limited to, 10u L, 10uL. polycaprolactone, plasticized cellulose materials, polyvinyl 107 uL. 10 uL, 10u L, 10u L, 10u L, 0.01 uL, 0.1 uL. 65 chloride, polyvinylidene fluoride, polyurethane isocyanate, 1 uL. 10 uL. 100 uL and 1000 uL. In an aspect, the liquid polyalginate, polysulfone, polystyrene, polyvinyl alcohol, volume capacity can be approximately up to 10 uL. up to polyacrylonitrile, polymethylene oxide, polyethylene oxide, US 8,682,619 B2 13 14 polytetrafluoroethylene, polymethyl methacrylate, polya layers. In illustrative embodiments, in Coscinodiscus sp. the mide, polyether-polyamino copolymer, thermoplastic cribellum is the outer porous layer, followed by a second copolymer, polyanhydride, polydioxanone, polyorthoester, porous layer the cribrum, which is connected to a third layer, poly(propylene fumarate), polyesteramide, polyamido a porous silica plate containing the foramen holes. The pores amine, polythioester, nylon, polyacrylamide, acrylamide, of the cribellum (approx. 45 nm) are smaller than the pores of nylon, urethane, polytetrafluoroethylene, polyurethane, dim the cribrum (approx. 192 nm), which are smaller than the ethylsiloxane/methylvinylsiloxane copolymer, acetate, poly foramen holes (approx. 1150 nm). In illustrative embodi ester, dextran, calcium, silicon, phosphorous, iron, magne ments, in Thalassiosira eccentrica, the foramen pores (ap sium, manganese, Sodium, potassium, chromium, titanium, prox. 770 nm) are part of the external layer, while the internal nickel, Zinc, copper, cobalt, tungsten, silver, gold, platinum, 10 layer includes pores of approx. 43 nm, similar to the cribellum enzyme, acid, amino acid, peptide, polypeptide, protein, oli layer, above (Methods for identifying the layers, and retain gonucleotide, nucleic acid, ribonucleic acid, oligosaccharide, ing one or more of the layers are known in the art (J. Nanosci. monosaccharide, polysaccharide, glycopeptide, glycolipid, and Nanotech. (2006) 6:982–989). lipoprotein, sphingolipid, glycosphingolipid, glycoprotein, In an embodiment, diatom cages include a rigid, semi peptidoglycan, lipid, carbohydrate, metalloprotein, pro 15 rigid, or other structural Support with at least one external teoglycan, chromosome, adhesion molecule, cytokine, wall, and at least one internal cavity including at least part of chemokine, immunoglobulin, antibody, antigen, platelet, one or more of a diatom cell wall, valve, or frustule. In an extracellular matrix, blood plasma, cell wall, hormone, embodiment, the internal cavity includes at least one of a organic compound, inorganic compound, salt, cell ligand, cell semi-permeable membrane, one or more living cells or tis receptor, or chlorophyll. Sues, or one or more biologically active molecules. In an embodiment, the semi-permeable barrier includes a In an embodiment, a method for making a diatom cage diatom barrier. In an embodiment, the diatom barrier includes includes, for example, micromanipulation or nanomanipula a diatom cage. Diatom cages may include various parts or tion (e.g., micromachining or nanomachining). In an embodi whole diatom skeletons from which organic matter has been ment, Surface organic materials are removed from the frus removed. Diatoms exhibit a multitude of shapes and patterns 25 tules of diatoms, and the paired frustules make up the diatom in their siliceous valve structure (J. Nanosci. and Nanotech. cage. The paired frustules may be sealed using one or more (2005) 5: 108-119). Shape and form are inherited in diatoms standard methods, including but not limited to, at least one of as a result of auxospore formation and vegetative reproduc sealants such as glue, bands, wraps or attachments such as tion. Diatom shapes include, but are not limited to cylindrical, screws, nails, hook and eye or any other closure methods ellipsoid, cubic and needle-shaped, and include features Such 30 (optionally removable) used by a medical professional Such as, but not limited to, radial, concentric, and/or traversal ribs, as Sutures. honeycomb structures, Warren trusses, radial and/or concen In an embodiment, the diatom cage has a shape selected tric undulations, hollow spines, and/or bulging, dome-shaped from the group consisting of cylindrical, ellipsoid, cubic, and protuberances as shown for example in Fragilariopsis, needle-shaped. The diatom skeleton, or cage may optionally Thalassiosira, AsterOmphalus, Coscinodiscus, Arachnoidis 35 include features such as, but not limited to, radial, concentric, cusm, AsterOmphalus, Navicula, Chaetoceros, Chorethron, or transversal ribs, honeycomb structures, Warren trusses, and/or Odontella as well as most other diatoms. radial or concentric undulations, hollow spines, or bulging, Diatom frustules include pores, optionally surrounded by dome shaped protuberances. silica structures, presumably to allow efficient nutrient uptake In an embodiment, the size of the diatom skeleton, or cage (J. Nanosci. and Nanotech. (2005) 5: 108-119; J. Nanosci. and 40 is at least approximately 0.5um, at least approximately 1 um, Nanotech. (2006) 8:982–989). Illustrative pore sizes include at least approximately 2 um, at least approximately 3 um, at 0.8 um areolae, and cribra of 160 nm and 35 nm in Coscino least approximately 4 um, at least approximately 5 um, at discus wailesii. least approximately 6 Jum, at least approximately 7 um, at Diatom spines can be siliceous or made of chitan (J. least approximately 8 um, at least approximately 9 um, at Nanosci. and Nanotech. (2005) 5:108-119). The siliceous 45 least approximately 10 Jum, at least approximately 11 Jum, at spines can be extensive, and highly structured, including, for least approximately 12 Jum, at least approximately 15um, at example, barbs and lattice work. least approximately 18 Jum, at least approximately 20 Jum, at The geometry of the diatom siliceous structures is deter least approximately 25um, at least approximately 30 Jum, at mined by specialized organelles, the silica deposition vesicles least approximately 35 um, at least approximately at least (J. Nanosci. and Nanotech. (2005) 5:108-119). An organic 50 approximately 40 um, at least approximately 50 um, at least matrix of proteins/peptides and polysaccharides cause poly approximately 60 um, at least approximately 75 um, at least condensation of the silica, and remain an integral part of the approximately 90 um, at least approximately 100 um, at least 3-dimensional structure of the material formed. approximately 125 um, at least approximately 150 um, at Methods for removing some, most or all organic material least approximately 175um, at least approximately 200 um, from a diatom skeleton are known in the art and include, but 55 at least approximately 250 um, at least approximately 300 are not limited to, treatment with a variety of oxidizing um, at least approximately 350 um, at least approximately agents, such as KMnO and HCl or Sulfuric and nitric acid, as 400 um, at least approximately 500 um, at least approxi well as acid treatment to according to the method of Von mately 600 um, at least approximately 750 um, at least Storsch as described in Hasle and Syvertsen (Marine Diatoms approximately 1000 um, at least approximately 1250 um, at (1997) Ed. Tomas. Academic Press. San Diego, Calif., Chap 60 least approximately 1500 um, at least approximately 1750 ter 2: J. Nanosci. and Nanotech. (2005) 5:131-139; J. um, at least approximately 2000 um, at least approximately Nanosci. and Nanotech. (2005) 5: 108-119; J. Nanosci. and 2500 um, at least approximately 3000 um, at least approxi Nanotech. (2006) 6:982-989). mately 3500 um, at least approximately 4000 um, at least In an embodiment, the diatom skeleton includes one or approximately 4500 um, or at least approximately 5000 um. more of three porous layers, such as but not limited to, the 65 In an embodiment, the diatom cage is at least partially cribellum, the cribrum, and a foramen layer. The pore sizes of isolated from a naturally-occurring diatom, cultured diatom, the layers differ among diatoms, as does the order of the or modified diatom (a physical, genetic, or chemical modifi US 8,682,619 B2 15 16 cation, for example). In an embodiment, the diatom includes about 270 nm, about 280 nm, about 290 nm, about 300 nm, at least one of centric or pinnate. In an embodiment, the about 320 nm, about 340 nm, about 360 nm, about 380 nm, diatom includes at least one of eucentric, eccentric, araphid, about 400 nm, about 420 nm, about 440 nm, about 460 nm, eunotioid, monoraphid, naviculoid, cymbelloid, epithemoid, about 480 nm, about 500 nm, about 520 nm, about 540 nm, nitzschioid, or Surirelloid. In an embodiment, the diatom about 560 nm, about 580 nm, about 600 nm, about 700 nm, includes at least one of Fragilariopsis, Thalassiosira, Aster about 800 nm, about 900 nm, about 1000 nm, about 1500 nm, Omphalus, Coscinodiscus, Arachnoidiscusm, Asteromphalus, about 2000 nm, or any value less than or therebetween. Navicula, Chaetoceros, Chorethron, or Odontella. In an embodiment, the diatom cage porosity is approxi Methods for obtaining diatoms from nature are known in mately 1%, approximately 3%, approximately 5%, approxi the art. For example, diatoms can be obtained from a wide 10 mately 10%, approximately 15%, approximately 20%, variety of habitats including, but not limited to, bodies of water including salt water (e.g. oceans) and fresh water (e.g. approximately 30%, approximately 40%, approximately ponds), as well as soil (Appl. Microbiol. Biotechnol. (2003) 50%, approximately 60%, approximately 70%, approxi 60:612-623). Centric diatoms are essentially planktonic mately 80%, approximately 90%, or any value greater than or therebetween. algae, and are found in all open water masses. Pennate dia 15 toms are frequently, but not exclusively, benthic, and are In an embodiment, the diatom cage is at least partially found growing on sediments or attached to rocks or microal non-immunogenic. In general, diatoms are chemically inert gae in their benthic form. Some species are also found in soil. and bio-compatible (PLoS Biol. (2004) 2010):e306: 1512 Diatoms are abundant in the beginning of spring and autumn 1515). Methods of modifying the immunogenicity of the when nutrients are not limiting, and light intensity and day diatom cage are known in the art, and include but are not length are optimal for diatom photosynthesis. limited to, modifications relating to the immunogenicity of Methods for culturing diatoms are known in the art, and associated tissue (such as, for example, the semi-permeable include, for example, laboratory scale and commercial scale component, bone tissue, other tissue, or cells), biological cultivation using Small-scale, closely controlled photobiore materials (such as, immune modulatory compounds known in actors, and large-scale, open air systems, (Appl. Microbiol. 25 the art and discussed herein); or genetic modifications. Biotechnol. (2003) 60:612-623; Appl. Microbiol. Biotech In an embodiment, the diatom is modified (e.g., physically, nol. (2003) 60:624–632). Diatoms cultured in small-scale, chemically, or genetically modified). Biotechnological meth closely controlled bioreactors include, but are not limited to, ods of genetic and non-genetic modification and manipula Phaeodactylum tricornutum, Skeletonema costatum, Chaeto tion of nucleic acids and proteins are known in the art (Sam ceros spp., Nitzchia laevis, Cyclotella cryptica, or Haslea 30 brook and Russell. (2001) Molecular Cloning: A Laboratory Ostrearia. Diatoms cultured in large-scale, open air systems Manual 3rd edition. Cold Spring Harbor Laboratory Press; include, but are not limited to, Skeletonema, Thalassiosira, Sambrook, Fritsch, Maniatis. Molecular Cloning: A Labora Phaeodactylum, Chaetoceros, Cylindrotheca, Bellerochea, tory Manual 3' edition. includes aspiral bound, 3 volume set, Actinocyclus, Nitzschia, or Cyclotella. Although most associated with a web site as an on-line laboratory manual (on microalgae are obligate photoautotrophs and their growth is 35 the worldwide web at MolecularCloning.com). dependent on the generation of photosynthetically derived Methods for genetic engineering are known in the art for a energy, genetically engineered diatoms may be grown infer variety of diatom species, including but not limited to, P menterS. tricornutum, Cyclotella cryptica, Cylindrotheca fusiformis, In some embodiments, the diatom skeleton or cage is Navicula saprophila, and Thalassiosira weissflogii (J. Phy designed or treated to, at least partially or completely, prevent 40 col. (1995) 31:1004-1012: Mol. Gen. Genetics (1996) 252: or reduce restructuring. In some embodiments, the diatom 572-579; Appl. Biochem. Biotechnol. (1996)57/58:223-231; skeleton, structure or cage is designed or treated to be at least J. Phycol. (1999) 35:113-120; Mar. Biotech. (1999) 1:239 partially, or completely, restructured. Diatom restructuring 251; Science (2001) 292:2073-2075; Appl. Microbiol. Bio includes, but is not limited to, influx and growth of the sub technol. (2003) 60:612-623; J. Nanosci. and Nanotech. jects cells (including, but not limited to, bone cells) in or on 45 (2005) 5:5-14: FEBS J. (2005) 272:3413-3423; J. Physiol. the diatom skeleton, structure or cage. Mechanisms of (2006) 42:1059-1065). Recombinant methods include, but restructuring, treatments to modify restructuring, genes gov are not limited to, mutagenesis and transformation tech erning restructuring and methods for detecting and measuring niques, including transient and stable transfection. Stable changes in bone are known in the art and described herein. transfection has been achieved in both inducible and consti In illustrative embodiments, compounds to mediate depo 50 tutive manners. Co-transformation is effective in allowing sition and/or resorption of cells (e.g. bone) can be adminis introduction of a non-selective gene as well as a selection tered locally by being applied to, or made part of the diatom marker gene of which a variety are available for use in dia skeleton, structure, and/or cage either globally, or in localized toms. Transfection methods include, for example, micropar areas, depending on, for example, whether complete or partial ticle bombardment. restructuring is desired. In illustrative embodiments, com 55 In an embodiment, cell wall and membrane proteins were pounds can be administered by incorporation in the diatom targeted and covalently modified by heterologous expression skeleton, structure and/or cage as one of the one or more in Cylindrotheca fusiformis (J. Phycol. (1999) 35:113-120). biologically active molecules and/or part of the living cells In an embodiment, P. tricornatum was transformed with a and/or tissues, as discussed herein. gene encoding a glucose transporter (glut 1 from human In an embodiment, the pore sizes of a diatom cage range 60 erythrocytes or hup 1 from the microalga Chlorella kessleri) from about 1 nm, about 5 nm, about 10 nm, about 25 nm, that allowed growth on exogenous glucose in the absence of about 30 nm, about 50 nm, about 40 nm, about 50 nm, about light (Science (2001) 292:2073-2075), and C. cryptica was 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, genetically engineered for further biodeisel production about 110 nm, about 120 nm, about 130 nm, about 140 nm, (Appl. Biochem. Biotechnol. (1996) 57/58:223-231). In an about 150 nm, about 160 nm, about 170 nm, about 180 nm, 65 embodiment, diatom multipartite plastid targeting signals about 190 nm, about 200 nm, about 210 nm, about 220 nm, were characterized in vivo (J. Cell Sci. (2002) 115:4061 about 230 nm, about 240 nm, about 250 nm, about 260 nm, 4069; Plant J. (2005) 41: 175-183). US 8,682,619 B2 17 18 The complete genome sequence is known for at least one desired treatment, such locations are well-known to health diatom species, Thalassiosira pseudonana, and is in progress care workers including, but not limited to, physicians and for at least one other diatom species, Phaeodactylum tricor nurses, as well as Veterinary, animal husbandry, fish, game, nutum (Science (2004)306:79-86: Plant Physiol. (2005) 137: Zoo, bird, reptile, and exotic animal officials. 500-513; J. Nanosci. and Nanotech. (2005) 5:5-14). In addi 5 In an embodiment, the device includes at least one of an tion, EST databases of nonredundant cDNAs have been infusion pump, osmotic delivery system, peristaltic pump, or created and at least partially analyzed for at least P tricorna simple diffusion delivery system for administering the at least tum and Thalassiossira pseudonana (Plant Physiol. (2002) one therapeutic agent. In an embodiment, at least two thera 129:993-1002: J. Nanosci. and Nanotech. (2005) 5:5-14). peutic agents are administered by way of the device. In an In an embodiment, the diatom is genetically engineered to 10 embodiment, the device includes separate chambers config alter at least one of growth under fermentation conditions, ured to hold multiple different microorganisms. Such that glucose uptake, or expression of a glucose transporter. each chamber includes a different agent, or a different altered Although most microalgae are obligate photoautotrophs and microorganism. See, for example, U.S. Pat. App. Pub. No. their growth is dependent on the generation of photosyntheti 20090202608, which is incorporated herein by reference. cally derived energy, diatoms may be genetically engineered 15 In an embodiment, the device includes one or more gears. (or undergo mutagenesis) to allow growth under conditions For example, published reports indicate that a solution of suitable for fermentation cultivation. In an example, Phaeo Bacillus subtilis are capable of rotating gears that are approxi dactylum tricornutum was genetically engineered to express mately 380 microns in size. See, for example, Sokolov, et al., a glucose transporter through gene transfer of glut 1 or hup 1 ABSTRACT, PNAS, vol. 106, no. 51, online at 0913015107 and was cultured with the addition of exogenous glucose in (2009), which is incorporated herein by reference. As the absence of light in a microbial fermenter (Science (2001) described in the publication, the bacteria Suspended in a solu 292:2073-2075). tion can turn microgears by manipulation of oxygen in the In an embodiment, the diatom frustules, silica nanostruc solution. Also as reported, since the bacteria utilized were ture, cell wall, or membrane is modified through genetic or aerobic, decreasing the level of oxygen resulted in a decrease non-genetic engineering. In an embodiment, the diatom silica 25 in movement of the gears, while increasing the oxygen level nanostructure is modified through genetic or non-genetic resulted in increasing movement of the gears. In an embodi engineering. In an embodiment, the diatom is genetically ment, at least one gear. Such as described herein, is utilized to engineered to express one or more modified cell wall pro operate a pump, such as a peristaltic pump, for dispensing the teins, membrane proteins, or transporters. In an embodiment, at least one therapeutic agent. the diatom is genetically engineered to express one or more 30 Altered Microorganisms modified Silaffins, Silaffin-related genes, polyamines, The compositions, methods, devices, and systems polyamine-related genes, or silicic acid transporters. A vari described herein relate to at least one altered microorganism ety of diatom cell wall and membrane proteins as well as their (e.g., an auxotrophic microorganism) that is configured to encoding nucleic acid sequences are known in the art, includ deliver at least one agent (e.g., a therapeutic agent) to at least ing but not limited to, polyamines, Silaffins, and silicic acid 35 one Substrate (e.g., biological tissue). In an embodiment, the transporters (ChemBioChem (2006) 7:1419-1427; Mol. & at least one agent is produced by at least one nucleic acid Cell. Proteomics (2006) 5:182-193: FEBS Lett. (2005) 579: construct included in the at least one altered microorganism. 3765-3769; J. Nanosci. and Nanotech. (2005) 5:146-157; J. In an embodiment, the device provides at least one metabolite Nanosci. and Nanotech. (2005) 5:158-166). In an embodi required by the at least one altered microorganism. In an ment, cell wall and membrane proteins were targeted and 40 embodiment, the at least one altered microorganism includes covalently modified by heterologous expression in Cylin at least one inducible genetic element configured to initiate drotheca fusiformis (J. Phycol. (1999) 35:113-120). death of the altered microorganism. In an embodiment, a method for non-genetic modification In an embodiment, an altered microorganism includes a of diatoms includes cell wall modifications, are known in the microorganism that has been chemically, physically, or art (Protist (2006) 158:21-28; J. Nanosci. and Nanotech. 45 genetically modified from a naturally occurring microorgan (2005) 5:92-95; J. Nanosci. and Nanotech. (2005) 5:68-78; J. ism, or a microorganism that has been artificially synthesized. Nanosci. and Nanotech. (2005) 5:41–49). In an embodiment, In an embodiment, the altered microorganism includes an cell wall silification can be modified by changes in the growth auxotrophic microorganism. For example, in an embodiment, environment, including, but not limited to, pH, nutrient lev an auxotrophic microorganism includes at least one modifi els, light levels, temperature, and grazing. In an embodiment, 50 cation that renders it dependent on at least one metabolite or cell wall silification can be modified by Zeolitisation. In an other essential nutrient, that the microorganism is unable to embodiment, cell wall silification can be modified by culture manufacture for itself, and instead must be provided to the conditions including metals other than silica, including but microorganism (e.g., by the device). Examples of particular not limited to, germanium. metabolites are described herein, and include but are not In an embodiment, the device may be implanted by stan 55 limited to, energy sources or other metabolic pathway dard methods known in the art including, but not limited to, requirements. Surgery, injection, Suppository, and inhalation. The device In an embodiment, the growth of the altered microorgan may be placed, for example, Subcutaneously, intra-muscu ism can be directed. In one example, by allowing selective larly, intra-peritoneally, intra-venously, intra-arteriolar, in growth of the microorganism, or selective agent production capillary beds, Subdermally, intradermally, orally, rectally, 60 (e.g., lactic acid Suppressor gene), the microorganism can be nasally, into the cerebrospinal fluid space, intracranially, directed or controlled. In another example, the altered micro intraperitoneal, transurethral, or transvaginal. The device organism includes a recombinant microorganism with a may be implanted during a Surgical procedure, or may be mutation that renders the microorganism dependent on an injected using, for example, a hollow bore needle, Such as external factor for Survival (e.g., thymidylate synthase gene). those used for biopsies. Alternatively, injection may be by a 65 In another example, the altered microorganism contains an gun, Such as those used for anesthetic darts. The device can be essential gene and a control sequence that regulates expres implanted in any location in a Subject appropriate for the sion Such that an essential gene is expressed in a permissive US 8,682,619 B2 19 20 environment and not expressed in a nonpermissive environ lactis, Lactobacillus divergens, Lactobacillus farcininis, ment. In another example, the altered microorganism con Lactobacillus fermentum, Lactobacillus fomicalis, Lactoba tains a lethal gene and a control sequence that regulates cillus fructivorans, Lactobacillus fructosus, Lactobacillus expression Such that the lethal gene is expressed in a nonper gallinarum, Lactobacillus gasseri, Lactobacillus graminis, missive environment, and not expressed when in a permissive Lactobacillus halotolerans, Lactobacillus hamsteri, Lacto environment. See, for example, U.S. Patent Application Pub bacillus helveticus, Lactobacillus heterohiochii, Lactobacil lication No. 20080253990, and U.S. Patent Application Pub lus hilgardii, Lactobacillus homohiochii, Lactobacillus iners, lication No. 20080254014, each of which is incorporated Lactobacillus intestinalis, Lactobacillus jensenii, Lactoba herein by reference. cillus johnsonii, Lactobacillus kandleri, Lactobacillus kefiri, In an embodiment, the at least one altered microorganism 10 Lactobacillus kefiranofaciens, Lactobacillus kefirgranum, includes at least one of a prokaryote or a eukaryote. In an Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillus embodiment, the at least one altered microorganism includes leichmannii, Lactobacillus lindneri, Lactobacillus malefer at least one of bacteria, protozoa, rotifers, algae, or fungi. In mentans, Lactobacillus mali, Lactobacillus maltaronicus, an embodiment, the at least one altered microorganism Lactobacillus manihotivorans, Lactobacillus minor; Lacto includes at least one of a non-pathogenic strain, transgenic 15 bacillus minutus, Lactobacillus mucosae, Lactobacillus microorganism, magnetotactic microorganism, anaerobic or murinus, Lactobacillus nagelii, Lactobacillus Oris, Lactoba aerobic microorganism, food grade strain, obligate microor cillus panis, Lactobacillus parabuchneri, Lactobacillus ganism, attenuated microorganism Strain, facultative anaer paracasei, Lactobacillus paracasei Subsp. paracasei, Lacto obe, non-invasive strain, probiotic, colonizing microorgan bacillus paracasei Subsp. tolerans, Lactobacillus parakefiri, ism, element-modifying microorganism, or photosynthetic Lactobacillus paralimentarius, Lactobacillus paraplan microorganism. In an embodiment, the at least one element tarum, Lactobacillus pentosus, Lactobacillus perolens, Lac modifying microorganism includes at least one of a nitrogen tobacillus piscicola, Lactobacillus plantarum, Lactobacillus fixing microorganism, nitrifying microorganism, denitrifying pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lac microorganism, hydrocarbon-utilizing microorganism, tobacillus rimae, Lactobacillus rogosae, Lactobacillus rumi dechlorinating microorganism, or a Sulfate-reducing micro 25 nis, Lactobacillus sakei, Lactobacillus sakei Subsp. carnosus, organism. Lactobacillus sakei Subsp. sakei, Lactobacillus salivarius, In an embodiment, the at least one altered microorganism Lactobacillus salivarius Subsp. salicinius, Lactobacillus Sali includes at least one of Bifidobacterium, Lactococcus, Lac varius Subsp. salivarius, Lactobacillus Sanfranciscensis, tobacillus, Salmonella, Clostridium, Escherichia, Listeria, Lactobacillus sharpeae, Lactobacillus Suebicus, Lactobacil Streptococcus, Staphlococcus, Bacillus, Marinobacter; 30 lus trichodes, Lactobacillus uli, Lactobacillus vaccinoster Micrococcus, Dietzia, Oceanobacillus, Citriococcus, Geor cus, Lactobacillus vaginalis, Lactobacillus viridescens, Lac genia, Microbacterium, Stappia, Isoptericola, Cellulomonas, tobacillus vitulinus, Lactobacillus xylosus, Lactobacillus Rhizobia, Frankia, Klebsiella, Nocardioform Actinomycetes, yamanashiensis, Lactobacillus vananashiensis Subsp. mali, Cytophagacia, Corynebacterium, Vibrionacia, Cyanobacte Lactobacillus vananashiensis Subsp. Yamanashiensis, Lac ria, Pseudomonas, Rhastonia, Sphaerotilus, Shewanella, 35 tobacillus zeae, Clostridium novyi, Clostridium sordellii, Wolbachia, or Azotobacter, Azospirillum. In an embodiment, Bifidobacterium longum, Escherichia coli, Salmonella typh the at least one altered microorganism includes at least one of imurium, Salmonella paratyphi, Salmonella pneumoniae, Saccharomyces, Candida, Brettanomyces, Zygosaccharomy Salmonella enterica, Streptococcus pneumoniae, Streptococ ces, Yarrowia, Schizosaccharomyces, Torulaspora, Neoty cus pyogenes, Streptococcus group B, Streptococcus mutans, phodium, or Cryptococcus. 40 Streptococcus sobrinus, Streptococcus equi, Staphylococcus Some non-limiting examples of microorganisms that can ssp., Erysipelothrix rhusiopathiae, Bacillus anthracis, List be modified according to various embodiments described eria monocytogenes, Mycobacterium leprae, Mycobacterium herein include: Lactococcus garvieae, Lactococcus lactis, tuberculosis, Clostridium tetani, Clostridium botulinum, Lactococcus lactis Subsp. Cremoris, Lactococcus lactis Clostridium difficile, Clostridium perfringens, Corynebacte Subsp. hordniae, Lactococcus lactis, Lactococcus lactis 45 rium diphtheriae, Mycoplasma ssp., Rhodococcus, Nocardia, Subsp. Lactis, Lactococcus piscium, Lactococcus plantarum, Gordona, Jensenia, Shewanella, Shewandella Oneidensis, Lactococcus raffinolactis, Lactobacillus acetotolerans, Lac Dehalococcoides, Burkholderia Zenovorans, Comamonas, tobacillus acidophilus, Lactobacillus agilis, Lactobacillus Cupriavidus, Sphingomonas, Acidovorax, Desulfovibrio, algidus, Lactobacillus alimentarius, Lactobacillus amy Anabaena cylindrica, Plectonema, Nostoc commune, Rhodo lolyticus, Lactobacillus amylophilus, Lactobacillus amylo 50 bactersphaeroides, Rhodopseudomonaspalustris, or Rhodo vorus, Lactobacillus animalis, Lactobacillus aviarius, Lac bacter capsulatus, Escherichia coli Nissle 1917, Salmonella tobacillus aviarius Subsp. araffinosus, Lactobacillus aviarius typhimurium 14028, Salmonella typhimurium SL1344, Sal Subsp. aviarius, Lactobacillus bavaricus, Lactobacillus monella typhi, Salmonella abortus-ovi, Salmonella abortus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, equi, Salmonella Dublin, Salmonella gallinarum, Salmonella Lactobacillus bulgaricus, Lactobacillus carnis, Lactobacil 55 pullorum, Shigella fexneri, Shigella sonnei, Haemophilus lus casei, Lactobacillus casei Subsp. alactosus, Lactobacillus influenzae, Bordetella pertussis, Nisseria meningitides, Nis casei Subsp. casei, Lactobacillus casei Subsp. pseudoplan seria gonorrohia, Pasteuralla multocida, Yersinia pestis, tarum, Lactobacillus casei Subsp. rhamnosus, Lactobacillus Escherichia coli 4608–58, Salmonella flexneri 2a SC602, casei Subsp. tolerans, Lactobacillus catenaformis, Lactoba Escherichia coli CFTO73, Escherichia coli Top 10, Escheri cillus cellobiosus, Lactobacillus collinoides, Lactobacillus 60 chia coli MC1000, Escherichia coli NF1815, Escherichia confusus, Lactobacillus coryniformis, Lactobacillus coryni coli NF1830, Escherichia coli HB101, Escherichia coli formis Subsp. coryniformis, Lactobacillus coryniformis BD3364, Escherichia coli BD3364, Escherichia coli HfrC, Subsp. torquens, Lactobacillus Crispatus, Lactobacillus cur Escherichia coli BD3342, Escherichia coli BD3346, vatus, Lactobacillus curvatus Subsp. curvatus, Lactobacillus Escherichia coli XAC, Escherichia coli BD71, Escherichia curvatus Subsp. melibiosus, Lactobacillus delbrueckii, Lac 65 coli BD76, Escherichia coli 517.1, Lactococcus lactis tobacillus delbrueckii Subsp. bulgaricus, Lactobacillus del MG 1363, Lactococcus lactis-Thy 12, Clostridium novyi-NT, brueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. Lactobacillus plantarum NCIMB8826, Lactobacillus plan US 8,682,619 B2 21 22 tarum NCIMB8826Intl. Lactobacillus fermentum KLD, Lac least one biological tissue, Subject, or other Substrate (which tobacillus plantarum MD007, Lactobacillus plantarum may be the same or different substrate as the source of the MD007Intó, Lactococcus lactis NZ3900, MC-1 magnetotac microorganism). tic bacteria, Lactococcus lactis PH3960, Streptococcus gor In an embodiment, the altered microorganism includes at donii, Lactobacillus zeae, Streptococcus mutans, Bacteroides least one microorganism strain that naturally resides in the gut ovatus, Bacteroides fragilis, PrevOtella, Saccharomyces bou ofa subject, or has been modified to survive and/or proliferate lardii, Firmicutes, Gammaproteobacteria, PrevOtellaceae, in the gut of a Subject. In an embodiment, the semi-permeable Archaea, Listeria innocua, Staphylococcus xylosus, Staphy barrier is implanted into the gut of a subject for delivery of at lococcus carnosus, Listeria monocytogenes, Klebsiella pneu least one therapeutic agent produced by the microorganism. moniae, Azotobacter vinlandii, Anabaena cylindrica, Plec 10 tonema, Nostoc commune, Rhodobacter sphaeroides, In an embodiment, the microorganism or the device Rhodopseudomonas palustris, Rhodobacter capsulatus, includes at least one inducible genetic element configured to Clavibacter, Alcaligenes, Sphingobacterium, Phyllobacte initiate death in the at least one microorganism. For example, rium, Aeromonas, Stenotrophomonas, Acidovorax, Comamo in an embodiment, the microorganism includes inducible Sui nas, Desulfo vibrio, Steintrophomonas, Serratia, Pseudomo 15 cide genetic elements that are configured to initiate or induce nas aeruginosa, Steintrophomonas maltophilia, Serratia death in the microorganism upon encountering at least one marsescens, Variovorax, Chryseobacterium, Comamonas, inducer that regulates the genetic elements. In an embodi Acidovorax, Stenotrophomonas, Sphingobacterium, Xanth ment, the genetic elements that regulate the induction of death Omonas, Frateuria, Zoogloea, Alcaligenes, Flavobacterium, of the microorganism are incorporated as part of the microo Derxia, Lampropedia, Brucella, Xanthobacter; Therms, ganism's own chromosomal or genetic constitution. In an Thermonicrobium, Halomonas, Alteromonas, Serpens, Jan embodiment, the genetic elements that regulate the induction thinobacterium, Bordetella, Paracoccus, Beijerinckia, Fran of death of the microorganism are included as part of a vector cisella, Eubacteria, Actinomycetes, Nocardia, Rhodococcus, (e.g., plasmid, cosmid, etc.). See, for example, U.S. Patent Gordona, Nocardioides, Saccharopolyspora, Application No. 20050276788, which is incorporated herein Micropolyspora, Promicromonospora, Intrasporangium, 25 by reference. Pseudonocardia, Oerskovia, Stomatococcus, Planococcus, In an embodiment, the device itself is configured to induce Aerococcus, Peptococcus, Peptostreptococcus, Coprococcus, apoptosis or necrosis in the at least one altered microorgan Gemella, Pediococcus, Leuconostoc, Ruminococcus, ism. For example, in an embodiment, the device (e.g., semi Sarcina, Aeromonas, Photobacterium, Vibrio, Plesiomonas, permeable barrier) is configured to include or release at least Zymomonas, Chronobacterium, Cardiobacterium, Calym 30 one molecule that induces apoptosis or necrosis in the at least matobacterium, Streptobacillus, Eikenella, Gardnerella, one altered microorganism. Phyllobacterium, Rhizobium, Bradyrhizobium, Agrobacte In an embodiment, a conditional lethal system for eukary rium, Cytophaga, Flexibacter; Saprospira, Flexithrix, Herpe otic cells (e.g., fungal cells such as yeast, or other eukaryotic to siphon, Capnocytophaga, Sporocytophaga, Aureobacte cells) is utilized by providing intracellular production of the rium, Agromyces, Arachnia, Rothia, Acetobacterium, 35 Serratia marcescens nuclease in the cell, which destroys the Actinomyces, Arthrobactera, Arcanobacterium, Lachno genetic material in the cell. See, for example, Balan and spira, Propionibacterium, Eubacterium, Butyrivibria, Brevi Schenberg, Yeast, Vol. 22, pp. 203-212 (2005), which is incor bacterium, Bifidobacterium, Microbacterium, Caseobacter, porated herein by reference. As reported, under normal con or Thermoanaerobacter, Enterobacter sp. 638, or Burkhold ditions, the nuclease, encoded by the nucA gene, is secreted eria cepacia BU72, or other strain. 40 into the extracellular medium. Cloning it without the signal In an embodiment, the at least one altered microorganism sequence, however, results in killing the yeast cell upon glu includes MC-1 magnetotactic bacteria. For example, magne cose depletion from the medium. Id. The conditional lethal totactic bacteria have flagella that provide for mobilization, system also disfavors horizontal gene transfer from recombi and can be combined with nanoparticles of magnetite or nant yeast cells to other microorganisms found in the envi magnetosome chains embedded in the bacteria for directing 45 ronment. Id. the microbes. See, for example, Felfoul, et al., IEEE Xplore In an embodiment, a lac-hok cassette is utilized for induc Abstract, issue 22-26, pp. 1463-1466 (2007), which is incor ing death, wherein the hok gene from plasmid R1 belongs to porated herein by reference. a family of genes encoding Small polypeptides (about 50 In an embodiment, the altered microorganism includes at amino acids) which, when overexpressed, collapse the mem least one microorganism with the designation of 'generally 50 brane potential and lead to cell death. See, for example, Con regarded as safe” (GRAS) status in the food industry. treras, et al., App. Env. Microbiol. Vol. 57, no. 5, pp. 1504 Many microorganism strains are registered with the Ameri 1508 (1991), which is incorporated herein by reference. In an can Type Culture Collection (ATCC, Rockville, Md., USA), embodiment, a gef system, a chromosomally encoded E. coli and can be adapted for use with various embodiments gene highly homologous to hok, is utilized for inducing death described herein. 55 in the altered microorganism. For example, the altered micro In an embodiment, any microorganism for which at least a organism Survives only in the presence of effectors of the portion of the genome has been sequenced is utilized. For meta-cleavage pathway encoded by the TOL plasmid of P example, by sequencing the genome of a particular microor putida. Id. For example, in an embodiment, microorganisms ganism, regulatory elements and sites for chromosomal inser configured to degrade Substituted benzoates utilize a Lad tion can be identified. Furthermore, bioinformatics and pro 60 protein (Lac repressor) expressed from a Pm::lacI fusion moter-trapping strategies can assist in locating endogenous represses transcription from a Ptac::gef cassette in the pres promoters to further regulate production of the at least one ence of XylS effectors (coding for the regulator necessary to therapeutic agent. activate transcription from Pm, in the presence of an effector In an embodiment, at least one altered microorganism Such as 3-methylbenzoate, in this example), whereas in the includes a microorganism that is isolated from at least one 65 absence of XylS effectors, expression of the gef gene is no biological tissue, environmental medium, Subject, or other longer repressed, leading to cell killing. Id. Substitution of Substrate. The microorganism is modified, and placed into at XylS for another protein expands the range of response. Id. US 8,682,619 B2 23 24 Thus, in an embodiment, a similar construct is developed for As described herein, in an embodiment the at least one regulation of production of a particular therapeutic agent in a composition includes at least one essential nutrient, Such as a altered microorganism, as described herein. metabolite, that is required by the at least one altered micro In an embodiment, the at least one altered microorganism organism. In an embodiment, the altered microorganism has includes at least one inducible genetic element configured to a strict requirement for at least one metabolite that is not initiate death of the at least one microorganism. In an embodi present or is present at low concentrations in the external ment, the inducible genetic element includes at least one environment (e.g., auxotrophic). For example, the at least one secretory signal sequence. Various mechanisms can be essential nutrient includes, but is not limited to, at least one of employed to initiate death of the microorganism. For an organic or inorganic Small molecule, nucleic acid, amino 10 acid, peptide, polypeptide, protein, glycopeptide, glycopro example, the inducible genetic element can be configured to tein, glycolipid, lipopolysaccharide, peptidoglycan, pro initiate programmed cell death, to initiate autophagocytosis teoglycan, lipid, fatty acid, metalloprotein, metal, liposome, of the at least one microorganism, to lyse the at least one carbohydrate, or radiation. In an embodiment, the at least one microorganism, or by other means. essential nutrient includes at least one of arabinose, lactose, In an embodiment, the at least one inducible genetic ele 15 maltose, Sucrose, glucose, Xylose, galactose, rhamnose, fruc ment configured to initiate death of the at least one altered tose, melibiose, starch, inunlin, lipopolysaccharide, arsenic, microorganism includes at least one of programmed cell cadmium, hydrocarbon, chromium, ultra-violet radiation, death 1 gene (PDCD1), programmed cell death 2 gene infrared radiation, electromagnetic radiation, visible radia (PDCD2), programmed cell death 3 gene (PDCD3), pro tion, antibiotic, oxygen, carbon dioxide, nitrogen, Xylan, or grammed cell death 4 gene (PDCD4), programmed cell death nisin. Other non-limiting examples of essential nutrients 5 gene (PDCD5), programmed cell death 6 gene (PDCD6), include at least one of L-arabinose, allolactose, D-glucose, programmed cell death 7 gene (PDCD7), programmed cell D-Xylose, D-galactose, amplicillin, tetracycline, penicillin, death 8 gene (PDCD8), programmed cell death 9 gene pristinamycin, retinoic acid, or interferon. In an embodiment, (PDCD9), programmed cell death 10 gene (PDCD10), pro the at least one essential nutrient includes the at least one grammed cell death 11 gene (PDCD11), programmed cell 25 therapeutic agent. In certain instances, the at least one essen death 12 gene (PDCD12), caspase gene, rel gene, hok gene, tial nutrient is also supplied or produced by the at least one Sok gene, diaminopimelate gene, nuclease gene, methylase biological tissue. gene, DNA ligase gene, DNA gyrase gene, toxin-antitoxin In an embodiment, the at least one nucleic acid construct module, relF gene, triclosan, lysine, or lysine-holin. In an includes at least one regulatory sequence. In an embodiment, embodiment, the toxin-antitoxin module includes at least one 30 the at least one regulatory sequence includes at least one of a of masEF, chpBIK, relBE, yefM-yoeB, dinJ-yafl, barnase promoter, enhancer, or repressor. In an embodiment, the at barstar, or ecnA-ecnB. In an embodiment, the at least one least one regulatory element is regulated by at least one inducible genetic element configured to lyse the at least one inducer. In an embodiment, the at least one inducer includes auxotrophic microorganism includes at least one of a at least one quorum-sensing molecule. Particular non-limit nuclease gene, or lysis gene E. 35 ing examples of quorum-sensing molecules are described In an embodiment, the at least one inducible genetic ele herein. ment configured to initiate death of the altered microorganism In an embodiment, the at least one essential nutrient is includes at least one of extracellular death factor, maZF, or configured to be supplied or provided by at least one other maZEF. For example, toxin-antitoxin modules are generally modified biological cell located in the device. The modified gene pairs specifying for a toxin and its antitoxin, and are 40 biological cell can include, for example, at least one of bac found on the chromosomes of many bacteria. For example, in teria, protozoa, rotifers, algae, or fungi. In an embodiment, E. coli, maZF encodes a stable toxin (MazF), and mazE the modified biological cell includes at least one prokaryote encodes a labile antitoxin (mazE) which prevents the lethal cell or eukaryote cell. In an embodiment, the modified bio effect of MazF. See, for example, J. Bacteriol. Vol. 186, no. logical cell includes at least one altered microorganism. In an 24, pp. 8295-8300, (2004), which is incorporated herein by 45 embodiment, the modified biological cell includes at least one reference. As published, the quorum sensing peptide, Extra blood cell, muscle cell, nerve cell, fibroblast, adipose cell, cellular Death Factor, is a signal molecule required for stem cell, pluripotent cell, epithelial cell, Secretory cell, skin maZEF-mediated cell death. Id. In an embodiment, a micro cell, neoplastic cell, or other biological tissue or organ cell. In bial biofilm is desired, and the device includes means for an embodiment, the modified biological cell includes at least measuring biofilm formation. In an embodiment, a microbial 50 one autologous cell or modified autologous cell. In an biofilm is not desired, and the device includes means for embodiment, the modified biological cell is at least part of at inhibiting biofilm formation (e.g., Ga(NO), coating mate least one cell mass. In an embodiment, the at least one cell rials such as fluoroalkylated acrylic acid oligomer). See, e.g., mass includes at least one tumor, scar, pore, pit, eschar, granu Peeters, et al., J. Antimicrob. Chemo. Vol. 61, pp. 1062-1065 loma, keloid, artheromatous plaque, abscess, pustule, Scaling (2008); and Okada et al. Dental Mat. J. Vol. 27, no. 4, pp. 55 (e.g., psoriasis or eczema), infected tissue, hair follicle, 565-572 (2008), each of which is incorporated herein by necrotic tissue, stratum corneum, wrinkle, wound, tumor, reference. skin structure, nevus, cyst, lesion, callus, neoplastic tissue, As illustrated in FIG. 8, an example of an inducible genetic gangrenous tissue, or cellular deposit. element, including an inducible promoter 800 capable of In an embodiment, the modified biological cell is modified regulating expression of at least one gene 810. In the absence 60 for at least one of a commensal or cooperative relationship ofan inducer 820, the gene 810 is not transcribed (as indicated with the at least one altered microorganism. In an embodi by the “X”). However, in the presence of the inducer 820, the ment, the modified biological cell is modified for obligatory promoter 800 directs transcription of the gene 810, resulting cooperation with the at least one altered microorganism. in production of at least one transcript 830. Likewise, in the Developing a modified biological cell that is modified to be presence of a repressor 840, the promoter 800 does not sup 65 in a cooperative relationship can be conducted using routine port gene transcription of the gene 810 (as indicated by the laboratory procedures. For example, populations of Saccha “X”). romyces cerevisiae have been modified to create obligatory US 8,682,619 B2 25 26 cooperation by mutating each strain in Such a manner as to In an embodiment, the at least one altered microorganism render each strain nutritionally deficient without the other. is in a syntrophic relationship with at least one other altered See, for example, Shou, et al. PNAS, vol. 104, no. 6, pp. microorganism. See, for example, Marx, Science Vol.324, pp. 1877-1882 (2007), which is incorporated herein by reference. 1150-1151 (2009), which is incorporated herein by reference. For example, a first S. cerevisiae strain was modified in order For example, one altered microorganism may convert the to require adenine to grow and overproduce lysine, while a primary resource to an intermediate that can be used by second S. cerevisiae strain was modified to require lysine to another altered microorganism. In an embodiment, one grow and overproduce adenine. Id. These modified, nonmat altered microorganism provides motility for another altered ing yeast strains compose a synthetic obligatory cooperative microorganism, which may in turn provide a nutrient source. system, termed COSMO (cooperation that is synthetic and 10 Id. mutually obligatory) by providing an essential metabolite to In an embodiment, the altered microorganism includes at the other strain. Id. As published, persistent cooperation could least one nucleic acid construct, including a nucleic acid be established with the modified yeast strains, and was math construct that includes at least one artificial operon. In an ematically and experimentally shown to be viable over a wide 15 embodiment, the at least one artificial operon encodes at least range of initial conditions, with oscillating population ratio one polycistronic mRNA transcript. settling to a value predicted by nutrient Supply and consump In an embodiment, the altered microorganism includes at tion. Id. In another example, as described herein, quorum least one nucleic acid construct encoding at least one thera sensing molecules are utilized for regulating production of peutic agent. In an embodiment, the nucleic acid construct the at least one therapeutic agent. includes at least one inducible promoter or enhancer. In an Furthermore, even in the absence of explicitly engineered embodiment, the at least one nucleic acid construct includes mechanisms to stabilize cooperation, the system can consis at least one promoter, enhancer, or repressor that is regulated tently develop increased ability to Survive reductions in popu by at least one inducer. In an embodiment, the at least one lation density. Id. For example, members of a microorganism inducer includes at least one quorum-sensing molecule. consortium can exert both positive and negative control over 25 Microorganisms produce, release, exchange, and detect one another's activities by exchanging metabolic intermedi quorum-sensing molecules in response to population density, ates that either assist or compromise the growth of their and respond to alteration in gene expression. See, for neighbors. In one example, engineered acyl-HSL communi example, Bassler and Losick, Cell, vol. 125, pp. 237-246 cation has been used in biological "circuits’ that coordinate (2006), which is incorporated herein by reference. In an population-wide behaviors ranging from population density 30 embodiment, quorum-sensing molecules regulate at least one dependent fluorescence, cell Suicide, and invasion of cancer of competence, bioluminescence, virulence factor secretion, cells, to pattern formation. Id. In one example, upon induction biofilm formation, sporulation, morphological differentia of the biological circuit that encodes the communication and tion, secondary metabolite production, cell-to-cell signaling, the programmed cellular response, one population (predator) or protein production. Id. dies out win the absence of the other (prey). Communication 35 between the two populations directs the prey to rescue the In an embodiment, the quorum-sensing signal is produced predator, but once the predator recovers to a sufficiently high in response to an environmental signal or growth event. In an density, it begins to kill the prey. Id. embodiment, the quorum-sensing signal is secreted and rec In an embodiment, a cooperative relationship includes, but ognized by other microorganisms. In an embodiment, the is not limited to biofilm formation, colonization, virulence, 40 quorum-sensing signal accumulates to a threshold concentra proliferation, communication, and other activities. See, for tion before it stimulates a response. In an embodiment, the example, Brenner, et al., Trends Biotech. Vol. 26, no. 9, pp. response includes a concerted response. In an embodiment, 483-489 (2008), which is incorporated herein by reference. the response includes a cellular response that is in addition to For example, the at least one altered microorganism can par or instead of metabolic or detoxification processes. See, for ticipate in at least one microbial consensus consortium for 45 example, Diggle, et al., Curr. Biol. Vol. 117, no. 21, pp. cross-talk and cell-cell signaling. See, for example, Brenner, R907-R910 (2007), which is incorporated herein by refer et al., PNAS, vol. 104, no. 44, pp. 17300-17304 (2007), which CCC. is incorporated herein by reference. In an embodiment, the at least one quorum-sensing mol Published studies describe that synthetic ecosystems of at ecule includes thiolactone. In an embodiment, the at least one least one microbial population can communicate bi-direc 50 quorum-sensing molecule includes homoserine lactone. In an tionally through quorum sensing and regulate gene expres embodiment, the at least one quorum-sensing molecule sion and Survival of other population members, by way of includes at least one of N-acylhomoserine lactone, or Y-buty engineered gene circuits. See, for example, Balagadde, et al., rolactone. In an embodiment, the at least one quorum-sensing Mol. Sys. Biol. vol. 4, no. 187, pp. 1-8 (2008), which is molecule includes at least one of 3-oxo-acylhomoserine lac incorporated herein by reference. As discussed herein, in an 55 tone, N-(3-oxoacyl)homoserine lactone, 3-hydroxy-acylho embodiment, the predator cells kill the prey by inducing moserine lactone, N-3-hydroxyacyl) homoserine lactone, expression of a killer protein in the prey, while the prey rescue 2-isocapryloyl-3-hydroxy-methyl-Y-butyrolactone, furano the predators by eliciting expression of an antidote protein in Sylborate ester, Pseudomonas quinolone signal, 2-heptyl-3- the predator. Thus, extinction, coexistence and oscillatory hydroxy-4-(1H)-quinolone, paraoXonase, methyl dodecenoic dynamics of the predator and prey populations are possible 60 acid, hydroxyl-palmitic acid methyl ester, N-(3-oxohex depending on the operating conditions, which can be deter anoyl)homoserine lactone (3-oxo-C6-HSL), N-(3-oxodode mined mathematically. Id. In an embodiment, the predator canoyl)-L-homoserine lactone, nisin, dihydroxyacetone includes the at least one modified biological cell, and the prey phosphate, glyceraldehydes 3-phosphate, or 2-alkyl-4-qui includes the at least one altered microorganism. In an nolone. embodiment, the predator includes the at least one altered 65 The following non-limiting examples indicate particular microorganism, and the prey includes the at least one modi structures of homoserine lactone (HSL) compounds utilized fied biological cell. by various microorganisms as quorum-sensing molecules. US 8,682,619 B2 27 28 See, for example, the worldwide web at nottingham.ac.uk/ -continued quorum/AHLS.htm, the content of which is incorporated O O herein by reference. O N 1.--~~~ 5 H. H. O 3-oxo-C12-HSL O N HH O C14-HSL O O O N ---~~~~ HH O 2O 3-oxo-C14-HSL O O N --~-N--- HH 25 O 7-cis-C14-HSL O OH O N ------~~ 30 HH O 3-hydroxy-7-cis-C14-HSL 35 Additional examples of chemical structures of particular quorum-sensing molecules are provided below. 40 O 3-oxo-C6-HSL 45 3-oxo-C8-HSL HO O O O N 1--~~ H. H. 50 O O O A-Factor C1O-HSL O O O OH 55 O N ---~~ H. H. N O H 3-oxo-C10-HSL PQS O O O N 1--~~~~ H. H. 60 CN O 65 H C12-HSL HHQ US 8,682,619 B2 29 30 -continued -continued OH O COOH DSF 5 OH O O OH DPD --N---~~~~ A-Factor is 2-isocapryloyl-3-hydroxy-methyl-y-butyro 3OH-PAME 10 lactone, POS is Pseudomonas quinolone signal (2-heptyl-3- O hydroxy-4-(1H)-quinolone), DSF is “diffusible factor' (me ~N/ SN Me thyl dodecenoic acid), AIP-1 is “autoinducing peptide 1 S (thiolactone peptide), DPD is 4,5-dihydroxy-2,3-pentanedi one, HHO is 4-hydroxy-2-heptylquinoline, and PAME is Asp MNH 15 hydroxyl-palmitic acid methyl ester. See, for example, the H-Tyr-Ser-Thr-N -M H ph Me worldwide web atgcat.davidson.edu, the content of which is incorporated herein by reference. O Table 1 below is a non-limiting list of examples of specific ALP-1 acylhomoserine lactone (AHL) compounds utilized by par ticular bacteria. TABLE 1 LuxRI homologues GenBank with links Accsession Major Bacterium to Swissprot Number AHL Phenotype Reference Aeromonas AhyR, AhyI X89469 C4-HSL Extracellular Swift etal 1997, hydrophila protease, biofilm 1999b, c, formation Lynch et al 1999 Aeromonas AsaR, AsaI U65741 C4-HSL Extracellular Swift et al 1997 Saimonicida protease Agrobacterium TraR, Tra.I L17024, 3-oxo- Conjugation Fuqua etal 1994, tumefaciens L222O7 C8-HSL Piper et al 1999, Burkhoideria CepR, CepI AF330018, C8-HSL Protease, Lewenza et al 1999 cepacia AF330012 siderophore Chronobacterium CviR, CviI no link C6-HSL Antibiotics, McClean et al 1997, violacetim available violacein, Chernin et al 1998 exoenzymes, cyanide Enterobacter EagR, Eag X743OO 3-oxo- Unknown Swift et al 1993 agglomerans C6-HSL Erwinia CarR, ExpR X74299, 3-oxo- Carbapenem Bainton et al 1992, Caioio'Oia Expl (Carl) X80475, C6-HSL antibiotic, Swift etal 1993, Subsp carotovora X72891 exoenzymes Pirhomen et al 1993 Erwinia ExpR, Expl X96440 3-oxo- Pectinases Nasser et a 1998 chrysanthemi (EchR, EchI) C6-HSL Escherichia coi SdiA AE005414 Unknown Cell division Sitnikov et al 1996 Nitrosomas Unknown 3-oxo- Emergence from Batchelor et al 1997 europaea C6-HSL agphase Obesumbacterium OprR, OprI no link 3-oxo- Unknown Swift et al 1999 proteus available C6-HSL Panioea Stewari EsaR, Esa L32183, 3-oxo- Exopolysaccharide Beck von Bodman L321.84 C6-HSL & Farrand 1995 Pseudomonas LasR, Las MS942S 3-oxo- Exoenzymes, Xcp, Chapon-Hervé et al 1997, aeruginosa C12-HSL biofilm formation, Gambello & Iglewski 1991, RhlR, cell-cell Passador et al 1993, spacing. Glessner et al 1999 L08962, C4-HSL Exoenzymes, cyanide, Latifietal 1995, 1996, U11811, RpoS, lectins, Winson et al 1995, U15644 pyocyanin, rhamnolipid, Pearson et al 1997, type 4 pili. Glessner et al 1999. Pseudomonas PhzR, Phz L32729, C6-HSL Phenazine Pierson et al 1994, attreofaciens L33724 antibiotic Wood et al 1997 Pseudomonas PhzR, Phz L48616 Unknown Phenazine Shaw etal 1997 fittorescens antibiotic Pseudomonas PsyR, Psy U398O2 Unknown Unknown Swift et al 1999 Syringae pv. tabaci Raistonia SolR, SolI AFO21840 C8-HSL Unknown Flavier et al 1997 Soianacearum Rhizobium eti RaiR, RaiI U92713 Unknown Restriction of Gray et al 1996, nodule number Rosemeyer et al 1998 US 8,682,619 B2 31 32 TABLE 1-continued LuxRI homologues GenBank with links Accsession Major Bacterium to Swissprot Number AHL Phenotype Reference Rhizobium RhiR M9883S 3-hydroxy- Nodulation, Gray 1997, leguminosarum 7-cis- bacteriocin, Rodelas et al 1999, C14-HSL stationary phase Thorne and Williams survival 1999 Rhodobacter CerR, CerI AFO16298 7-cis- Community escape Puskas et al 1997 sphaeroides C14-HSL Serraia SwrR, SwrI U22823 C4-HSL Swarming, protease Eberlet al 1996, liquefaciens Givskov et al 1997 Lindum et al 1998 Vibrio VanR, VanI U69677 3-oxo- Unknown Milton et al 1997 anguillartin C1O-HSL Vibrio LuxR, LuxI M19039, 3-oxo- Biolumine fischeri M96844, C6-HSL SC(Ce M25752 Xenorhabdits Unknown 3-hydroxy- Virulence, Dunphy et al 1997 nematophilus C4-HSL bacterial lipase or an agonist Yersinia YenR, Yen I X76082 C6-HSL Unknown Throup et al 1995 enterocolitica Yersinia pestis YpeR, Ype.I AFO714O1 Unknown Unknown Swift et al 1999a, b Yersinia YpsR, YpsI AFO79973 3-oxo- Motility, clumping Atkinson et al 1999 pseudotuberculosis C6-HSL YtbR,YtbI AFO79136 C8-HSL Unknown Atkinson et al 1999 Yersinia ruckeri YukR, YukI AFO7913S Unknown Unknown Atkinson et al 1999 See, for example, the worldwide web at nottingham.ac.uk/ The pH level of a particular biological tissue can affect the quorum/table.htm, the Subject matter of which is incorpo inducibility of the pH inducible promoter. For example, the rated herein by reference. In an embodiment, the at least one 30 gastric pH of humans is between 1.5 (fasting state), and nucleic acid construct includes at least one neutral, base or 3.0-5.0 (following feeding). Id. Likewise, human urine has a acid inducible promoter. Examples of acid inducible promot pH of approximately 6.0, human skin has a pH of approxi ers include, but are not limited to HVA1 promoter (plant mately 5.5, human ear canal has a pH of approximately 4.5, cells), P170, P1, or P3 (Lactococcus), baiA1, baiA3 (Eubac 35 while blood and cerebrospinal fluid tend to have a pH of teria), lipF promoter (Mycobacteria), FF-ATPase promoter approximately 7.3-7.4. See, for example, Boron, et al., Medi (Lactobacillus, Streptococcus, or Enterococcus), gadC, gad cal Physiology: A Cellular and Molecular Approach. D (Lactococcus, Shignella), glutamate decarboxylase pro Elsevier/Saunders. (2004), ISBN 1-4160-2328-3, which is moter (Mycobacteria, Clostridium, Listeria, Lactobacillus), incorporated herein by reference. Furthermore, dental caries or similar operons. See, for example, Cotter and Hill, Micro 40 and tooth demineralization is initiated at a pH of approxi biol. and Mol. Biol. Rev. vol. 67, no. 3, pp. 429–453 (2003); mately 5.2. Cotter and Hill, Ibid. Thus, a composition that is Hagenbeek, et al., Plant Phys., vol. 123, pp. 1553-1560 activated at various pH levels is useful for particular embodi (2000); Madsen, et al., Abstract, Mol. Microbiol. Vol. 56, no. ments. In an embodiment, the pH inducible promoter is 3, pp. 735-746 (2005); U.S. Pat. No. 6.242,194; Richter, et al., induced at a particular pH (e.g., chewing gum containing the Abstract, Gene, vol.395, no. 1-2, pp. 22-28 (2007), Mallonee, 45 composition described herein which is inducible at low pH in et al., J. Bacteriol., vol. 172, no. 12, pp. 701 1-7019 (1990); the mouth). The pH level of plant leaves is approximately 4.2. each of which is incorporated herein by reference. Examples See, for example, Sargent and Blackman, Abstract, J. Exp. of base inducible promoters include, but are not limited to, Botany, Vol. 21, no. 1, pp. 219-227 (2005), which is incorpo alkaline phosphatase promoters. rated herein by reference. In an embodiment, the acid inducible genetic element is 50 As described herein for various inducible genetic elements, inducible at a pH of approximately 0.0, approximately 0.5, the inducible genetic elements can include at least one induc approximately 1.0, approximately 1.5, approximately 2.0, ible promoter, enhancer, or repressor. As previously approximately 2.5, approximately 3.0, approximately 3.5. described, in an embodiment, the inducible genetic element is approximately 4.0, approximately 4.5, approximately 5.0, regulated by an inducer, Such as a quorum-sensing molecule. 55 Other examples of the at least one inducer include, but are not approximately 5.5, approximately 6.0, approximately 6.5, limited to, at least one of radiation, temperature change, alco approximately 6.6, approximately 6.7, approximately 6.8. hol, antibiotic, steroid, metal, salicylic acid, ethylene, ben approximately 6.9, or any value therebetween or less. Zothiadiazole, or other compound. In an embodiment, the at In an embodiment, the base inducible genetic element is least one inducer includes at least one of arabinose, lactose, inducible at a pH of approximately 7.1, approximately 7.5, 60 maltose, Sucrose, glucose, Xylose, galactose, rhamnose, fruc approximately 8.0, approximately 8.5, approximately 9.0, tose, melibiose, starch, inulin, lipopolysaccharide, arsenic, approximately 9.5, approximately 10.0, approximately 10.5, cadmium, chromium, temperature, light, antibiotic, oxygen approximately 11.0, approximately 11.5, approximately level, Xylan, nisin, L-arabinose, allolactose, D-glucose, 12.0, approximately 12.5, approximately 13.0, approxi D-Xylose, D-galactose, amplicillin, tetracycline, penicillin, mately 13.5, approximately 14.0, or any value therebetween 65 pristinamycin, retinoic acid, or interferon. Other examples of or greater. In an embodiment, the neutral inducible genetic inducers include, but are not limited to, at least a portion of element is inducible at a pH of approximately 7.0. one of an organic or inorganic Small molecule, clathrate or US 8,682,619 B2 33 34 caged compound, protocell, coacervate, microsphere, Janus In at least one embodiment, the at least one biological particle, proteinoid, laminate, helical rod, liposome, macro tissue is at least partially located in a subject. In an embodi scopic tube, niosome, sphingosome, toroid, vesicular tube, ment, as discussed herein, a subject includes, but is not lim vesicle, Small unilamellar vesicle, large unilamellar vesicle, ited to, a vertebrate or invertebrate, including a fish, reptile, large multilamellar vesicle, multivesicular vesicle, lipid mammal, amphibian, or bird. In at least one embodiment, the layer, lipid bilayer, micelle, organelle, nucleic acid, peptide, Subject includes at least one human. In an embodiment, the at polypeptide, protein, glycopeptide, glycolipid, lipoprotein, least one subject includes at least one of livestock, pet, Zoo lipopolysaccharide, Sphingolipid, glycosphingolipid, glyco animal, undomesticated herd animal, wild animal, aquatic protein, peptidoglycan, lipid, carbohydrate, metalloprotein, plant or animal, or product animal. 10 In an embodiment, the at least one subject includes at least proteoglycan, chromosome, nucleus, acid, buffer, protic Sol one of a sheep, goat, frog, dog, cat, rat, mouse, Vermin, vent, aprotic solvent, nitric oxide, vitamin, mineral, nitrous monkey, horse, cow, pig, chicken, shellfish, fish, turkey, oxide, nitric oxide synthase, amino acid, micelle, polymer, llama, alpaca, bison, buffalo, ape, primate, ferret, wolf, fox, copolymer, monomer, prepolymer, cell receptor, adhesion coyote, deer, rabbit, guinea pig, yak, chinchilla, mink, rein molecule, cytokine, chemokine, immunoglobulin, antibody, 15 deer, elk, camel, fox, elk, deer, raccoon, donkey, or mule. In antigen, extracellular matrix, cell ligand, Zwitterionic mate an embodiment, the at least one subject includes at least one rial, cationic material, oligonucleotide, nanotube, piloxymer, anthozoan species. In an embodiment, the at least one subject transfersome, gas, element, contaminant, radioactive particle, includes at least one of a sea anemone, coral, mollusk, fish, radiation, hormone, virus, quantum dot, temperature change, whale, dolphin, porpoise, seal, otter, beaver, seabird, gull, thermal energy, or contrast agent. See, for example, Theys, et pelican, albatross, duck, Swan, or goose. In an embodiment, al., Abstract, Curr. Gene Ther. Vol. 3, no. 3 pp. 207-221 the at least one subject includes at least one insect (e.g., fly, (2003), which is incorporated herein by reference. In an mosquito, beetle, moth, butterfly, etc.). In an embodiment, the embodiment, the at least one inducer is produced by at least at least one Subject includes at least one arachnid. In an one microorganism. embodiment, the at least one Subject includes at least one In an embodiment, the at least one inducible genetic ele 25 Crustacean. ment is temperature-inducible. For example, various heat In an embodiment, the Subject includes a plant. In an shock protein promoters have been isolated from microorgan embodiment, the at least one biological tissue includes one or isms, animal and plant cells (including maize and Soybeans), more of a stalk, stem, leaf, root, plant, or tendril. In an and other temperature-inducible promoters have been iso embodiment, the at least one biological tissue includes at least lated from various organisms and microorganisms. Some 30 one food product. In an embodiment, the at least one food non-limiting examples of promoters induced by a change in product includes one or more animal, plant, fungal or other temperature include hsp70 (e.g., animal cells, maize), biological food product. In an embodiment, the food product Gmhsp17.5-E (e.g., soybeans), Gimhsp17.6-L, HS6871 (e.g., includes meat. In an embodiment, the at least one biological soybeans), HSP18.2 (Arabidopsis), HSP18.1 (Arabidopsis), tissue includes at least one cell mass or wound. Hsp70B, P2 (Bacillus), P7 (Bacillus), PhS (E. coli), Tetrahy 35 In an embodiment, the at least one composition is self mena therophila promoter (protozoan), Hansenula polymor administered by the at least one Subject. In an embodiment, pha promoter (yeast), Schizosaccharomyces pombe promot the at least one composition is ingested by the at least one ers (yeast), or P(lambda phage). See, for example, Taylor, et Subject. In an embodiment, the at least one biological tissue al, Cell, Abstract, vol. 38, no. 2, pp. 371-381 (1984); U.S. Pat. includes at least one implantable or transplantable biological No. 4,352.581; U.S. Pat. No. 6,852,511, Wang, et al., Bio 40 tissue. In an embodiment, the at least one biological tissue is chem. and Biophys. Res. Commun. Abstract, Vol. 358, no. 4, transplanted or implanted into at least one subject. In an pp. 1148-1153 (2007), U.S. Pat. No. 7,462,708, each of which embodiment, the at least one biological tissue is from at least is incorporated herein by reference. one donor or recipient. In an embodiment, the at least one In an embodiment, the implantable device is configured to biological tissue includes at least one bodily orifice of a be implanted into at least one biological tissue or organ. In an 45 Subject. embodiment, the at least one biological tissue or organ is Selection of the particular microorganism can be based on located in at least one of in vitro, in vivo, in situ, in utero, ex calculations of Sub-populations in a population, a particular Vivo, in planta, or in silico. In an embodiment, the at least one Subject, a particular state of health of the Subject, a particular biological tissue includes at least one of one mucosal Surface. organ, a particular biological tissue, or a particular biological In an embodiment, the at least one biological tissue includes 50 cell type. For example, the intestinal microflora can contrib at least one of cartilage, skin, Scalp, hair, nail, nail bed, teeth, ute to pathogenesis in Susceptible subjects, or it can contrib eye, ear, ovary, Oviduct, tongue, tonsil, adenoid, liver, bone, ute to the metabolism and overall health of the subject. See, pancreas, Stomach, duct, valve, Smooth muscle, appendix, for example, O'Hara and Shanahan, EMBO reports, Vol. 7, blood vessel, bone marrow, heart, lung, brain, breast, kidney, no. 7, pp. 688-693 (2006), which is incorporated herein by bladder, urethra, ureter, gallbladder, uterus, prostate, testes, 55 reference. Microorganism populations vary according to age, vas deferens, fallopian tubes, large intestine, Small intestine, disease state or health state, and diet. Id. For example, Lac esophagus, oral cavity, nasal cavity, otic cavity, connective tobacilli are dominant among microflora associated with the tissue, muscle tissue, adipose tissue, or mucosa-associated urogenital tract of healthy women but are almost completely lymphoid tissue (MALT). In an embodiment, the device is absent in patients who develop most forms of urinary tract configured to be implanted in at least one body cavity of a 60 infections. See, for example, Hanniffy, et al., Adv. Applied Subject. For example, in an embodiment, the at least one body Microbiol., vol. 56, pp. 1-64 (2004), which is incorporated cavity includes at least one of an otic cavity, oral cavity, herein by reference. vaginal cavity, anal cavity, nasal cavity, esophagus, or eyelid. Published reports indicate that the human gut microbiota In an embodiment, the at least one body cavity includes the affects nutrition, development, metabolism, pathogen resis gastrointestinal tract of a Subject. In an embodiment, the 65 tance, and regulation of immune responses; and the micro gastrointestinal tract includes at least one of a stomach, large biota community changes with antibiotic use. See, Dethlef intestine, Small intestine, appendix, or any part thereof. sen, et al., PLOS Biol., vol. 6, no. 11, pp. 2383-2400 (2008), US 8,682,619 B2 35 36 which is incorporated herein by reference. For example, death of the at least one altered microorganism, and also approximately 3300-5700 bacterial taxa were identified as generate the at least one therapeutic agent. accounting for over 99% of the variable region sequence tags Vectors suitable for microbiological applications are well obtained. Id. Antibiotic use of ciprofloxacin influenced the known in the art, and are routinely designed and developed abundance of about one third of the bacterial taxa in the gut. for particular purposes. Some non-limiting published Id. examples of vectors that have been used to transform bacterial Further published reports indicate high diversity of micro strains include the following plasmids: pMW211, pBAD organisms located in the human mouth, with a small majority DEST49, pIDONRP4-P1R, pFNTR-P pENTR-DUAL, in common amongst any particular geographical region pENTR-term, pBR322, plDESTR4-R3, pBGS18-N9uc8, 10 pBS24Ub, pUbNuc, pIXY 154, pBR322DEST, group. See, for example, Myles, et al., Abstract, BMC Med. pBR322DEST-P-DUAL-term, p.JIM2093, pTG2247, Gen. Vol. 2, no. 45 (2009), which is incorporated herein by pMEC10, pMEC46, pMEC 127, p.TX, pSK360, p.ACYC184, reference. For example, the populations of individual groups pBO E93, pBR327, plW205, pKCL11, pKK2247, pMR60, of bacteria located in the saliva of one person tested were not pOU82, pR2172, pSK330, pSK342, pSK355, puHE21-2, largely common with other people located in a similar geo 15 pEHLYA2-SD. See, for example, Stritzker, et al. Intl. J. Med. graphical region. Id. Microbiol. Vol. 297, pp. 151-162 (2007); Grangette et al., In an embodiment, at least one microorganism (or a popu Infect. Immun. Vol. 72, pp. 2731-2737 (2004), Knudsen and lation thereof) is obtained from at least one subject, modified, Karlstrom, App. and EnV. Microbiol. pp. 85-92, Vol. 57, no. 1 and returned to the at least one Subject, and possibly others (1991), Rao et al., PNAS pp. 11993-11998, vol. 102, no. 34 (e.g., members of a household). In an embodiment, the at least (2005), each of which is incorporated herein by reference. one altered microorganism (or a population thereof) is Therapeutic Agents returned to approximately the same location of the Subject In an embodiment, the at least one altered microorganism from which it was extracted. In an embodiment, the at least produces at least one therapeutic agent. In an embodiment, one altered microorganism (or a population thereof) is the at least one therapeutic agent includes at least a portion of returned to a different location of the subject source. For 25 one of an organic or inorganic Small molecule, proteinoid, example, at least one microorganism can be extracted from nucleic acid, peptide, polypeptide, protein, glycopeptide, gly the intestine of a subject, modified, and returned to the oral colipid, lipoprotein, lipopolysaccharide, sphingolipid, gly cavity, otic cavity, Stomach, or intestine of that same Subject. cosphingolipid, fatty acid, glycoprotein, peptidoglycan, lipid, In an embodiment, at least one microorganism is extracted, carbohydrate, metalloprotein, proteoglycan, Vitamin, min modified, and returned to the Subject at approximately the 30 eral, amino acid, polymer, copolymer, monomer, prepolymer, same location from where it was extracted, and the altered cell receptor, adhesion molecule, cytokine, chemokine, microorganism is able to translocate to another location immunoglobulin, antibody, antigen, extracellular matrix within the Subject or to another Subject (e.g., maternal trans component, cell ligand, oligonucleotide, element, chromo fer). In an embodiment, at least some genetic sequence infor Some, nucleus, acid, base, buffer, protic solvent, aprotic Sol mation from the microorganism is obtained prior to or Sub 35 vent, hormone, metabolite, or contrast agent. sequent to modifying it. In an embodiment, the at least one In an embodiment, the fatty acid includes at least one of a altered microorganism is amplified prior to reinstating it in short chain fatty acid, long chain fatty acid, unsaturated fat, or the at least one biological tissue, or subject. saturated fat. In an embodiment, the at least one lipid includes Published studies indicate that the human gut contains at least one of a monounsaturated fat, polyunsaturated fat, distinct differences in the microflora of obese subjects, nor 40 trans fat, cis fat, omega fatty acid. In an embodiment, the at mal weight Subjects, and Subjects who have undergone gastric least one fatty acid includes at least one of an arachidic fatty bypass Surgery. See, for example, Zhang, et al., PNAS, pp. acid, Stearic fatty acid, palmitic fatty acid, erucic fatty acid, 2365-2370; available online at 10.10973/pnas.0812600106 linoleic fatty acid oleic fatty acid, or linolenic fatty acid. In an (printed 2009, available 2008), which is incorporated herein embodiment, the fatty acid includes at least one of myristoleic by reference. Further, these studies indicate a shift in micro 45 acid, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, biota populations following gastric bypass Surgery. Id. alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, In an embodiment, a microorganism is selected from at erucic acid, or docosahexaenoic acid. For example, it has least one subject based on a desired state of health, modified, been published that oleic acid protects against the develop and placed in the same or different subject. Selection of a ment of ulcerative colitis. See, for example, Punyanganie, et particular microorganism can be conducted utilizing conven 50 al., no. 100 AGA Abstracts, S-18 (2010). tional techniques, including but not limited to barcoded 16S In an embodiment, the polymer or co-polymer includes at pyrosequencing. See, for example, Andersson, et al., PLOS least one of polyester, polylactic acid, polyglycolic acid, cel One vol. 3, no. 7, pp. 1-8 (2008), which is incorporated herein lulose, nitrocellulose, urea, urethane, or other polymer. For by reference. The relatively small genome size of microor example, in an embodiment, at least one microorganism that ganisms, and the availability of high-throughput sequencing 55 is capable of synthesizing at least one thermoplastic polymer facilities has allowed the sequencing of more than 90 bacte (e.g., polyester) is utilized. See, for example, U.S. Pat. No. rial genomes for the public domain. Id. 5,663,063, which is incorporated herein by reference. For In an embodiment, the at least one altered microorganism example, as described in U.S. Pat. No. 5,663,063, E. coli includes at least one vector including at least one genetic strains can be modified to produce polyhyroxybutyrate and element for producing an agent, or initiating death in the 60 polyhydroxyalkanoate polyesters. Id. microorganism. Various vectors can be utilized, as described In an embodiment, at least one microorganism that is herein, including at least one of a plasmid, bacteriophage, capable of degrading at least one polymer (e.g., aliphatic cosmid, artificial chromosome, or other vector. In an embodi polyester) is utilized. See, for example, Suyama, et al., App. ment, the altered microorganism includes multiple vectors, Env. Microbiol. vol. 64, no. 12, pp.5008-5011 (1998), which some of which can be the same or different from the other 65 is incorporated herein by reference. For example, in an vectors. In an embodiment, the altered microorganism embodiment, the at least one microorganism is capable of includes at least one vector that is able to regulate initiation of degrading at least one of poly(beta-hydroxyalkanoate), poly US 8,682,619 B2 37 38 (epsilon-caprolactone), or poly(hexamethylene carbonate). until conditions change that result in alteration of the pH of Id. In an embodiment, the polymer or co-polymer includes at the location (or the altered microorganism travels to another least one of polyester, polylactic acid, polyglycolic acid, cel location with a different pH), and the agent becomes active. lulose, nitrocellulose, urea, urethane, or other polymer, as The activity of the agent can be gradual (along a gradation), or described herein. In an embodiment, the at least one thera Sudden. peutic agent includes one or more of anti-tumor agent, anti In an embodiment, the at least one therapeutic agent is microbial agent, anti-coagulant, anti-Viral agent, analgesic, capable of modulating at least one immune response. In an antiseptic, anesthetic, diagnostic agent, anti-inflammatory embodiment, wherein the at least one immune response agent, vaccine, cell growth inhibitor, cell growth promoter, includes at least one allergic or autoimmune response. In an chemical debridement agent, immunogen, antigen, radioac 10 embodiment, the at least one therapeutic agent is capable of tive agent, apoptosis promoting factor, angiogenic factor, inducing apoptosis in one or more cells of the at least one anti-angiogenic factor, hormone, enzymatic factor, enzyme, biological tissue. In an embodiment, the at least one admin papain, collagenase, protease, peptidase, elastase, urea, Vita istered therapeutic agent modulates the viability, prolifera min, mineral, nitrite, nitrate, nutraceutical, histatin, honey, tion, or metastasis of at least one tumor cell in the at least one alcium alginate, angiogenic factor, hormone, vitamin, min 15 biological tissue. eral, nutraceutical, cytokine, chemokine, probiotic, Sterol, In an embodiment, the at least one therapeutic agent contraceptive, coagulant, anti-coagulant, phage, prodrug. includes at least one of insulin, clacitonin, lutenizing hor prebiotic, blood Sugar stabilizer, Smooth muscle cell activa mone, parathyroid hormone, Somatostatin, thyroid stimulat tor, epinephrine, adrenaline, neurotoxin, neuro-muscular ing hormone, vasoactive intestinal polypeptide, tumor necro toxin, Botulinum toxin type A, microbial cell or component sis metabolite, endostatin, angiostatin, anti-angiogenic thereof, or virus or component thereof. In an embodiment, the antithrombin II, fibronectin, prolactin, thrombospondin I, nutraceutical includes one or more of a flavonoid, antioxi laminin, procollagen, collagen, integrin, steroid, corticoster dant, beta-carotene, anthocyanin, alpha-linolenic acid, oid, virus antigen, microorganism antigen, trefoil protein, or omega-3 fatty acids, plant products, or animal products. lipase. In an embodiment, the analgesic oranesthetic includes one 25 The trefoil peptide can include, but not be limited to, TFF1 or more of any aminoamid or aminoester local anesthetic, (also known as pS2, or breast cancer estrogen inducible ibuprofen, morphine, codeine, aspirin, acetaminophen, gene), TFF2 (also known as SP, or spasmolytic peptide), or lidocaine/lignocaine, ropivacaine, mepivacaine, benzocaine, TFF3 (also known as ITF, or intestinal trefoil factor). See, for chloroprocaine, cocaine, cyclomethycaine, dimethocaine/ example, U.S. Patent Application Publication Nos. larocaine, propoxycaine, procaine/novocaine, proparacaine, 30 200701 10723, and 20070122427; each of which is incorpo tetracainefamethocaine, articaine, bupivacaine, carticaine, rated herein by reference. See also, U.S. Pat. No. 7,220,418, cinchocaine/dibucaine, etidocaine, levobupivacaine, piper which is incorporated herein by reference. The vaccine can ocaine, prilocalne, trimecaine, saxitoxin, or tetrodotoxin. In include but not be limited to antigenic peptides, proteins, or an embodiment, the nutraceutical includes one or more of a carbohydrates. For example, the vaccine can include but not flavonoid, antioxidant, beta-carotene, anthocyanin, alpha-li 35 be limited to envelope proteins, capsid proteins, Surface pro nolenic acid, omega-3 fatty acids, plant products, or animal teins, toxins, polysaccharides, oligosaccharides, or enzymes products. needed to make at least one thereof. In an embodiment, the at least one therapeutic agent In an embodiment, the virus antigen includes at least one includes one or more of an antimicrobial agent, and includes antigen from one or more of a double-stranded DNA virus, at least one of an anti-fungal agent, antibiotic agent, anti 40 single-stranded DNA virus, double-stranded RNA virus, (+) bacterial, anti-parasitic agent, or anti-worm agent. In an single-strand RNA virus, (-) single-strand RNA virus, single embodiment, the at least one therapeutic agent includes one strand RNA-Reverse Transcriptase virus, or double-stranded or more of penicillin, cephalosporin, polymixin, Sulfona DNA-Reverse Transcriptase virus. In an embodiment, the mide, beta-lactam antibiotic, beta-lactamase inhibitor, ene virus antigen includes at least one antigen of a virus from one diynes, lincosamide antibiotic, nitroimidazole antibiotic, 45 or more of the family of Adenoviridae, Arenaviridae, Bun pleuromutilin antibiotic, polyketide antibiotic, polymyxin yaviridae, Calciviridae, Circoviridae, Coronaviridae, Filov antibiotic, polypeptide antibiotic, antimicrobial peptides, iridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orth quinolone antibiotic, rifamycin antibiotic, Sulfonamide anti omyxoviridae, Papovaviridae, Papillomaviridae, biotic, tetracycline antibiotic, aminoglycoside antibiotic, Polyomaviridae, Paramyxoviridae, Parvoviridae, Picor macrollide, tetracycline, cyclic lipopeptide, glycylcycline, 50 naviridae, Poxyiridae, Reoviridae, Retroviridae, Rhabdoviri oxazolidinone, amoxicillin, tobramycin, levofloxacin, gati dae, Pseudoviridae, or Togaviridae. In an embodiment, the floxacin, moxifloxacin, streptomycin, oxytetracycline, virus antigen includes at least one antigen from one or more of chloramphenicol, or amplicillin. human immunodeficiency virus (HIV) type I, HIV-type 2, In an embodiment, the therapeutic agent includes at least simian immunodeficiency virus (SIV), or feline leukemia one of calcium, carbon, nitrogen, Sulfur, nitrate, nitrite, cop 55 virus. In an embodiment, the virus antigen includes at least per, magnesium, selenium, boron, Sodium, aluminum, phos one antigen from one or more of respiratory syncytial virus phorus, potassium, titanium, chromium, manganese, iron, (RSV), influenza (flu), adenovirus, rhinovirus, enterovirus, nickel, Zinc, silver, barium, lead, Vanadium, tin, strontium, or poliovirus, rubella virus, paramyxovirus, herpes simplex molybdenum. virus type I (HSV-1), Herpes simplex virus 2 (HSV-2), rotavi In an embodiment, the at least one therapeutic agent 60 rus, neurotropic virus, coxsackie virus, hepatitis virus type A, includes at least one enzyme able to convert at least one hepatitis virus type B, hepatitis virus type C, or oncovirus. In prodrug or precursor compound into an active state. In an an embodiment, the at least one antigen includes at least one embodiment, the at least one enzyme includes at least one of antigen from one or more of Brucella, Chlamydia, Citro beta glucuronidase, cytosine deaminase, or nitroreductase. bacter; Coxiella brunetii, Escherichia coli, Francisella tulla In an embodiment, the at least one therapeutic agent is pH 65 rensis, Haemophilus, Legionella, mycobacterium, Neisseria, dependent. Thus, in certain embodiments, the agent can be Pseudomonas, Rickettsia, Salmonella, Shigella, Staphyllo administered to one particular location and remain inactive coccus, Streptococcus, Candida, Cryptococcus, Aspergillus, US 8,682,619 B2 39 40 Blastomyces, Histoplasma, Paracoccidioides, Nosema, din-1-yl)-2,4-dinitrobenzamide. In an embodiment, the at Encephalitozoon, Torulopsis, Pneumocystis, Trypanosoma, least one therapeutic agent includes at least one prodrug. In an Leishmania, Theileria, Plasmodium, Cryptosporidium, or embodiment, at least one altered microorganism delivers at Toxoplasma. least one prodrug, while at least one other altered microor In an embodiment, the at least one therapeutic agent ganism delivers at least one enzyme capable of converting the includes at least one vaccine. In an embodiment, the at least at least one prodrug into active form. For example, published one vaccine includes at least one of an antigenic peptide, studies demonstrating strains of recombinant Saccharomyces antigenic protein, or antigenic carbohydrate. In an embodi cerevisiae expressing plant P450 73A1 show that the enzyme ment, the at least one vaccine includes at least one of an is active and able to convert trans-cinnamic acid into p-cou envelope protein, capsid protein, Surface protein, toxin, 10 maric acid in vivo. See, for example, Garrait, et al., Applied polysaccharide, oligosaccharide, or enzyme needed to make Env. Microbiol. vol. 73, no. 11, pp. 3566-3574 (2007), and at least one thereof. In an embodiment, the vaccine compo Blanquet, et al., Applied EnV. Microbiol. Vol. 69, no. 5, pp. sition further comprises at least one adjuvant. In an embodi 2884-2892 (2003), each of which is incorporated herein by ment, the at least one therapeutic agent includes at least one reference. anti-inflammatory cytokine. 15 In an embodiment, the at least one therapeutic agent In an embodiment, at least one therapeutic agent includes includes at least one factor related to digestion of food, for at one of Interleukin-1, Interleukin-2, Interleukin-3, Interleu example insulin resistance, blood glucose regulation, caloric kin-4, Interleukin-5, Interleukin-6, Interleukin-7. Interleu use or caloric intake of a subject. For example, in an embodi kin-8, Interleukin-9, Interleukin-10, Interleukin-11, Interleu ment the therapeutic agent includes at least one of fasting kin-12, Interleukin-13, Interleukin-14, Interleukin-15, induced adipocyte factor (FIAF or ANGPTL4), peroxisome Interleukin-16, Interleukin-17, Interleukin-18, Interleukin proliferator-activated receptor-Y (PPARY), or similar agents. 19, Interleukin-20, Interleukin-21, Interleukin-22, Interleu See, for example, Jia, et al., Nature Rev. Vol. 7, pp. 123-129 kin-23, Interleukin-24, Interleukin-25, Interleukin-26, Inter (2008), which is incorporated herein by reference. leukin-27, Interleukin-28, Interleukin-29, Interleukin-30, In an embodiment, the at least one altered microorganism Interleukin-31, Interleukin-32, Interleukin-33, Interleukin 25 delivers at least two different therapeutic agents. In certain 34, Interleukin-35, Interleukin-36, Interleukin-37, Interleu cases, the at least two different therapeutic agents are regu kin-38, Interleukin-39, Interleukin-40, Interleukin-41, Inter lated by at least one different promoter. In an embodiment, at leukin-42, Interferon-Y, Interferon-C. Interferon-?3. least one therapeutic agent includes at least one antigen, and Transforming Growth factor, Granulocyte Macrophage at least one cytokine. In an embodiment, the at least one Colony Stimulating Metabolite, Macrophage-Colony Stimu 30 antigen includes tetanus toxin fragment. In an embodiment, lating Metabolite, Scarecrow, Erythropoietin, Granulocyte the at least one cytokine includes at least one of IL-2 or IL-6. Colony Stimulating Metabolite, Leukemia Inhibitory In an embodiment, the antigen is contained within the at least Metabolite. Oncostatin M, Ciliary Neurotrophic Metabolite, one altered microorganism, while the cytokine is secreted. Growth Hormone, Prolactin, Fibroblast Growth factor, Nerve See, for example, Steidler, et al. Infect. Immun. pp. 3183 Growth factor, Platelet Derived Growth factor, Epidermal 35 3189, vol. 66, no. 7 (1998), and U.S. Pat. No. 6,605.286; each Growth factor, Fas, Fas ligand, CD40, CD27, CD4, CD8, of which is incorporated herein by reference. In another CD2, CD3, Tumor Necrosis Metabolite-C, or Tumor Necro example, published studies show secretion of human myelin sis Metabolite-f. basic protein (hMBP) or hMBP as a fusion protein with Chemokines are biochemical signaling molecules that act beta-glucuronidase from E. coli. The heterologous products to attract other particular molecules, including but not limited 40 are produced by L. casei. See, for example, Maassen, et al., to cells, to a specific site. In at least one embodiment, the Vaccine, Abstract, vol. 17, no. 17, pp. 21.17-2128 (1999), therapeutic agent includes one or more chemokines. In at which is incorporated herein by reference. least one embodiment, the one or more chemokines include at The at least one altered microorganism includes at least one least one of a CC chemokine, CXC chemokine, C chemokine, nucleic acid construct encoding at least one therapeutic agent or CX3C chemokine. In an embodiment, the at least one 45 by at least one of mode, including but not limited to synthe therapeutic agent includes at least one of CCL1, CCL2. sizing the at least one agent, expressing the at least one agent CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10, on its Surface, or extracellular secretion of the at least one CCL11, CCL12, CCL13, CCL14. CCL15, CCL16, CCL17, therapeutic or agent. CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, In an embodiment, the at least one altered microorganism CCL25, CCL26, CCL27, CCL28, CCL29, CXCL1, CXCL2, 50 includes at least one nucleic acid construct encoding at least CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, one therapeutic agent and expresses at least a portion of the CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14. agent (e.g. antibody) on its Surface. In an embodiment, the at CXCL15, CXCL16, CXCL17, CXCL18, CXCL19, least one altered microorganism expresses at least a portion of CXCL20, CXCL21, CXCL22, XCL1, XCL2, XCL3, XCL4, a carbohydrate-binding motif on its Surface. In one example, XCL5, CX3CL1, CX3CL2, or CX3CL3. 55 the at least one altered microorganism expresses at least one In an embodiment, administration of the at least one thera of a heparin or mannose binding motif its Surface. peutic agent results in at least one of nucleic acid transcrip For example, as illustrated in FIG.9, in an embodiment, a tion, protein translation, cell division, apoptosis, necrosis, vector 900 including at least one nucleic acid construct 910 is cytoskeletal rearrangement, or secretion in the at least one placed into at least one microorganism 920 by methods biological tissue. 60 known in the art (e.g., electroporation, transformation, etc.). In an embodiment, the at least one therapeutic agent Once incorporated into the microorganism 920, the nucleic includes at least one prodrug or precursor compound. For acid construct 910 is transcribed, resulting in production of at example, the at least one prodrug or precursor compound least one transcript 930, which is converted intracellularly includes at least one glucuronide prodrug, Such as at least one into at least one protein 940. In an embodiment, the protein glucuronide of epirubicin, 5-fluorouracil, 4-hydroxycyclo 65 can remain intracellularly 940 or be secreted into a periplas phosphamide, or 5-fluorocytosine. In another example, the at mic space (not shown). Thus, the protein is obtained through least one prodrug or precursor compound includes 5-(aziri lysis of the microorganism 920. In an embodiment, the pro US 8,682,619 B2 41 42 tein is expressed on the surface 950 of the microorganism predominant route for protein export. Id. Proteins exported 920. In an embodiment, the protein is secreted extracellularly via Sec are translocated across the membrane in an unfolded 96.O. state through a membrane-embedded translocon, to which In an embodiment, the at least one altered microorganism they are targeted by cleavable N-terminal signal peptides. Id. includes at least one synthetic protein scaffold positioned to A second export pathway designated Tat (for twin-arginine modulate the stoichiometry of the synthesis of the at least one translocation), transports prefolded protein substrates. Id. therapeutic agent. For example, in an embodiment, the at least Proteins are targeted to the Tat pathway by tripartitie N-ter one synthetic protein scaffold is positioned to spatially or minal signal peptides, which contain a conserved twin-argi temporally recruit at least one protein or fragment thereof. nine motif in the N region of Tatsignal peptides. Id. The motif For example, synthetic protein scaffolds that spatially recruit 10 is described as R-R-X-d-do, where did represents a hydropho metabolic enzymes can be designed and modulate the stochi bic amino acid. The consecutive arginine residues are almost ometry of a biosynthetic pathway. See, for example, Dueber invariant and believed to be important for transport by this et al., Nat. Biotech. vol. 27, no. 8, pp. 753-759 (2009), which pathway. Id. The Tat System is capable of producing large is incorporated herein by reference. In one example, at least proteins, and proteins with lipid anchors, which are some one flux of an enzymatic reaction of a particular biosynthetic 15 times difficult to produce by way of the Sec system. Id. pathway can be balanced to, for example, limit the accumu According to published studies, the twin arginine transloca lation of intermediates, optimize production levels, prevent tion system has been demonstrated to be usable in various loss of intermediates, protect unstable intermediates from microorganisms. See, for example, Maillard, et al., PNAS, degradation, circumvent unfavorable equilibria and kinetics vol. 104, no. 40, pp. 15641-15646; U.S. Pat. No. 7,447,595: imposed by bulk-phase metabolite concentration, etc. Id. In each of which is incorporated herein by reference. an embodiment, Scaffolds provide a means for controlling In an embodiment, the at least one altered microorganism design by physically separating catalytic activities from bind is inducible to produce the at least one agent by at least one ing element, which allows modification (i.e., single interac prepro signal sequence. See, for example, Eiden-Plach, et al., tion to each enzyme) to the enzyme needed for attaining App. and EnV. Microbiol., vol. 70, no. 2, pp. 961-966, which modular control over complex formation. Id. Designing Such 25 is incorporated herein by reference. In an embodiment, the at scaffolds can be performed by tethering a short peptide ligand least one altered microorganism is inducible to produce the at to the enzyme, for example an SH2 domain, SH3 domain, least one agent by at least one Aga2p display. See, for GTPase binding domain, etc. In one particular embodiment, a example, U.S. Pat. No. 6,696,251, which is incorporated synthetic complex is designed by attaching a varying number herein by reference. In an embodiment, the displayed agent is of SH3 interaction ligands to the C terminus of one enzyme 30 cleaved from the yeast by at least one protease. (e.g., synthase), and attaching an SH3 domain to the N ter In an embodiment, the at least one altered microorganism minus of another enzyme (e.g., reductase). Id. includes a microorganism having one or more non-reverting In an embodiment, the at least one altered microorganism mutations. For example, a non-reverting mutation can involve includes at least one riboregulated transcriptional cascade a polynucleotide of greater than about 1 nucleotide, about 2 counter. See, for example, Friedland, et al., Science, vol. 324, 35 nucleotides, about 3 nucleotides, about 4 nucleotides, about 5 pp. 1199-1202, (2009), which is incorporated herein by ref nucleotides, about 10 nucleotides, about 15 nucleotides, erence. In an embodiment, the at least one riboregulated about 20 nucleotides, or any value therebetween or greater. In transcriptional cascade counter is positioned to produce at an embodiment, the non-reverting mutation blocks at least least one product upon exposure to at least one inducer. one biosynthetic pathway of the microorganism. For Various inducers are described herein. Several non-limit 40 example, the non-reverting mutation can include a deletion, ing examples include physical or chemical inducers, such as insertion, inversion, or any combination of these. Developing radiation, temperature, carbohydrate, peptide, protein, alco altered microorganisms containing non-reverting mutations hol, antibiotic, steroid, metal, salicylic acid, ethylene, ben is a routine practice, and is based on several non-limiting Zothiadiazole, or other compound. In an embodiment, the at factors, including the ability to mutate a particular gene with least one antibiotic includes at least one of amplicillin, tetra 45 out destroying the viability of the microorganism, the nature cycline, penicillin, pristinamycin, or other antibiotic. of the at least one therapeutic agent that is to be delivered by In an embodiment, the at least one riboregulated transcrip the altered microorganism, the nature of the microorganism, tional cascade counter is positioned to produce at least one and in some cases, the nature of the subject that will host the sequence of products upon exposure to at least one sequence altered microorganism. For example, in an embodiment the of inducers. In an embodiment, the at least one riboregulated 50 mutated gene prevents production of at least one enzyme that transcriptional cascade counter is positioned to produce at is required for a biosynthetic pathway of a metabolite needed least one product that is different than at least one other for replication or protein synthesis, without sacrificing viabil consecutive product. In an embodiment, the at least one ribo ity of the microorganism (e.g., aro genes, pab genes, pur regulated transcriptional cascade counter is positioned to pro genes, etc.). See, for example, U.S. Pat. No. 4,837,151, which duce at least one first product upon exposure to at least one 55 is incorporated herein by reference. Standard methods for first inducer. In an embodiment, the at least one riboregulated making non-reverting mutants include, but are not limited to, transcriptional cascade is inducible to produce at least one introducing transposable elements, site directed mutagenesis, second product upon exposure to at least one second inducer. congugational crossing, or other form of mutagenesis. The In an embodiment, the at least one riboregulated transcrip altered microorganism is produced by transduction, transfor tional cascade is inducible to produce at least one third prod 60 mation, or other means. uct upon exposure to at least one third inducer, etc. In an embodiment, the at least one therapeutic agent is able In an embodiment, the at least one altered microorganism to translocate to at least one other location in the biological is inducible to produce the at least one agent by at least one tissue or Subject. For example, in an embodiment, the at least twin-arginine translocation system. See, for example, Wid one altered microorganism delivers at least one therapeutic dick, et al., PNAS, vol. 103, no. 47, pp. 17927-17932 (2006), 65 agent to the gastro-intestinal region of a Subject, where it is which is incorporated herein by reference. For example, in absorbed and utilized at a distant location in the same subject most bacteria, the general Secretory pathway (Sec) is the (e.g., lungs, heart, etc.). In an embodiment, the at least one US 8,682,619 B2 43 44 therapeutic agent translocates systemically. In an embodi lose materials, polyvinyl chloride, polyvinylidene fluoride, ment, the at least one therapeutic agent translocates selec polyurethane isocyanate, polyalginate, polysulfone, polysty tively to a location (e.g., by utilizing particular cell ligand rene, polyvinyl alcohol, polyacrylonitrile, polymethylene receptor interactions, oxygen or other gas levels, magnetic oxide, polytetrafluoroethylene, polymethyl methacrylate, particles tagged to the microorganism, or other mechanism polyamide, polyether-polyamino copolymer, thermoplastic for directing the microorganism, etc.). copolymer, polyesteramide, polyamidoamine, polythioester, In an embodiment, the altered microorganism produces at nylon, polyacrylamide, acrylamide, nylon, urethane, polytet least one treatment or therapeutic agent catalyzes the conver rafluoroethylene, polyurethane, dimethylsiloxane/methylvii stion of a prodrug to its active state. For instance, in an nylsiloxane copolymer, acetate, polyester, dextran, calcium, embodiment, the at least one anaerobic microorganism is 10 silicon, phosphorous, iron, magnesium, manganese, Sodium, administered to a tumor that has a largely anaerobic environ ment. The anaerobic microorganism is allowed to proliferate potassium, chromium, titanium, nickel, Zinc, copper, cobalt, and produce an enzyme or other factor. Prior to, during, or tungsten, silver, gold, platinum, enzyme, acid, amino acid, after administration of the at least one anaerobic microorgan peptide, polypeptide, protein, oligonucleotide, nucleic acid, ism, at least one prodrug is administered to the tumor (e.g., by 15 ribonucleic acid, oligosaccharide, polysaccharide, glycopep way of a microorganism or other delivery method). The tide, glycolipid, lipoprotein, Sphingolipid, glycosphin enzyme or other factor secreted by the at least one anaerobic golipid, glycoprotein, peptidoglycan, lipid, carbohydrate, microorganism is convert the at least one prodrug to an active metalloprotein, proteoglycan, chromosome, adhesion mol state (e.g., to a cytotoxic agent). For example, the at least one ecule, cytokine, chemokine, immunoglobulin, antibody, anti enzyme includes at least one of beta glucuronidase or gen, platelet, extracellular matrix, blood plasma, cell wall, cytosine deaminase. In an embodiment, the at least one pro hormone, organic compound, inorganic compound, salt, cell drug includes at least one glucuronide prodrug. In an embodi ligand, cell receptor, or chlorophyll. ment, the at least one glucuronide prodrug includes at least In an embodiment, the semi-permeable barrier includes at one glucuronide of epirubicin, 5-fluorouracil, 4-hydroxycy least one pore or port component 1035 configured to selec clophosphamide, or 5-fluorocytosine. See, for example, U.S. 25 tively limit access between the interior and the exterior of the Pat. No. 6,652,849, which is incorporated herein by refer semi-permeable barrier 1017. In an embodiment, the semi CCC. permeable barrier includes a homogenous Surface or con In another example, the enzyme includes nitroreductase, struction material. In an embodiment, the semi-permeable and the prodrug includes 5-(aziridin-1-yl)-2,4-dinitrobenza barrier includes a heterogenous Surface or construction mate mide (CB1954). See, for example, U.S. Pat. No. 6,416,754, 30 rial. As described herein, bone cages, diatom cages, and other which is incorporated herein by reference. semi-permeable barriers discussed herein may be naturally As illustrated in FIG. 10, an implantable device 1000 com porous. In an embodiment including a semi-permeable bar prises an semi-permeable barrier 1017 structured to substan rier, pores or ports are utilized to restrict access to the interior tially enclose at least one altered microorganism 1020, the or internal cavity of the device. In an embodiment, the pores semi-permeable barrier 1017 defining an interior region 1015 35 or ports can be naturally occurring, manufactured in the and an exterior region or external wall 1016 of the device device, or both. In an embodiment, the device includes mul 1000. In an embodiment, the at least one altered microorgan tiple ports 1035 at various locations. In an embodiment, the ism 1020 includes at least one nucleic acid construct encod semi-permeable barrier 1017 includes at least one size exclu ing at least one therapeutic agent. In an embodiment, produc sion component 1050 (larger view of optional component of tion of the at least one therapeutic agent 1025 (indicated as 40 the port 1035). In an embodiment, the at least one size exclu concentric circles emanating from the at least one altered sion component 1050 includes at least one size exclusion microorganism 1020), results in release of the therapeutic membrane, Screen, or filter. agent to the interior 1015 of the semi-permeable barrier 1017. In an embodiment, the semi-permeable barrier 1017 In an embodiment, the at least one therapetuica gent 1025 is includes at least one charge exclusion component 1040 transported to the exterior of the semi-permeable barrier 45 (larger view of optional component of the port 1035). In an 1017. In an embodiment, the device 1000 further comprises at embodiment, the at least one charge exclusion component least one anchoring component 1010 from which to secure 1040 includes at least one charge exclusion membrane. In an the device to at least one biological tissue or organ 1095. In an embodiment, the semi-permeable barrier includes at least one embodiment, the anchoring component 1010 includes at least semi-permeable component (indicated by dotted lines). In an one latch, hook or tether. In an embodiment, the anchoring 50 embodiment, the device 1000 includes at least one internal component 1010 includes at least one adhesive or Velcro port component 1036 configured to selectively limit access attachment. between layers or internal modules located in the interior of As described herein, in an embodiment, the implantable the device 1000. In an embodiment, the at least one internal device 1000 is biocompatible. In an embodiment, the port component 1037 traverses one or more semi-permeable implantable device 1000 is biodegradable. In an embodiment, 55 barriers and actively or passively transports the therapeutic the semi-permeable barrier includes at least one of a bone agent 1025 from an internal compartment 1060 containing cage, diatom cage, polymer or co-polymer cage, protein cage, the at least one altered microorganism 1020, to the exterior of element cage, Vitamin cage, mineral cage, chloroplast cage, the device 1016. In an embodiment, the device 1000 includes or carbohydrate cage, as described herein. In an embodiment, at least one end cap 1030. In an embodiment, the at least one the semi-permeable barrier includes one or more collagen, 60 semi-permeable component includes at least one semi-per elastin, albumin, gelatin, extracellular matrix, acrylate, meth meable membrane. In an embodiment, the at least one semi acrylate, fibrinogen, thrombin, fibrin, cellulose, poly(hy permeable component includes at least one microporous or droxyalkanoate), poly(e-caprolactone), polysaccharide, nanoporous material. In an embodiment, the semi-permeable polythioester, starch, amylase, polylactone, polyphosp barrier 1017 includes means to deter or restrict ingress (e.g., haZene, polycyanaoacrylate, poly(lactic acid), poly(glycolic 65 a semi-permeable membrane, nanopore membrane, etc.) of at acid), polycaprolactone, polyanhydride, polydioxanone, least one of an immune cell or immune-system related mate polyorthoester, polypropylene fumarate), plasticized cellu rial from the subject. US 8,682,619 B2 45 46 In an embodiment, the at least one altered microorganism permeable barrier 1017. In an embodiment, the semi-perme is located in a compartment that is at least partially separated able barrier 1017 is configured to alter the at least one thera from another compartment of the device (e.g., the compart peutic agent 1025 by at least one of altering at least one ments can be linearly arranged, concentrically arranged, or physical or chemical property of the at least one therapeutic any other arrangement). The compartments can be separated, 5 agent 1025. In an embodiment, the at least one semi-perme for example, by a semi-permeable component, where it is able barrier 1017 includes at least one of an emulsion, lipid, desired to allow movement of the contents of one compart dispersion, or micelle. In an embodiment, the at least one ment to another. In an embodiment, the at least one therapeu semi-permeable barrier 1017 includes one or more cells. In an tic agent is allowed to pass through to another compartment or embodiment, the at least one semi-permeable barrier 1017 to the exterior of the device, while the at least one altered 10 includes one or more cell junctions. In an embodiment, the microorganism producing the at least one therapeutic agent is one or more cell junctions include at least one of a tight prohibited from leaving its compartment. Regulatory control junction, Zonula adherens, macula adherens, or gap junction. of the therapeutic agent or altered microorganism can be In an embodiment, the semi-permeable barrier 1017 is at least implemented, for example, by a semi-permeable component, one of expandable, compressible, rigid, or pliable. size exclusion component, or other means. 15 In an embodiment, the device 1000 further comprises at In an embodiment, the flow rate of fluid through the device least one detection material 1086. In an embodiment, the at can be regulated. For example, in an embodiment the flow rate least one detection material 1086 includes at least one of a can be altered depending on at least one of the materials contrast agent, or electronic identification device. In an utilized for the device, use of a pump, or varying the number embodiment, the at least one electronic identification device of pores orports in the device. In an embodiment, the flow rate includes at least one radio frequency identification device. In offluid through the device can be adjusted to alter the delivery an embodiment, the at least one detection material 1086 of the at least one therapeutic agent. includes at least one of a radioactive, luminescent, colorimet In an embodiment, the device 1000 further comprises at ric or odorous Substance. In an embodiment, the at least one least one sensor 1070. In an embodiment, the at least one detection material 1086 includes at least one of a diamagnetic sensor 1070 includes an optical density sensor. Optical den 25 particle, ferromagnetic particle, paramagnetic particle, Super sity sensors are well known in the art, and include, for paramagnetic particle, particle with altered isotope, or other example, TruCellTM or TruCell2TM, that are molded optical magnetic particle. In an embodiment, the device 1000 further sensors for measuring cell counts in cell cultures or other comprises at least one transmitter 1087 that can send signals media. In an embodiment, the device 1000 further includes at to a receiver 1088 (that can be located in the interior or least one pump 1080. In an embodiment, the at least one pump 30 exterior of the device 1000). In an embodiment, the device 1080 includes at least one osmotic or peristaltic pump. In an 1000 further comprises at least one circuit 1090 operably embodiment, the osmotic pump includes a semi-permeable coupled to one or more of the components of the device. membrane incorporated into a portion of the wall of the As described elsewhere herein, the semi-permeable barrier device (or wall of the compartment, ifutilized to move thera structured with an angle of enclosure of approximately 10 peutic agent from an inner compartment to an outer compart 35 degrees, approximately 20 degrees, approximately 30 ment of the device). In an embodiment, an osmotically active degrees, approximately 40 degrees, approximately 50 agent is mixed with the therapeutic agent. In an embodiment, degrees, approximately 60 degrees, approximately 70 the pump 1080 is connected to at least one component (e.g., a degrees, approximately 80 degrees, approximately 90 port 1035), by a channel 1085, for example. In an embodi degrees, approximately 1OO degrees, approximately 110 ment, no connection 1085 is required between the pump 1080 40 degrees, approximately 120 degrees, approximately 130 and the at least one component. degrees, approximately 140 degrees, approximately 150 In an embodiment, the semi-permeable barrier is config degrees, approximately 160 degrees, approximately 170 ured to actively or passively transport at least a portion of the degrees, approximately 18O degrees, approximately 190 at least one therapeutic agent 1025 to the exterior of the degrees, approximately 2OO degrees, approximately 210 semi-permeable barrier 1017 (e.g., via a port 1035). In an 45 degrees, approximately 22O degrees, approximately 230 embodiment, the semi-permeable barrier is configured to degrees, approximately 240 degrees, approximately 250 actively or passively transfer at least a portion of the at least degrees, approximately 260 degrees, approximately 270 one therapeutic agent 1025 based on at least one chemical degrees, approximately 28O degrees, approximately 290 gradient. In an embodiment, the semi-permeable barrier 1017 degrees, approximately 3OO degrees, approximately 310 is configured to actively or passively transfer at least a portion 50 degrees, approximately 320 degrees, approximately 330 of the at least one therapeutic agent 1025 based on one or degrees, approximately 340 degrees, approximately 350 more of pH differential, salt concentration gradient, electrical degrees, approximately 360 degrees, or any value less than or charge gradient, therapeutic agent concentration gradient, therebetween. endocytosis, exocytosis, diffusion, altered microorganism In an embodiment, the semi-permeable barrier is struc concentration gradient, or condensation. For example, in an 55 tured to be permeable to material less than or approximately embodiment, the semi-permeable barrier allows certain mol equal to 50 nm, less than or approximately equal to 40 nm, ecules or ions to pass through it depending on at least one of less than or approximately equal to 30 nm, less than or the pressure, concentration, or temperature of the molecules approximately equal to 20 nm, less than or approximately or solutes on either side, as well as the permeability of the equal to 10 nm, less than or approximately equal to 5 nm, less barrier to each solute. In an embodiment, the semi-permeable 60 than or approximately equal to 1 nm, less than or approxi barrier includes at least one lipid bilayer, thin film composite mately equal to 100 um, less than or approximately equal to membrane, cellulose ester membrane, charge mosaic mem 50 um, less than or approximately equal to 25um, less than or brane, bipolar membrane, anion exchange membrane, alkali approximately equal to 10 Jum, less than or approximately anion exchange membrane, or proton exchange membrane. equal to 5um, less than or approximately equal to lum, or any In an embodiment, the semi-permeable barrier 1017 is 65 value therebetween. configured to alter the at least one therapeutic agent 1025 In an embodiment, the at least one altered microorganism prior to, during, or after transfer to the exterior of the semi 1020 includes at least one auxotrophic microorganism. In an US 8,682,619 B2 47 48 embodiment, the device 1000 includes at least one essential ing, binder, filler, compression aid, encapsulation aid, preser nutrient required by the at least one auxotrophic microorgan Vative, granulation agent, spheronization agent, stabilizer, ism. In an embodiment, the at least one essential nutrient is in adhesive, pigment, Sorbent, nanoparticle, microparticle, or limited Supply. In an embodiment, the at least one altered gel. microorganism 1020 is responsive to at least one of pH, light, In an embodiment, the device is configured to be included or heat. with at least part of one or more of dentures or other oral In an embodiment, the device is configured to induce apo implants, contact lens or other ocular implants, orifice insert, ptosis in the at least one altered microorganism. In an embodi orifice spray or inhaler, prosthetics, Sutures, Surgical staples, ment, the device is configured to induce apoptosis in the at dental floss, Stents, shunts, bandages, absorbable mesh, or least one altered microorganism 1020 by at least one of alter 10 oral implant. ing pH of the interior of the semi-permeable barrier, or alter As depicted in FIG. 11, other embodiments of the device ing the temperature of the interior of the semi-permeable 1000 disclosed herein include devices that are external to a barrier. Thus, in an embodiment, the device is configured to biological tissue, or reside on the Surface of the Subject (e.g., increase or decrease the pH of the interior by release of an bandages, etc.). As depicted in FIG. 11, the semi-permeable acidic or basic Substance, respectively. For example, in an 15 membrane 1017 at least partially encloses at least one altered embodiment, the semi-permeable barrier includes poly(lac microorganism 1020 that has been modified to deliver at least tic-co-glycolic) acid (PLGA). Published reports indicate that one agent (e.g., therapeutic agent). the degradation of PLGA results in a reduction in the local pH The therapeutic agent described herein may be formulated when implanted into a biological tissue. See, for example, neat or may be combined in the device with one or more Liu, et al. Int’l J. Nanomed. Vol. 1(4), pp. 541-545 (2006), acceptable carriers, diluents, excipients, and/or vehicles Such which is incorporated herein by reference. Furthermore, the as, for example, buffers, Surfactants, preservatives, solubiliz rate of degradation (and thus, the pH) can be regulated by ing agents, isotonicity agents, and stablilizing agents as adding titanium nanoparticles to the barrier. Id. For example, appropriate. A pharmaceutically acceptable carrier, for the addition of titanium nanoparticles slows the rate of deg example, may be approved by a regulatory agency of the state radation of the PLGA device, and thus, does not drop the 25 and/or Federal government such as, for example, the United localized pH as rapidly. Id. States Food and Drug Administration (US FDA) or listed in In an embodiment, the device includes heating capacity the U.S. Pharmacopeia or other generally recognized phar (e.g., battery powered electrical heat) that can be remotely macopeia for use in animals, and more particularly in controlled. In an embodiment, the device includes power humans. Conventional formulation techniques generally generated from the biological tissue or subject for which it 30 known to practitioners are described in Remington: The Sci serves. For example, in an embodiment the device is config ence and Practice of Pharmacy; 20" Edition; Cover page (3 ured to increase the local temperature near the device at least pgs.); printed on Mar. 19, 2010; Lippincott Williams & about one degree Celsius, at least about two degrees Celsius, Wilkins, Baltimore, Md. which is incorporated herein by at least about three degrees Celsius, at least about four degrees reference. Celsius, at least about five degrees Celsius, at least about six 35 Acceptable pharmaceutical carriers can be present in the degrees Celsius, at least about seven degrees Celsius, at least same compartment as the at least one altered microorganism, about eight degrees Celsius, at least about nine degrees Cel or in a different compartment (e.g., the at least one altered sius, at least about ten degrees Celsius, or any value therebe microorganism can be located in an inner compartment and tween or greater. In an embodiment, this increase temperature the at least one carrier can be located in an outer compart is held for a matter of about a few seconds, about a few 40 ment). Examples of pharmacetulical carriers include, but are minutes, about a few hours, or about a few days. not limited to, the following: Sugars, such as lactose, glucose In an embodiment, the at least one altered microorganism and Sucrose; starches, such as corn starch and potato starch; 1020 includes at least one inducible genetic element config cellulose, and its derivatives, such as Sodium carboxymethyl ured to initiate death of the at least one altered microorganism cellulose, ethyl cellulose, bovine serum albumin, keyhole 102O. 45 limpet hemocyanin, tetanus toxoid, cellulose acetate, and In an embodiment, the device is configured for administra hydroxymethylcellulose; polyvinylpyrrolidone; cyclodextrin tion to at least one biological tissue by at least one route and amylose; powdered tragacanth; malt, gelatin, agar and including peroral, topical, transdermal, epidermal, intrave pectin; talc; oils, such as mineral oil, polyhydroxyethoxylated nous, intraocular, tracheal, transmucosal, intracavity, Subcu castor oil, peanut oil, cottonseed oil, safflower oil, Sesame oil, taneous, intramuscular, inhalation, fetal, intrauterine, intra 50 olive oil, corn oil and soybean oil; polysaccharides, such as gastric, placental, intranasal, interdermal, intradermal, alginic acid and acacia; fatty acids and fatty acid derivatives, enteral, parenteral, Surgical, or injection. In an embodiment, Such as Stearic acid, magnesium and sodium Stearate, fatty the intracavity route includes at least one of oral, vaginal, acid amines, pentaerythritol fatty acid esters; and fatty acid uterine, rectal, nasal, peritoneal, Ventricular, or intestinal. The monoglycerides and diglycerides; glycols, such as propylene delivery may include inhalation, depot injections, implants, 55 glycol; polyols, such as glycerin; Sorbitol, mannitol and poly or other mode of delivery by way of an apparatus. ethylene glycol; esters, such as ethyl oleate and ethyl laurate; In an embodiment, a device includes a time-release formu buffering agents, such as magnesium hydroxide, aluminum lation. In at least one embodiment, a device includes at least hydroxide and sodium benzoate/benzoic acid; water, isotonic one of an Suspension, mixture, Solution, Sol, clathrate, col saline: Ringer's solution; ethyl alcohol; phosphate buffer loid, emulsion, microemulsion, aerosol, ointment, capsule, 60 Solutions; other non-toxic compatible Substances employed micro-encapsule, powder, tablet, Suppository, cream, device, in pharmaceutical compositions. The pharmaceutical compo paste, resin, liniment, lotion, ampule, elixir, spray, syrup, sitions are generally formulated as sterile, Substantially iso foam, pessary, tincture, detection material, polymer, biopoly tonic and in full compliance with all Good Manufacturing mer, buffer, adjuvant, diluent, lubricant, disintegration agent, Practice (GMP) regulations of the U.S. Food and Drug Suspending agent, solvent, light-emitting agent, colorimetric 65 Administration. agent, glidant, anti-adherent, anti-static agent, Surfactant, In an embodiment, the at least one sensor includes at least plasticizer, emulsifying agent, flavor, gum, Sweetener, coat one biosensor. In an embodiment, the at least one biosensor US 8,682,619 B2 49 50 includes at least one altered microorganism. In an embodi TABLE 2-continued ment, the at least one altered microorganism detects the at least one agent. For example, in an embodiment, a biosensor Disease or Condition Therapeutic Agent microorganism is express green fluorescent protein (or Alzheimer's disease Metal protein attenuation another measurable material) upon exposure to the at least compounds, Ion channel blockers, Oligomeric amyloid beta formation inhibitors one agent. See, for example, Hansen et al., App. EnV. Micro Alzheimer's disease RAGE inhibitors biol. Vol. 67, no. 1, pp. 239-244 (2001), which is incorporated (Receptor for Advance Glycation herein by reference. In an embodiment, the biosensor micro endproducts), Antibodies targeting amyloid organism is expresses a measurable material upon exposure beta Alzheimer's disease GSK-3B Kinase 10 inhibitors, CdkSp25 Kinase inhibitors, to at least one byproduct of the altered microorganism. For Extracellular signal-regulated kinase 2 example, as set forth in Hansen, et al., a altered microorgan (ERK2) inhibitors, C-abl Kinase inhibitors, ism expresses green fluorescent protein in the presence of MARK Kinase inhibitors, Protein phosphate tetracycline, which is produced by a naturally occurring bac promoters (PP-2A) Alzheimer's disease terial strain. Id. As indicated, testing was conducted by inocu Modulators of Amyloid beta production (e.g., 15 Secretase inhibitor, gamma Secretase lating soil with the biosensor microorganisms, and detecting inhibitor, gamma Secretase modulators, or the biosensor microoganisms by flow cytometry. Id. For alpha secretase stimulators), Inhibitors of example, induced biosensor bacteria were isolated using fluo inhibit amyloid beta aggregation (e.g., Metal protein attenuation compounds, Ion channel rescence-activated cell sorting (FACS) and examined by epi blockers, or Oligomeric amyloid beta fluorescence microscopy. Id. formation inhibitors), Amyloid beta load In an embodiment, a method of treating a disease or con reducer (e.g., RAGE inhibitors (Receptor for dition in a subject comprises administering at least one Advance Glycation endproducts), or Antibodies targeting amyloid beta), Tau implantable device including an semi-permeable barrier related microtubule destabilization inhibitors structured to Substantially enclose at least one altered micro (GSK-3B Kinase inhibitors, CdkSp25 organism, the structure defining an interior region and an Kinase inhibitors, ERK2 Kinase inhibitors, exterior region of the device; wherein the at least one altered 25 C-abl Kinase inhibitors, MARK Kinase inhibitors, Protein phosphate promoters (PP microorganism includes at least one nucleic acid construct 2A)) encoding at least one therapeutic agent; and wherein the at Bone Fractures Receptor activator of NF-kappaB ligand (RANKL) least one implantable device dispenses to the Subject an effec activators, Bone Morphogenetic Protein-7 tive amount of the at least one therapeutic agent. (BMP-7) Bone loss during cancer (RANKL 30 activator and Bisphosphonates) and In an embodiment, the subject is afflicted with or suspected (Aromatase treatment inhibitors for breast of being afflicted with at least one disease or condition. As cancer OR Anti-androgens for prostate described herein, the at least one disease or condition may cancer OR luteinizing hormone-releasing include one or more of a pathogenic infection, parasitic infec hormone (LHRH) agonists for prostate tion, autoimmune disease, sepsis, Systemic inflammatory cancer) 35 Rheumatoid Arthritis an anti-CD20 agent, a JAK3 (anus kinase 3) response syndrome, septic shock, multiple organ dysfunction inhibitor, a CCR1 (chemokine c-c motif syndrome, allergic reaction, or cancer. In at least one embodi receptor 1) antagonist, a Syk (spleen tyrosine ment, the at least one inflammatory disease or condition kinase) inhibitor, a P38 MAP kinase inhibitor, CTLA-4 (cytotoxic T-lymphocyte includes one or more of anaphylaxis, viral infection, bacterial antigen 4), a Tumor necrosis factor (TNF)- infection, plasmodium infection, protozoan infection, nema alpha antagonist, a TNF-alpha ligand, a tode infection, or other worm infection. In at least one 40 steroid, an inhibitor of the IL-12 Superfamily embodiment, the at least one inflammatory disease or condi of cytokines (IL-1, 6, 12, 15, 17, 18, 32) Cancer (Vascular endothelial growth factor tion includes malaria. In at least one embodiment, the para (VEGF) blocker or VEGF receptor ligand), sitic infection includes at least one infection or infestation of (Platelet-derived growth factor (PDGF) one or more of a phytoparasite, Zooparasite, ectoparasite, blocker or PDGF receptor ligand), a receptor endoparasite, or one or more of parasitic cysts, larvae, or eggs. 45 tyrosine kinase inhibiting (rtki) compound Cancer (a RAS kinase inhibitor or a RAF Particular non-limiting examples of diseases or conditions kinase inhibitor or a MEK kinase inhibitor or are listed in Table 2 below. an ERK kinase inhibitor) and (an AKT kinase inhibitor or an inhibitor of the TABLE 2 mammalian target of rapamycin (mTOR) 50 kinase or S6k1 or 4E-BP1), angiogenesis Disease or Condition Therapeutic Agent inhibitor (e.g., VEGF blocker, VEGF receptor ligand, PDGF blocker, PDGF Obesity/Diabetes Ghrelin antagonists, PYY, Leptin, receptor ligand, EGF blocker, a receptor Obestatin, GLP-1, Exendin, Amylin, G tyrosine kinase inhibiting (rtki) compound), a protein coupled receptor GRP 119 agonists, tumor cell pathway inhibitor (e.g., a RAS selective Melanin Concentrating Hormone 55 kinase inhibitor or a RAF kinase inhibitor or (MCH) receptor blockers, Cannabinoid-1 a MEK kinase inhibitor, an ERK kinase agonists, Lipase inhibitors, Neuropeptide Y inhibitor, a RAS kinase inhibitor or a RAF (NPY) blocker, Oxymodulin, Silent Mating kinase inhibitor or a MEK kinase inhibitor, Type Information Regulation 2 homolog-1 an ERK kinase inhibitor), inhibitors of (SIRT-1, sirtuin) activators, Oxyntomodulin, chromatin modification (e.g., an inhibitor of Cholecystokinin (CCK) agonists, Gastric Histone Deacetylase (HDAC), an inhibitor of Inhibitory Polypeptide (GIP) agonists 60 histone acetyltransferase (HAT)), TNF-alpha Hepatitis interferons; protease inhibitors, e.g., antagonist, a TNF-related apoptosis inducing Telaprevir (VX-950; Vertex Pharmaceuticals, igand (TRAIL) antibody, Wnt inhibitor, a Inc.); antibodies (e.g., monoclonal, Hh (hedgehog) inhibitor, PI-3 Kinase humanized, polyclonal, single-chain) inhibitor, a MEK kinase inhibitor, PI-3 Alzheimer's disease secretase inhibitor, gamma Secretase Kinase inhibitor, an mTOR kinase inhibitor, inhibitor, gamma Secretase modulators, alpha 65 epidermal growth factor receptor tyrosine secretase stimulators, serotonin inhibitors kinase inhibitor (EGFr-TKI), a B-cell US 8,682,619 B2 51 52 TABLE 2-continued VCH, Weinheim, 409–470, 2007, which is incorporated herein by reference. Particles encapsulating cells can be Disease or Condition Therapeutic Agent greater than 1 mm and as Small as a single cell. For example, leukemia/lymphoma 2 (BCL-2) blocker, Breguet et al., describe the encapsulation of Chinese hamster TRAIL receptor antibody, a traditional ovary (CHO) cells into alginate/poly-L-lysine particles of cytotoxic compound, P38 MAP kinase inhibitor and (a Rafkinase inhibitor, a MEK 500 um and 800 um with as many as 17,000 cells perparticle. kinase inhibitor, or an ERK kinase Breguet, et al., Cytotechnology 53:81-93, 2007, which is inhibitors), EGFr TKI, an ERB2 inhibitor incorporated herein by reference. The amount of immunogen used for immunization can be 10 See, for example, U.S. Patent App. Pub. No. 20090202608, an integer microgram (ug) from approximately 0.1 ug to which is incorporated herein by reference. 1,000 ug including approximately, but not limited to approxi In an aspect, the immunogen and/or adjuvant can be pro mately, 0.1 ug, 0.5 Lig, 1 Jug. 5ug. 10 ug. 25 Jug, 30 ug. 35ug. duced by cells, e.g., COS cells, incorporated into the internal 40 ug, 45 ug, 50 ug. 100 ug, 250 ug, 500 ug, and 1000 ug. In cavity of the semi-permeable barrier. The number of cells 15 an aspect, the amount of immunogen can be approximately up producing the immunogen and/or adjuvant can be approxi to 0.1 ug, up to 0.5 Lig, up to 1 Lig, up to 5 Jug, up to 101g, up mately up to 10 cells, up to 10 cells, up to 10 cells, up to 10° to 25ug, up to 50 ug, up to 100 ug, up to 250 g. up to 500 ug, cells, up to 107 cells, up to 10 cells, or up to 10 cells. In an or up to 1000 ug. As an example, GARDASILR) for immuni aspect, the number of cells incorporated into the semi-perme Zation against the human papillomavirus includes 20 Jug of able barrier can be dependent upon the amount of immunogen recombinant HPV 6 L1 protein, 40 g of recombinant HPV and adjuvant needed for immunization, the efficiency of the 11 L1 protein, 40 ug of recombinant HPV 16 L1 protein, and incorporated cells, and the size of the semi-permeable barrier 20 ug of recombinant HPV 18 ul proteins. See, e.g., GAR cavity. For example, studies describe synthesis and secretion DASILRHPV vaccine Prescribing Information, U.S. Food & of recombinant tick-borne encephalitis virus protein E in Drug Administration. In an aspect, the total amount of immu COS cells in yields ranging from 1 to 5ug per 10°COS cells. 25 nogen needed for immunization can be incorporated into one Allison, et al., J. Virol. 69: 5816-5820, 1995, which is incor semi-permeable barrier and implanted into a Subject. In an porated herein by reference. As such, up to 5x10 cells would aspect, the total amount of immunogen needed for immuni be needed to generate 25ug of protein E for immunization. In zation can be distributed in a number of semi-permeable the case of a COS cell with an estimated cellular volume of barriers, all or part of which are implanted into a subject. 500 cubic um, the 5x10 cells could be accommodated in a 30 In an aspect, the immunogen can be a live, attenuated or semi-permeable barrier with an internal cavity measuring at dead pathogen or tumor cell. In an aspect, the amount of least 2.5x10 cubicum in volume with approximate dimen immunogen can be expressed as the number of cells used for sions of at least 1350 umx1350 Lumix 1350 um. The total num immunization. The number of cells can be approximately up ber of cells producing the immunogen and/or adjuvant can be to 10, up to 10, up to 10, up to 10°, up to 107, up to 10, or incorporated into one semi-permeable barrier for implanta 35 up to 10. For example, Berger, et al., describe using 7x107 tion or distributed in a number of semi-permeable barriers for autologous prostate tumor cells for immunization against implantation. locally advanced or metastatic prostrate cancer. Berger, et al., The internal cavity of the semi-permeable barrier can hold J. Pharm. Pharmaceut. Sci. 10:144-152, 2007, which is non-cellular particles of varying size. In an aspect, the par incorporated herein by reference. In an aspect, the amount of ticles are the immunogen, for example, virus-like particles. In 40 immunogen can be expressed as the number of infectious an aspect, the particles can incorporate and/or encapsulate an units, a measure of the concentration of live, attenuated immunogen that is a biomolecule or a live, attenuated or pathogen in a given amount of fluid. The number of infectious inactivated pathogen or tumor cell. In an aspect, the particles units can be approximately up to 10 IU, up to 10, up to 10, can encapsulate one or more cells that have been genetically up to 10, up to 10, up to 10, or up to 10. For example, the engineered to express an immunogen and/orantigen. The size 45 FluMist(R) influenzalive virus vaccine contains 107 infec of the particles depends upon the composition of the particles tious units of each of three influenza strains for the 2008-2009 and the intended contents thereofas well as the size of the season. See, e.g., FluMist(R), Prescribing Information, U.S. semi-permeable barrier cavity. Food & Drug Administration. The total number of cells and/ The diameter of the particles can be any integer nm from or infectious units needed for immunization can be incorpo approximately 1 nm to 107 nm including approximately, but 50 rated into a single semi-permeable barrier for implantation or not limited to, 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 250 distributed in a number of semi-permeable barriers for nm, 500 nm, 1000 nm, 10 nm, 10 nm, 10 nm, and 107 nm. implantation. In an aspect, the diameter of the particles can be approxi The external wall of the one or more semi-permeable bar mately up to 10 nm, up to 50 nm, up to 100 nm, up to 500 nm, riers can be porous. Porosity refers to the percentage of void up to 1000 nm, up to 10 nm, up to 10 nm, up to 10 nm, or 55 space in a solid. Adv. Colloid Interface Sci. 76-77:341-372, up to 107 nm. Virus-like particles derived from self-aggrega 1998, which is incorporated herein by reference. Porosity is a tion of genetically engineered viral proteins are in the Sub morphological property independent of the material. Porosity micromolar size range. For example, Tamura, et al., describe can be created by, for example, salt leaching, gas foaming, the preparation of viral particles derived from recombinant phase separation, freeze-drying, and sintering, depending on expression of the Norwalk virus capsid protein that are 38 nm 60 the material used to fabricate the access-limiting scaffold. in size. Tamura, et al., J. Virol. 74:11589-11597, 2000, which The porosity can be any integer percentage from approxi is incorporated herein by reference. Kallinteri & Carnett mately 1% to approximately 99% including, but not limited describe a number of polymeric nanoparticles used for drug to, approximately 2%. 3%, 4%, 7%, 10%, 12%, 15%, 20%, delivery including vaccine delivery ranging in average size 35%, 50%, 60%, 75%, and/or 90%. In an aspect, the porosity from 100 nm to over 600 nm Kallinteri & Garnett. Polymeric 65 can be approximately 1% to 99%, 1% to 15%, 3% to 12%.5% “Nanoparticle for Drug Delivery”. Nanomaterials for Medi to 10%, 40% to 95%, 50% to 90%, 60% to 75%, 3% to 90%, cal Diagnosis and Therapy. Ed. by Challa Kumar. Wiley 10% to 75%, 15% to 90%, and 25% to 90%. US 8,682,619 B2 53 54 The porosity may not be uniform throughout the barrier. Canada) for Surgical implantation in humans is prepared from The porosity of trabecular barrier can be approximately 50% bovine bone and has been used to augment autografts for hip to 90%, while that of cortical access-limiting can be approxi revision surgery. Acta Orthop. 76:544-549, 2005, which is mately 3% to 12%. Biomaterials 26:5474-5491, 2005, which incorporated herein by reference. Studies of the immunologi is incorporated herein by reference. 5 cal mechanisms underlying the rejection of pig organs The pore size of the one or more semi-permeable barriers implanted into primates has resulted in the development of can be any integer nm from approximately 1 nm to approxi novel lines of genetically engineered pigs that are more mately 10,000 nm including, but not limited to, approxi immunologically compatible with man.J. Nephrol. 16 (Suppl mately 2 nm, 3 nm, 4 nm, 5 nm, 8 nm, 10 nm, 12 nm, 15 nm, 7):S16-21, 2003, which is incorporated herein by reference. 20 nm, 25 nm, 50 nm, 100 nm, 200 nm,300 nm, 500 nm, 600 10 The bone cage can be comprised of anorganic bone. Anor nm, 800 nm, 1,000 nm, 2,000 nm, 5,000 nm, or 10,000 nm. In ganic bone or anorganic bone matrix has been used for bone an aspect, the pore size can be approximately 1 nm to 10,000 repair. Clin. Plast. Surg. 21:437-444, 1994; J. Long Term Eff. nm, 10 nm to 5,000 nm, 25 nm to 1,000 nm, 50 nm to 750 nm, Med. Implants 8:69-78, 1998, which are incorporated herein 100 nm to 500 nm, 10 nm to 100 nm, 5 nm to 50 nm, 1 nm to by reference. Anorganic bone or anorganic bone matrix 10 nm, 2 nm to 20 nm, 500 nm to 5,000 nm, 1,000 nm to 15 includes autologous, allogeneic, or Xenogeneic bone (with 10,000 nm, or 250 nm to 1,000 nm in width. In an aspect, the respect to a subject within whom the bone is implanted) that pore size can be approximately 10 Lum up to approximately has been deorganified. Examples of the use of Such tissues 100 um in width. In an aspect, the pore size may not be include, but are not limited to, Bio-Oss(R natural bone sub uniform throughout the structure. stitute (Geistlich Pharma Ag, Wolhusen, Switzerland), that is In an embodiment, the semi-permeable barrier is struc- 20 composed of anorganic bovine bone, or an anorganic bone tured to be permeable to biological or chemical material matrix. Arch Oral. Biol. (2005) July 29 Epub ahead of print): greater than or approximately equal to 50 Daltons, greater Biomaterials 26: 5648-5657, 2005, which are incorporated than or approximately equal to 100 Daltons, greater than or herein by reference. approximately equal to 101 Daltons, greater than or approxi The bone cage can be comprised of demineralized bone. mately equal to 102 Daltons, greater than or approximately 25 Demineralized bone has been used as allografts for bone equal to 103 Daltons, greater than or approximately equal to repair. Cell Tissue Bank 6:3-12, (2005) which is incorporated 104 Daltons, greater than or approximately equal to 105 herein by reference. Demineralized bone can include autolo Daltons, greater than or approximately equal to 106 Daltons, gous, allogeneic, or Xenogeneic bone (with respect to a Sub greater than or approximately equal to 107 Daltons, greater ject within whom the bone is implanted) that has been dem than or approximately equal to 108 Daltons, greater than or 30 ineralized. An example of the use of demineralized, freeze approximately equal to 109 Daltons, greater than or approxi dried bone together with anorganic bovine bone for maxillary mately equal to 110 Daltons, or any value greater than or sinus grafting is presented in Int. J. Oral Maxillofac. Implants therebetween. 18:556-60, 2003, which is incorporated herein by reference. Device Including One or More Bone Cages Derived from Once the organic, anorganic, freeze-dried and/or deminer Natural or Synthetic Materials 35 alized bone is obtained, the cage can be created by a variety of Natural Source of Bone Materials. techniques. The bone can be machined using, for example, Organic bone can encompass multiple kinds of bone microtomes such as the Leica SP2600 (or 1600) Saw Micro obtained from donors including cortical, trabecular and can tome (Leica Microsystems Nussloch GmbH, Postfach 1120, cellous. The bone can be autologous, allogeneic, or Xenoge Heidelberger Strasse 17-19, D-69226 Nussloch, Germany) neic, with respect to a subject within whom the bone is 40 that can slice bone to a finished thickness of approximately implanted. Autologous tissue can be excised from one part of 20-30 Lum. Lasers, such as the YAG laser rod, can be used to an individual and implanted into another part of the same cut bone with a minimum width of approximately 10 um for individual. Allogeneic tissue can be harvested from one indi deeper beam penetrations and less than 1 um for thin coatings vidual donorand implanted into a genetically different recipi (Laserod, Inc., Gardena, Calif.90247-5252). Micro tweezers, ent individual within one species. A xenogeneic tissue would 45 such as those from MEMS Precision Instruments, can be used be harvested from an individual in one species and implanted to assemble the pieces as necessary. Methods for preparing in an individual recipient from a different species. 2-50 um thick sections of undecalcified hard tissues, for In an aspect, the bone cage can be comprised of autologous example those found in e.g., Histochem Cell. Biol. 113:331 bone excised from, e.g., the iliac crest, skull, or fibula. Autolo 339, 2000 can be used. gous tissues do not typically elicit immune rejection. In an 50 An example of a method to make bone cages of FIG. 1 aspect, the bone cage can be comprised of allogeneic bone and/or FIG. 3A is described below. The bone cage can be harvested from a cadaver from any location in the body and constructed by excising a portion of cortical bone approxi optionally frozen prior to re-implantation to decrease immu mately 3 mm by 1 mm from the iliac crest of a subject using nogenicity. Examples of uses for allogeneic bone include, but a microsaw. This portion of bone is then micromachined to a are not limited to, Allogro(R) demineralized bone matrix, Allo- 55 desired size, for example about 30 um by 90 um, using a Source, Centennial Colo.; Orthoblast(R), demineralized bone microsaw. The shape is rectangular, or Smoothed to an matrix and cancellous bone in reverse phase medium, Isotis oblong, although other shapes may be implemented. The Orthobiologics, Irvine Calif.; Opteform R) demineralized interior cavity of the bone cage is hollowed using a microma bone matrix based allograft, Exactech, Inc., Gainesville Fla.: chining laser, leaving an approximately 5um thickbone wall. and Grafton demineralized bone matrix, Osteotech, Inc., Eat- 60 The bone wall can be perforated with approximately 1 to 2 um ontown N.J. Allograft bone substitutes can include allograft holes using a micromachining laser. A second piece of bone bone of a variety of material sources, both natural and Syn can be micromachined and shaped to form a bone cap or plug. thetic, or a composite. An example of a bone cage that can be constructed using Xenogeneic bone tissue can be obtained from animals and these techniques is shown in FIG. 2C. Bone formed in a can be used for implantation in humans. For example, Surgi- 65 tubular structure can be sliced into sections, for example bone(R), sterile, extracellular composite of hydroxyapatite and perpendicular to the tubular Haversian systems that make up collagen of bovine bone (Unilab, Inc., Mississauga, ON, cortically dense bone, to produce verythin bone rings. These US 8,682,619 B2 55 56 rings can then be further sectioned into barrel staves to form (2005); J. Artif. Organs 8:131-136, 2005, J. Biomed. Mater a barrel-shaped construct, laid side by side to form a tube Res. A. 68:725-734, 2005, J. Long Term Eff Med. Implants shaped construct, or overlapped to make Smaller portal struc 8:69-78, 1998, which are incorporated herein by reference. tures. Further holes and Smaller cutting can create joints to Examples include, but are not limited to, Norian(R) SRS(R) fast allow the various components to fit together and be assembled set putty, Norian Corp., Cupertino Calif.; ProCosteon(R) using micro tweezers. hydroxyapatite bone substitute, Biomet, Inc. Warsaw, Ind., In an alternative aspect, bone cages can be constructed by Osteograf R hydroxyapatite bone material, and Osteoset(R) excising a portion of bone, followed by micromachining to calcium sulfate bone substitute, Dentsply, UK; Wright Medi the desired size and/or shape. The orientation of the construct cal Technology, Inc., Arlington, Tenn. can be selected to align the natural pores of the bone to form 10 Organic materials of natural origin including collagen, a natural internal cavity for the bone cage. The interior cavity fibrin, and hyaluronic acid can be used, as can inorganic of the bone cage can be further refined using focused beam material of natural origin including, for example, coralline machining to enlarge or re-shape the interior cavity of the hydroxyapatite. A variety of metals can be used in artificial bone cage. Additional pores can be added as described herein, scaffolds for bone, including silicon, titanium and aluminum. if the natural porosity of the bone is not sufficient to allow the 15 J. Biomed. Mater. Res. A. 70: 206-218, 2004, J. Biomed. desired amount and/or type of nutrients and/or other materials Mater. Res. 56: 494-503, 2001, J. Biomed. Mater. Res. A. 72: to reach and/or elute from the internal cavity. 288-295, 2005, which are incorporated herein by reference. The methods for making a bone cage described herein are In addition to the methods for making bone cages dis illustrative and are not intended to be limiting. In addition, cussed above, design and prototyping of scaffolds can be these and other methods can be used in combination as well as performed digitally, and the material can be processed as separately. sponge-like sheets, gels, or highly complex structures with The bone cage can be comprised of biocompatible and/or intricate pores and channels. Int. J. Prothodont. 15: 129-132, implantable artificial bone Substitutes containing metals, 2002; Ann. NY Acad. Sci. 961: 83-95, 2002, which is incor ceramics and/or polymers. Artificial bone scaffolding can be porated herein by reference. A biocompatible three-dimen used in bone repair. Int. J. Oral Maxillofac. Surg. 33:325-332; 25 sional internal architectural structure with a desired material 2004, Int. J. Oral Maxillofac. Surg. 33:523-530, 2004, which Surface topography, pore size, channel direction and trabecu are incorporated herein by reference. Artificial bone includes lar orientation can be fabricated. Biomaterials 23: 4437 any bone Substitute composites or scaffolds with a structural 4447, 2002, which is incorporated herein by reference. Fab rigidity Substantially equal to or greater than that of cartilage, rication of scaffolding can be accomplished using and with pores that allow at least fluid passage. The pores can 30 conventional manual-based fabrication techniques or com allow passage of macromolecules, but not cells. The cells may puter-based solid free form fabrication technologies. Fron produce and secrete one or more immunogens and/or one or tiers in Tissue Engineering, New York, Elsevier Science 107 more adjuvants. In an aspect, the pores can allow passage of 120, 1998, J. Biomed. Mater. Res. 51: 376-382, 2000, J. cells as well as macromolecules. Passage refers to processing Biomater. Sci. Polymer. E., Vol. 7, No. 1, pp. 23-38, 1995, Br: including, but not limited to, diffusion, release, extrusion, 35 J. Plast. Surg. 53: 200-204, 2000, which are incorporated and/or migration. herein by reference. See, e.g., U.S. Application Numbers The mechanical properties of naturally occurring bone, 2007/0184088; 2007/0134346; 2007/0134345; 2007/ including stiffness and tensile strength, can be provided by 0.134225; 2007/0134224; 2007/0134223; 2007/0134.222; and the bone tissue “scaffold’ that contains significant amounts of 2007/0134216, which are incorporated herein by reference. non-living material. Such as organic minerals, as well various 40 Device Including One or More Bone Cages Generated Using proteins of the extracellular matrix. Hydroxyapatite Synthetic Source of Bone Materials. The device including one or more bone cages can be gen A variety of bone substitutes can be used in tissue engi erated using hydroxyapatite either alone or in combination neering to create scaffolds. Synthetic Biodegradable Polymer with other agents. Hydroxyapatite is synthesized by precipi Scaffolds (1997) Boston, Mass.: Birkhauser; J. Biomed. 45 tation after the mixing of a calcium-containing Solution and a Mater. Res. 54:162-171, 2001, Int. J. Oral Maxillofac. Surg. phosphate-containing solution (see, e.g., U.S. Pat. No. 5,858, 33:523-530, 2004, which are incorporated herein by refer 318: U.S. Pat. No. 6,592,989, which are incorporated herein CCC. by reference). For example, hydroxyapatite can be formed by These include, but are not limited to, synthetic organic combining solutions of calcium nitrate and ammonium phos materials such as clinically used nondegradable and biode 50 phate with a calcium to phosphate ratio of about 10:6. The pH gradable and bioresorbable polymers including polygly of the solution is adjusted to a pH of about 2.0 with dilute acid collide, optically active and racemic polylactides, polydiox or to about 10.0 with dilute base. The resulting precipitate is anone, and polycaprolactone, polymers under clinical collected by centrifugation at about 9,000 to 10,000 rpm, investigation including polyorthoester, polyanhydrides, and washed several times with distilled water, filtered and dried. polyhydroxyalkanoate, early stage polymeric biomaterials 55 In some instances, the slurry of hydroxyapatite granules in including poly(lactic acid-co-lysine), as well as biodegrad water can be extruded through a spray nozzle under pressure able polymer ceramic scaffolds. J. Mater. Sci. Mater. Med. and in the presence heat to form Smaller and more uniform 16:807-819; 2005, Biomaterials 19:1405-1412, 1998, which granules (see, e.g., U.S. Pat. No. 5,858.318, which is incor are incorporated herein by reference. Examples include, but porated herein by reference). The characteristics of the result are not limited to, CortoSSR Self-setting synthetic ceramic 60 ing hydroxyapatite powder are assessed using X-ray diffrac composite, Orthovita, Malvern, Pa.; 3D open cell polylactic tion, Fourier transform infrared spectroscopy (FTIR), acid (OPLA); and Immix(R) amorphous D, L-Polylactide-co scanning electron microscopy (SEM), and/or transmission glycolide synthetic bone graft scaffold, Osteobiologics Inc. electron microscopy (TEM). Alternatively, granular San Antonio, Tex. hydroxyapatite is purchased from a commercial Source Synthetic inorganic molecules can also be used in scaffold 65 (from, e.g., Clarkson Chromatography Products, Inc., South ing, including hydroxyapatite, calcium/phosphate compos Williamsport, Pa.). Hydroxyapatite granules of uniform size ites, calcium Sulfate, and glass ceramics. Biotechnol. Bioeng. can also be generated using any of a number of commercially US 8,682,619 B2 57 58 available milling and/or grinding systems (from, e.g., ence. The binding to hydroxyapatite can be enhanced by Hosokawa Micron, Summit, N.J.) followed by sizing through incorporating an acidic oligopeptide (e.g., six to eight resi a series of sub-millimeter mesh sieves. Particle size is dues of L-aspartic acid) into the binding biomolecule. See, assessed using laser light scattering instrumentation (e.g., e.g., Nishioka, et al., Mol. Genet. Metab. 88:244-55, 2006, Mastersizer 2000, Malvern Instruments, Inc., Malvern, which is incorporated herein by reference. Worcestershire, UK). The one or more immunogens and/or one or more adju The powdered hydroxyapatite can be shaped into appro vants can be linked to the bone cage through a chemical priate structures using slurry cast molding. See, e.g., Rum linkage between one or more components of the immunogen pler, et al., J. R. Soc. Interface 5:1173-1180, 2008, which is and/or adjuvant and one or mote components of the bone incorporated herein by reference. Casting molds can be 10 cage. Chemical linkers may be used to link together proteins, designed using computer-aided design (CAD) Software and carbohydrates, oligonucleotides, Small biomolecules, or produced using a three-dimensional wax printer (e.g., Model combinations thereof. In an aspect, the chemical linker can Maker II 3D modeling system, Solidscape, Merrimack, bind directly to the mineral component of the bone. N.H.). The molds are filled with a slurry of hydroxyapatite Chemical linkers such as homobifunctional, heterofunc particles, heated to 600° C. to remove the wax mold, and 15 tional, and/or photoreactive cross-linking agents can be used sintered at 1300° C. for 1 hour. to link the immunogen and/or the adjuvant to biomolecules Alternatively, hydroxyapatite spheres with an internal cav associated with a Surface of the bone cage. The immunogen ity are generated using methods described by Lee, et al., J. and/or the adjuvant can be linked to biomolecules associated Mater. Sci. Mater. Med. 19:3029-3034, 2008, which is incor with a surface of the bone cage through amine groups, Sulf porated herein by reference. Hydroxyapatite powder (20% by hydryl groups, carbohydrate groups, or a combination weight) is vigorously mixed in dichloromethane containing thereof. Examples of homobifunctional cross linkers include, polyvinyl buryral (5% by weight) and then dropped into a but are not limited to, primary amine/primary amine linkers water bath containing 2% polyvinyl alcohol. The slurry is such as BSOCES (bis(2-succinimidooxy-carbonyloxy stirred while the solvent is evaporated. The resulting micro ethyl) sulfone), DMS (dimethyl suberimidate), DMP (dim spheres (0.1 to 1 millimeter in diameter) are collected by 25 ethyl pimelimidate), DMA (dimethyladipimidate), DSS (dis filtration, dried overnight and then heat treated from several uccinimidyl suberate), DST (disuccinimidyl tartate), Sulfo hours at a temperature ranging from about 600° C. to about DST (sulfodisuccinimidyl tartate), DSP (dithiobis(succinim 1200° C. idyl propionate), DTSSP (3,3'-dithiobis(succinimidyl propi In some instances, the powdered hydroxyapatite is com onate), EGS (ethylene glycol bis(Succinimidyl Succinate)) pressed into blocks, discs or other structures that are further 30 and sulfhydryl/sulfhydryl linkers such as DPDPB (1,4-di-(3'- machined to form the bone cage. For example, hydroxyapa 2 pyridyldithio-propionamido) butane). Examples of het tite powder can be dry blended with ethylene vinyl acetate erofunctional cross linkers include, but are not limited to, (EVA) powder and low density polyethylene and pressed into primary amine/sulfhydryl linkers such as MBS (m-maleimi ceramic blocks by hot pressing at 150° C. for 5 minutes at a dobenzoyl-N-hydroxysuccinimide ester), Sulfo MBS pressure varying from 30–70 MPa. See, e.g., Velayudhan, et 35 (m-maleimidobenzoyl-N-hydroxysulfosuccinimide), GMBS al., Materials Letters 46:142-146, 2000, which is incorpo (N-y-maleimidobutyryl-oxysuccinimide ester), Sulfo GMBS rated herein by reference. Alternatively, preformed blocks, (N-y-maleimidobutyryloxysulfosuccinimide ester), EMCS discs, or other structures of hydroxyapatite can be purchased (N-(e-maleimidocaproyloxy) Succinimide ester), Sulfo from a commercial source (from, e.g., Clarkson Chomatog EMCS(N-(e-maleimidocaproyloxy) sulfo succinimide), raphy Products, Inc., South Williamsport, Pa.; Berkeley 40 SIAB (N-Succinimidyl(4-iodoacetyl)aminobenzoate), Advanced Biomaterials, Inc., Berkeley, Calif.). SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane One or more cavities are formed in the block of hydroxya 1-carboxylate), SMPB (succinimidyl 4-(rho-maleimidophe patite using a drill with a micro drill bit (e.g., drill bits ranging nyl) butyrate), Sulfo SIAB (N-sulfosuccinimidyl(4-io in size from 50-250 micrometers from Union Tools, Co., doacetyl)aminobenzoate), Sulfo SMCC (sulfosuccinimidyl Buena Park, Calif.). Micro-holes in the wall of the block of 45 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), Sulfo hydroxyapatite can be generated using a focused laser. A laser SMPB (sulfosuccinimidyl 4-(rho-maleimidophenyl)bu can be used to cut and shape hydroxyapatite. See, e.g., Teix tyrate), and MAL-PEG-NHS (maleimide PEG N-hydrox eira, et al., J. Biomed. Mater. Res. A, 81:920-929, 2007, which y succinimide ester); sulfhydryl/hydroxyl linkers such as is incorporated herein by reference. The block of hydroxya PMPI (N-rho-maleimidophenyl)isocyanate; sulfhydryl/car patite is further machined to create micro-holes as Small as 1 50 bohydrate linkers such as EMCH(N-e-maleimidocaproic micrometer in the wall of the block using a laser microma acid) hydrazide); and amine/carboxyl linkers such as EDC chining system with a pulsed diode pumped solid State laser (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro (from, e.g., Oxford Lasers, Oxon, UK). chloride). Cross-Linking an Immunogen and an Adjuvant to Bone Cage In an aspect, the one or more immunogens and/or the one or Device 55 more adjuvants can be linked directly to the bone cage A device including one or more bone cages can include the through a bisphosphonate linkage. Bisphosphonates bind to one or more immunogens and/or one or more adjuvants the mineral phase of bone and have been used in the treatment attached to a Surface of the one or more bone cages. The one of osteoporosis for antiresorptive therapy. An amino group or more immunogens and/or one or more adjuvants can be can be added to bisphosphonate by chemical synthesis and non-covalently attached to the bone cage by simple adsorp 60 used to functionalize the bisphosphonate with a heterologous tion. For example, hydroxyapatite, a bone replacement mate cross-linking agent Such as those described herein. The het rial with a crystal structure similar to the inorganic matrix of erologous cross-linking agent can be used to link the amino bone, can be used as an adsorption matrix. Studies describe bisphosphonate to a biomolecule. The biomolecule-modified the adsorption and release of a recombinant human protein to aminobisphosphonate can bind to the surface of bone as well a hydroxyapaptite-based implant for use in delivering a 65 as to the Surface of bone Substitutes, e.g., hydroxyapatite. See, therapy to a bone graft. Boix, et al., J. Inorg. Biochem. e.g., Ehrick et al. Bioconjugate Chem. 19:315-321, 2008, 99:1043-1050, 2005, which is incorporated herein by refer which is incorporated herein by reference. US 8,682,619 B2 59 60 In an aspect, the immunogen and/or the adjuvant can be mal stem cells can be reliably isolated and cultured in thera linked to the bone cage through an azide-alkyne mediated peutic quantities. Bone 13:81-88, 1992, which is incorporated linkage. The copper-catalyzed azide-alkyne cycloaddition is herein by reference. Several methods can be used to isolate a 1,3-dipolar cycloaddition between an azide and a terminal mesenchymal stem cells from, for example, bone marrow, alkyne to form a triazole. A copper-free cycloaddition reac- 5 adipose tissue, and muscle, based on the physical and immu tion has also been described for use in living cells. See, e.g., nological characteristics. Basic & Clinical Pharmacology & Baskin et al., Proc. Natl. Acad. Sci, USA. 104:16793-16797, Toxicology 95:209, 2004, Ann. Biomed Eng. 32:136-147, 2007, which is incorporated herein by reference. To link one 2004, which are incorporated herein by reference. Mesenchy or more components, one component is derivatized with mal stem cells can differentiate into various lineages includ azide while the other component is derivatized with alkyne. 10 ing osteoblasts in vitro. Science 284: 143-147, 1999; J Cell The components can be readily Snapped together using "click Sci. 113:1161-1166, 2000; Int. J. Oral Maxillofac. Surg. chemistry. Viral particles, oligonucleotides, carbohydrates, 33:325-332, 2004, which are incorporated herein by refer lipids, proteins, and peptides can be functionalized with azide CCC. and/or alkyne for use in “click chemistry” reactions in which The bone cage can be comprised of cells cultured in vitro building block components are readily 'snapped together. 15 on bone scaffolding. In an aspect, the bone scaffolding can be See, e.g., Heinet al., Pharm. Res. 25:2216-2230, 2008; Ming, degradable in vitro and/or in vivo. Porosity and pore size of et al., Nucleic Acids Symp. Ser: (Oxf). 52:471-472, 2008; Van the scaffold can play a role in bone formation, osteogenesis Dongen et al., Bioconjugate Chem. 20:20-23, 2009; Godeau, and osteoconduction in vitro and in vivo. Methods of mea etal, J. Med. Chem. 51:4374-4376, 2008, which are incorpo Suring and controlling porosity and pore size in artificial rated herein by reference. 2O scaffolds can be used. Biomaterials 26:5474-5491, 2005, Cellular Restructuring a Device Including One or More Bone which is incorporated herein by reference. Cages Stem cells and/or osteoblast progenitor cells can be propa The device including one or more bone cages can be con gated on scaffolds of a variety of shapes including, those structed utilizing cells cultured in vitro including, but not shown in FIG. 2. The cells are grown until fusion, or partially limited to, stem cells, fibroblasts, endothelial cells, osteo- 25 grown to result in a lattice shape. The bone cells cultured in blasts and/or osteoclasts. The in vitro cultured cells can be vitro include autologous, allogeneic, or Xenogeneic cells, configured to form the bone cage structure or configured to with respect to a subject within whom the bone cage is restructure the bone cage wallorinner compartment. Restruc implanted. A method of making a bone cage using mesenchy turing the device including one or more bone cages is useful mal stem cells is described herein and, for example, FIG.3B. to design compartments that can provide temporal release or 30 An artificial scaffold, for example, of degradable polymer, triggered release of the one or more immunogens and one or can be laid down in the desired open lattice-work shape of the more adjuvants from the bone cage. Restructuring or restruc two halves of the bone structure. Expanded mesenchymal tured, as it relates to the bone cage, refers to a change in the stem cells (autologous, allogeneic, or Xenogeneic) are cul physical structure of the bone cage, including, but not limited tured in the latticework shapes, in vitro, and encouraged to to, bone size, shape, architecture and quality. Bone restruc- 35 differentiate into osteoblasts. Once the cells have populated turing includes, but is not limited to, bone resorption, osteo the lattice structure, other optional components of the device conduction, or bone deposition. Timed release can depend including the one or more bone cages is added, and the device upon the size of the bone cage compartment and the thickness is implanted. of the bone cage wall. The bone cage wall can be slowly The bone cage can comprise living tissue. Living tissue degraded to release one or more of the one or more immuno- 40 refers to the presence of living bone cells such as, but not gens and the one or more adjuvants. In an aspect, the non limited to, osteoblasts, or osteoclasts within the bone scaf stem cells can be isolated from a Subject. Bone cell popula fold. Living tissue includes living bone cells in artificial bone tions can be derived from all bone surfaces by a variety of scaffolding. The living tissue can be autologous, allogeneic, techniques, including, but not limited to, mechanical disrup or Xenogeneic, with respect to a subject within whom the tion, explantation, and enzyme digestion. Tissue Eng. 1:301 - 45 bone cage is implanted. The bone cage can comprise dead 308, 1995, which is incorporated herein by reference. Meth tissue. “Dead tissue' refers to the absence of living bone cells, ods can be used to culture and/or propagate osteoprogenitor Such as, but not limited to, osteoblasts, or osteoclasts within cells and/or osteoblast-like cells in vitro. Int. J. Oral Maxil the bone scaffold. The dead tissue can be autologous, alloge lofac. Surg. 33:325-332, 2004, which is incorporated herein neic, or Xenogeneic, with respect to a subject within whom the by reference. Culture conditions can be used for manufactur- 50 bone cage is implanted. ing bone tissue including, but not limited to, temperature, The bone cage can be designed and/or treated, at least culture medium, biochemical and mechanical stimuli, fluid partially or completely, to prevent restructuring. In the case of flow and perfusion. Int. J. Oral Maxillofac. Surg. 33:523-530. a bone cage with artificial scaffolding, autologous, or non 2004, which is incorporated herein by reference. autologous bone, bone restructuring can include, but is not The non-stem cells can be differentiated from stem cells, 55 limited to, the influx and growth of the subject’s bone cells in including, but not limited to, fetal, embryonic, cord blood, the artificial, autologous, or non-autologous bone scaffold. mesenchymal and/or hematopoeitic stem cells. In an aspect, Mechanisms of restructuring, treatments to modify restruc the numbers of stem cells can be increased in culture in vitro turing, and genes governing restructuring can be used. Nature prior to differentiation. Methods can be used for isolation, 1:47-54, 2005, which is incorporated herein by reference. culturing and transplantation of stem cells. Fetal Diagn. Ther: 60 Methods for detecting and measuring changes in the device 19:2-8, 2004, Best Pract. Res. Clin. Obstet. Gynaecol. including one or more bone cages is described. The change 18:853-875, 2004, which are incorporated herein by refer can result, for example, from global or discrete increases or CCC. decreases in bone mass. Alternatively, the change can result, In an aspect, the stem cells can be mesenchymal stem cells. for example, from global or discrete increases or decreases in Mesenchymal stem cells are multipotent cells found in sev- 65 the relative ratios of cells, including, but not limited to, the eral, perhaps most, adult tissues. Blood 105:1815-1822, number of osteoblasts as compared with the number of osteo 2005, which is incorporated herein by reference. Mesenchy clasts. The change can also result, for example, from global or US 8,682,619 B2 61 62 discrete increases or decreases in bone pore size and/or poros tively, discrete portions of the bone cage can be coated to ity. Increase and/or decrease in bone mass, relative ratio of selectively prevent restructuring as discussed herein. cells, or pore size and/or porosity, for example, can be mea One or more hormones and/or related compounds, includ sured as any integer percent change from 1% to 99%, for ing, but not limited to, estrogen, growth hormone, calcitonin, example, 10%, 25%, 50%, 75%, and 95%, as compared with Vitamin D, and/or calcium, that encourage bone growth, can the original bone mass, relative ratio of cells, or pore size be administered before, during, or after implantation of the and/or porosity, respectively, either globally or in a discrete bone cage to partially or completely modify bone restructur location. ing. In an aspect, the bone cage can be treated globally or Bone restructuring, a combination of bone resorption by discretely with a thin layer of one or more of these hormones 10 to encourage bone growth throughout or in discrete locations. osteoclasts and bone deposition by osteoblasts, can be modi Anabolic therapies including, but not limited to, hormones fied. Resorption as it relates to the bone cage refers to a such as parathyroidhormone (PTH-(1-84)), teriparatide decrease in bone mass from either global or discrete reduc (PTH-(1-34)), and/or excess glucocorticoid, that can increase tions in, for example, the extracellular matrix and/or cells. bone turnover and porosity can be administered systemically Bone resorption can be mediated by osteoclasts, so treatments 15 to partially or completely modify restructuring. Osteoporosis that inhibit the activity of osteoclasts decrease bone resorp Int. 13:97-104, 2002. In an aspect, these hormones can be tion. Methods for detecting and measuring these changes are administered locally by treating the entire bone cage, or dis described. Biomaterials 26:5474-5491, 2005, which is incor crete portions of the bone cage, to allow selective restructur porated herein by reference. ing. These hormones can be administered by placing them In an aspect, restructuring of the bone cage can be partially inside the cage as one of the one of the one or more immu or completely reduced or prevented. In an aspect, the bone nogens and one or more adjuvants and/or one or more cells or can be designed and/or treated to be at least partially, or tissues. completely, restructured. Modifications of bone restructuring Bone resorption can be influenced by the administration of can result, for example, from administration of compounds cytokines that increase osteoclast activity including, but not that influence bone resorption and/or deposition, by the selec 25 limited to, interleukin-1, M-CSF, tumor nevrosis factor, and/ tion of the pore size and/or porosity of the bone, by the or interleukin-6. Bone resportion can be influenced by the selection of the type of bone, by the selection of the location administration of cytokines that decrease osteoclast activity of implantation, as a result of inherent, induced, or genetically including, but not limited to, interleukin-4, interferon-Y, and/ modified immunogenicity, and as a result of other genetic ortransforming growth factor-3. Inanaspect, bone resorption modification. In an aspect, the bone is partially or completely 30 can be influenced by other humoral factors including, but not resorbable. limited to, leukotrienes, arachidonic metabolites, and/or Compounds that influence bone restructuring through prostaglandins and their inhibitors and including 5-lipoxyge modifications in bone resorption and/or deposition can be nase enzyme inhibitors. applied before, during, or after implantation of the bone cage. Bone formation can be influenced by the administration of Compounds can be administered at the discretion of the 35 factors that promote osteoblast activity and proliferation health professional and depend on the desired timing and the including, but not limited to, insulin-like growth factors I and extent of the modification of a subjects bone restructuring. II, transforming growth factor-B, acidic and basic fibroblast Administration of the compounds can be systemic or local growth factor, platelet-derived growth factor, and/or bone ized. Systemic and local administration includes any method morphogenic proteins. used in the art for pharmaceutical administration. 40 Bone formation can be influenced by the administration of The device including one or more bone cages including one factors that promote osteoblast activity and proliferation or more immunogens and one or more adjuvants can be including, but not limited to, growth hormone, parathyroid administered locally by being applied in the subject in the hormone (PTH), bone morphogenetic protein (BMP), trans vicinity of the bone either globally, or in localized areas, forming growth factor-O. (TGF-C.), TGF-31, TGF-B2, fibro depending on whether complete or partial restructuring is 45 blast growth factor (FGF), granulocyte/macrophage colony desired. An example is the incorporation of the cell binding stimulating factor. (GM-CSF), epidermal growth factor peptide P-15 on anorganic bovine bone matrix. Biomaterials (EGF), platelet derived growth factor (PDGF), insulin-like 25:4831-4836, 2004, J. Biomed. Mater. Res. A. 74:712-721, growth factor (IGF), scatter factor/hepatocyte growth factor 2005, Biomaterials 26:5648–5657, 2005, which are incorpo (HGF), fibrin, collagen, fibronectin, vitronectin, hyaluronic rated herein by reference. Other examples include, but are not 50 acid, an RGD-containing peptide or polypeptide, an limited to, addition of TGF-B, platelet-derived growth factor, angiopoietin or vascular endothelial cell growth factor fibroblast growth factor, and bone morphogenic proteins. (VEGF). In an aspect, compounds can be administered by incorpo Bone pore size and porosity influence bone restructuring ration in the bone cage including one or more immunogens through modifications in bone resorption and/or deposition. and one or more adjuvants optionally in combination with 55 Since the size of the pores in the bone impacts new bone living cells and/or tissues, as discussed herein. growth, decreasing the pore size and/or the percent of poros Bis-phosphonates, used systemically to prevent bone ity of the bone in the cage reduces or prevents restructuring. In resorption can be applied before, during, or after implantation contrast, increasing the pore size and/or the percent porosity of the bone cage to partially or completely modify bone of the bone in the cage enhances restructuring. The bone cage restructuring. Osteoporos Int 13: 97-104, 2002, Curr. 60 can be constructed Such that the pore size and porosity is Osteoporos. Rep. 1: 45-52, 2003, which are incorporated approximately uniform through out the cage, or that the pore herein by reference. Such therapies can also be administered size and porosity varies depending on the location. Varying locally by treating the bone cage, or by placing them inside the pore size and/or porosity in discrete locations leads to the cage in combination with one of the one or more immu partial restructuring (either partial enhancement or partial nogens and one or more adjuvants, or optionally one or more 65 prevention). cells or tissues that produce the one or more immunogen In an aspect, the pore size of the one or more bone cages can and/or the one or more adjuvant, to elute over time. Alterna be approximately 1 nm to 10 nm, approximately 1 nm to 20 US 8,682,619 B2 63 64 nm, approximately 1 nm to 25 nm, approximately 1 nm to 50 the extent of restructuring. The bone cage can be implanted in nm, approximately 1 nm to 100 nm, approximately 1 nm to non-bone soft tissues including, but not limited to, liver, 150 nm, approximately 15 nm to 50 nm, approximately 50 nm muscle, lung, or fat. to 100 nm, approximately 25 nm to 100 nm, approximately 50 Immunogenicity of the bone cage influences bone restruc nm to 150 nm, or approximately 25 nm to 150 nm. In an turing through modifications in bone resorption and/or depo aspect, the pore size can be larger, for example, approxi sition by osteoblasts and osteoclasts, as well as through mately 150 nm to 500 nm, approximately 250 nm to 750 nm, immune mechanisms. Methods of influencing the immuno or approximately 500 nm to 1,500 nm, in one or more loca genicity of cells can be used. Examples include, but are not tions. This can allow partial restructuring in these one or more limited to, the immuno-compatibility of donor and recipient, locations. 10 the inherent immunogenicity of the bone material or cells, the In a further aspect, the pore size of the one or more bone presence of immune modulatory compounds, and genetic cages can be approximately 150 nm to 500 nm, approxi modifications. mately 250 nm to 750 nm, or approximately 500 nm to 1,500 The device including one or more bone cages can be par nm. In an aspect, the pore size can be Smaller, for example, tially or completely non-immunogenic with respect to a Sub approximately 1 nm to 20 nm, approximately 1 nm to 25 nm, 15 ject within whom the device is implanted, or alternatively, can approximately 1 nm to 50 nm, approximately 1 nm to 100 nm, be partially or completely recognized as self. In an aspect, the approximately 1 nm to 150 nm, approximately 15 nm to 50 one or more bone cages can be partially or completely immu nm, approximately 50 nm to 100 nm, approximately 25 nm to nogenic with respect to a subject within whom the device is 100 nm, approximately 50 nm to 150 nm, or approximately 25 implanted, or alternatively, can be partially or completely nm to 150 nm. This can prevent or reduce restructuring in recognized as non-self. Non-immunogenic means that the these one or more locations. immune response, if any, is not such that immune Suppressive The porosity can be approximately 1% to 15%, approxi drugs would be required following implantation of the bone mately 3% to 12%, approximately 5% to 10%, approximately Cage. 1% to 3%, approximately 1% to 5%, or approximately 1% to Bone cage restructuring and immunogenicity can be modi 10% in one or more locations. In an aspect, the porosity can be 25 fied by the immuno-compatibility of donor and recipient. In a a greater percentage in one or more locations, for example further aspect, bone cages completely or partially made from approximately 40% to 95%, approximately 50% to 90%, bone derived from a donor autologous to the recipient of the approximately 60% to 75%, approximately 15% to 90%, and bone cage, are non-immunogenic and recognized as self. approximately 25% to 90%. This can allow partial restructur Previously frozen allogeneic bone, as well as Xenogeneic or ing in these one or more locations. 30 allogeneic anorganic bone, is considered non-immunogenic. The type of bone used in the fabrication of the one or more The device including one or more bone cages can be com bone cages influences bone restructuring through modifica pletely or partially made from bone derived from a donor tions in bone resorption and/or deposition. Measurements of allogeneic to the recipient of the bone cage. In an aspect, in the influence on bone restructuring of each type of bone can which the bone is from cadavers, and frozen, de-mineralized, be performed in vitro, as well as in pre-clinical and clinical 35 and/or deorganified, immuno-Suppressive therapy is not gen studies. Different bone types and/or sources have a differen erally required although some recipients may develop anti tial ability to support restructuring. As a result, bone restruc HLA antibodies (The Merck Manual of Diagnosis and turing can be partially or completely reduced, or alternatively, Therapy. Sec. 12, Ch.149). In an aspect, in which the alloge partially or completely enhanced depending on the bone cho neic bone is not frozen, deorganified or demineralized, for sen. In addition, different bone types/sources can be used in 40 example, an immune response may result unless modified by discrete locations in the bone cage to enhance or prevent/ other means, such as immuno-Suppressive therapy. decrease bone restructuring. The device including one or more bone cages can be com Studies assessing the ability of new bone or bone cells to pletely or partially made from bone derived from a donor restructure a variety of artificial and/or anorganic bone in Xenogeneic to the recipient of the bone cage. In an aspect, in bone transplant patients or in vitro culture have shown, for 45 which the bone is anorganic bovine bone, for example, example, that implantation of Bio-OSSR anorganic bovine immuno-Suppressive therapy is not required, although some bone (Geistlich, Wolhusen, Switzerland) leads to limited, recipients may experience a transient macrophage infiltrate, reduced or absent restructuring compared with other artificial but no systemic or local immune response. J. Periodontol. or natural organic bone options such as Algipore(R) porous 65:1008-15, 1994, which is incorporated herein by reference. fluorohydroxyapatitic biomaterial. Clin. Oral Implants Res. 50 In an aspect, in which the bone cage is made from Xenogeneic 15:96-100, 2004, J. Mater. Sci. Mater. Med. 16:57-66, 2005, bone that is not anorganic or pre-frozen, the bone cage can be which are incorporated herein by reference. Since these stud immunogenic and not recognized as self. ies have also identified artificial bone that encourages restruc The device including one or more bone cages can be par turing, as does natural bone, the bone cage can be designed tially made from non-immunogenic bone including, but not with portions that are resistant to restructuring as well as 55 limited to, autologous bone and/or pre-frozen, de-organified, portions that encourage restructuring as desired. and/or demineralized allogeneic bone, and/or anorganic Bone restructuring can be modified by making the bone Xenogeneic bone, and partially made from immunogeneic cage from cortical bone, ortrabecular or cancellous bone. The bone including, not limited to, allogeneic bone that is not choice of bone will impact the extent of restructuring since pre-frozen, de-organified, and/or de-mineralized and/or cortical bone is generally less porous than trabecular or can 60 Xenogeneic bone that is not anorganic. The immunogenic cellous bone. In addition, discrete parts of the bone cage can bone can be placed in discrete locations to encourage restruc be formed from one type of bone or another to influence the turing. In an aspect, the non-immunogenic bone can be placed restructuring of discrete locations. in discrete locations to prevent or reduce restructuring. Bone restructuring can be modified by the location of Bone cage restructuring and immunogenicity can be modi implantation. Bone restructuring can be greater when the 65 fied by the inherent immunogenicity of the bone material or bone is implanted in bone rather than other locations. The cells. In an aspect, the one or more bone cages can be com type of bone the bone cage is implanted in will also influence pletely or partially made from stem cells including, but not US 8,682,619 B2 65 66 limited to, mesenchymal, fetal, cord blood, and/or hemato IL-21, and/or one or more selectins. Other appropriate com poietic stem cells. Bone cages can be completely or partially pounds can be used by health professionals and can be found, made from differentiated stem cells such as bone cells, for example, in the Physicians Desk Reference. including, but not limited to, osteoblasts and/or osteoclasts, Immunostimulatory and/or inflammatory molecules can fibroblasts, or endothelial cells. The cells can be autologous, be applied to discrete locations on the bone cage. This can allogeneic, or Xenogeneic as relates to a Subject in whom they result in partial or complete restructuring of the discrete area. are implanted. Immunosuppressive compounds can be applied to discrete The one or more bone cages can be composed of autolo locations on the bone cage. This can prevent or reduce gous, allogeneic, Xenogeneic and/or artificial bone in which restructuring of the bone cage in at least those locations. autologous, allogeneic, and/or Xenogeneic stem cells have 10 been cultured. The stem cells can be induced to differentiate The bone cage can comprise cells that have been geneti into, for example, bone cells including, but not limited to, cally modified. In an aspect, the genetically modified cells osteoblasts and/or osteoclasts. Stem cells can be cultured in include, but are not limited to, stem cells, bone cells, cells discrete areas of the bone cage. The autologous, allogeneic comprising the semi-permeable component, and/or one or more cells or tissues. and/or Xenogeneic mesenchymal stem cells can partially or 15 completely decrease the immunogenicity of part, or all, of the Genetic modification of cells can influence bone restruc bone cage. turing and/or immunogenicity. Genetic modification of cells Stem cells generally have decreased immunogenicity and influences bone resorption and/or deposition. In an aspect, can induce transplant tolerance. For example, hematopoietic genetic modification of cells stimulates or inhibits immune stem cells can induce tolerance as can embryonic stem cells. reactions. Genetic modification of cells influences the perme Expert Opin. Biol. Ther: 3:5-13, 2003, which is incorporated ability and/or the immuno-isolatory aspects of the semi-per herein by reference. In addition, transplanted allogeneic mes meable component. Genetic modification of cells results in enchymal stem cells demonstrate a lack of immune recogni the release, Secretion, diffusion and/or deposition of one or tion and clearance. Blood 105:1815-1822, 2005, Bone Mar more immunogens and one or more adjuvants. Genetic modi row Transplant (22)30:215-222, Proc. Natl. Acad. Sci. USA 25 fication of cells influences the binding of one or more immu 99:8932-8937, 2002 as well as being useful in graft-versus nogens and one or more adjuvants to the bone cage including, host disease (Lancet 363:1439-1441, 2004), which are incor but not limited to, the bone wall and/or the semi-permeable porated herein by reference. Mesenchymal stem cells do not component. activate alloreactive T cells even when differentiated into The bone cage comprises genetically modified stem cells various mesenchymal lineages (Exp. Hematol. 28:875-884. 30 including, but not limited to, embryonic, fetal, mesenchymal, 2000, Exp. Hematol. 31:890-896, 2003, and suppress prolif eration of allogeneic T cells in an MHC-independent manner. and/or hematopoietic stem cells. The stem cells can be non Transplantation 75:389-397, 2003, Blood 105:1815-1822, differentiated. The stem cells can be stimulated to differenti 2005, which are incorporated herein by reference. ate. The stem cells can be non-differentiated mesenchymal The one or more bone cages can be composed of autolo 35 stem cells. The mesenchymal stem cells can be differentiated gous, allogeneic, Xenogeneic and/or artificial bone in which into cells including, but not limited to, osteoblast, osteoclast autologous, allogeneic, and/or Xenogeneic bone cells have or endothelial cells. been cultured. The bone cells can include, but are not limited Cells can be genetically modified to increase or decrease to osteoblasts and osteoclasts. In an aspect, the bone cells can bone restructuring. Stem cells, such as mesenchymal stem be cultured in discrete areas of the bone cage. Bone cages 40 cells, can be genetically modified to be more or less osteo created from autologous, allogeneic, Xenogeneic and/or arti conductive when differentiated into osteoblasts or other com ficial bone, in which allogeneic or Xenogeneic (to a Subject in ponents of bone. Methods for genetic modification of mes which it is to be implanted) bone cells can be propagated, enchymal stem can be used. Ann. Biomed. Eng. 32:136-47, increases the immunogenicity of the bone cage when 2004, Cloning Stem Cells &: 154-166, 2005, which are incor implanted in the Subject. 45 porated herein by reference. Bone cage restructuring and/or immunogenicity can be Methods for modifying the osteoconduction of cells are modified by the presence of immuno-modulatory com described. For example, bone morphogenetic protein-2 pounds. These include immuno-Suppressive as well as (BMP-2) an osteoinductive agent, up-regulates the expres immuno-stimulatory compounds. Immuno-Suppressive com sion of osteogenic phenotypes, and induces bone nodule for pounds decrease immunogenicity and hence decrease 50 restructuring, while immuno-stimulatory compounds mation in a dose-dependent manner. Spine 29:960-5, 2004, increase immunogenicity and hence increase restructuring. which is incorporated herein by reference. Ciz, an inhibitor of The immuno-modulatory compounds can be administered osteoblast differentiation, interferes with bone morphogenic systemically to a subject before, during and/or after implan protein signaling, that leads to increased bone mass. In a tation of the bone cage using methods known in the art. The 55 further aspect, a BMP and/or Ciz gene can be transduced into compounds can be adsorbed onto the surface of the bone cells and/or its expression up-regulated. Alternatively, a BMP cage, placed inside it as the one or more immunogens and one and/or Ciz gene can be deleted from the cells by genetic or more adjuvants, or secreted from the one or more cells or knockout, RNAi, orantisense nucleic acid, and/or its expres tissues. In an aspect in which the one or more immunomodu sion down-regulated by genetic inactivation methods. latory compounds can be adsorbed onto the bone cage, they 60 Cells can be genetically modified to increase or decrease can be adsorbed to one or more discrete locations on the bone immunogenicity and/or an immune response. In an aspect, Cage, the cells including, but not limited to, stem cells, bone cells, The immunosuppressive compounds include, but are not cells of the semi-permeable component, and/or the one or limited to, corticosteroids, such as prednisolone or methyl more cells or tissues, can be genetically modified to express prednisolone. In an aspect, the immune stimulatory and/or 65 immune recognition markers of the host, to secrete and/or inflammatory molecules include, but not limited to, tumor express anti-inflammatory molecules, and/or to express or necrosis factor C., interferon Y, interleukin 2 (IL-2), IL-12, secrete immune-stimulatory molecules. US 8,682,619 B2 67 68 Device Including One or More Bone Cages Including a Semi permeable component can be designed to at least partially or permeable Component completely enhance restructuring. The bone cage partially or completely surrounds and/or is The semi-permeable component can be a semi-permeable Surrounded by a semi-permeable component. The bone cage membrane. The semi-permeable membrane can include, but partially or completely encloses and/or is enclosed by a semi is not limited to, artificial membranes, biological membranes, permeable component. The semi-permeable component can and/or a combination of artificial and biologically-derived be partially or completely comprised of the bone wall of the components. The manufacture and use of artificial semi-per bone cage. The semi-permeable component can be partially meable membranes can be used. Cell Transplant 10:3-24, or completely external to the bone wall of the bone cage. The 2001, which is incorporated herein by reference. Artificial semi-permeable component can be partially or completely 10 internal to the bone wall or the bone cage. The semi-perme semi-permeable membranes include, but are not limited to, able component partially or completely encloses one or more hydrogel membranes. Biochim. Biophys. Acta 804:133-136, immunogens and one or more adjuvants, optionally in com 1984, Journal of Biomedical Materials Research 26:967 bination with one or more cells or tissues that produce the one 977, 1992. J. Biomed. Mater. Res. 26:967–977, 1992 and or more immunogen and/or the one or more adjuvant. The 15 ultrafiltration membranes Diabetes 45:342-347, 1996, J. semi-permeable component can include, but is not limited to, Clin. Invest. 98:1417-1422, 1996, Transplantation 59:1485 artificial membrane, cells with tight junctions, plasma mem 1487, 1995, J. Biomech. Eng. 113: 152-170, 1991, both of brane, micelles, liposomes, Virosomes, intracellular mem which can be employed in the immuno-isolation of branes, red blood cells, red blood cell ghosts, or aggregated xenografts, for example Ann. NYAcad. Sci. 875:7–23, 1999. platelets. In an aspect, the device including one or more bone The membranes can be made, for example, from polymer cages can include red blood cells loaded with one or more films and thermoplastic hollow fibers. In addition, biological immunogens and/or one or more adjuvants. Red blood cells semi-permeable membranes can be used to encapsulate islet loaded with one or more immunogens and/or one or more cells followed by implantation. World J. Gastroenterol. adjuvants can be useful in treatment of HIV infection by 11:5714-5717, 2005, which is incorporated herein by refer targeting immunogen and adjuvant to viral reservoirs carry 25 CCC. ing replication-competent HIV, including monocytes/mac The semi-permeable component can be partially or com rophages. Cervasi et al., J. Virol. 80: 10335-10345, 2006; M. pletely composed of cells with tight junctions. Tight junction Magnani, Erythrocyte Engineering for Drug Delivery and or Zonula occludens refers to the intercellular junction that Targeting, Landes Bioscience, Austin Tex., 2002; "Erythro regulates diffusion between cells and allows the formation of cyte-based drug delivery'. Expert Opinion on Drug Delivery, 30 barriers that can separate compartments of different compo 2: 311-322, 2005, which are incorporated herein by reference. sition. The intercellular gate formed by tightjunctions is size Semi-permeable component refers to a selective impedi and ion selective, among other things. ment to the passage offluids and/or Substances in the fluids. In The cells with tight junctions can include, but are not an aspect, the semi-permeable component can prevent the limited to, epithelial and/or endothelial cells, or a combina passage of macromolecules and cells, but allows the passage 35 tion. Both epithelial cells and endothelial cells can form tight of oxygen and/or nutrients. The passage of one or more junctions between cells. Methods 30:228-234, 2003, which is immunogens and one or more adjuvants from the cage and/or incorporated herein by reference. products can be released by the one or more cells or tissues in The semi-permeable component can be comprised of cells the cage can be allowed. In an aspect, the passage of macro with tight junctions where the cells are stem cells, or are molecules, or macromolecules and cells can be allowed. 40 differentiated from stem cells. Stem cells can be cultured in The semi-permeable component can include, but is not vitro to confluency on the interior and/or exterior of a bone limited to, the bone wall, one or more semi-permeable mem scaffold of the desired shape and composition. The stem cells branes, cells with tight junctions, one or more plasma mem can include, but are not limited to, one or more of mesenchy branes, one or more intracellular membranes, one or more red mal, embryonic, fetal, or hematopoietic stem cells. The stem blood cell ghosts, and one or more aggregated platelets or 45 cells can be stimulated to differentiate. The stem cells can other cells. The semi-permeable component can be com differentiate into one or more of endothelial cells and epithe prised of cells that are autologous, allogeneic, or Xenogeneic lial cells. The stem cells can differentiate into bone cells, with respect to a subject within whom the semi-permeable including, but not limited to, osteoblasts or osteoclasts. The component may be implanted. stem cells do not differentiate into bone cells. In an aspect, part or all of the semi-permeable component 50 Methods for differentiating mesenchymal stem cells into can be partially or completely non-immunogenic and/or can endothelial cells Basic & Clin. Pharmacol. & Toxicol. be recognized as self by a subject within whom it is 95:209-214, 2004 and hematopoietic stem cells into epithe implanted. In an aspect, part or all of the semi-permeable lial stem cells can be used. Stem cells can be relatively non component can be partially or completely immunogenic and/ immunostimulatory, and to retain this characteristic follow or can be recognized as non-self by a subject within whom it 55 ing differentiation. is implanted. The semi-permeable component can be a plasma mem The semi-permeable component can be comprised of cells brane. The plasma membrane can be made from red cell that are cultured in vitro. The semi-permeable component can ghosts. Red cell ghosts can be created by removal of the be comprised of cells that are genetically engineered. In an erythrocyte cytoplasm by lysis followed by size-exclusion aspect, Some or all of the cells can be genetically engineered 60 chromatography. In an aspect, one or more red cell ghosts to release, secrete, deliver, diffuse, and/or provide one or encapsulate the one or more immunogens and one or more more immunogens and one or more adjuvants. In an aspect, adjuvants optionally in combination with the one or more Some orall of the cells can be genetically engineered to be less living cells and/or tissues. Methods of using red cell ghosts immunogenic or to be more immunogenic. In yet other for drug delivery have been described. Expert Opinion on aspects, some or all of the cells can be genetically engineered 65 Drug Delivery 2:311-322, 2005, Drug Delivery, 2003 to increase or decrease bone restructuring including, but not Taylor & Francis eds. 10(4):277-282, BioDrugs 18:189-198, limited to, bone deposition and bone resorption. The semi 2004, which are incorporated herein by reference. US 8,682,619 B2 69 70 The one or more red cells ghosts can be fused to form an cells and/or tissues can include, but are not limited to, bacte internal or external continuous or semi-continuous mem ria, yeast, islet cells, liver cells, thyroid cells, bone cells, brane. The fused red blood cell ghosts encapsulate the one or and/or neural cells. more immunogens and one or more adjuvants optionally in Other aspects include methods for delivering one or more combination with the one or more living cells and/or tissues. immunogens and one or more adjuvants to a subject. The one The semi-permeable component can include an aggregate or more immunogens and one or more adjuvants to be deliv of platelets. The bone cage is coated internally and/or exter ered to the subject are identified and/or selected, for example nally with a platelet aggregating compound on which plate by health care workers including, but not limited to, physi lets aggregate in vitro and/or in vivo. The platelet aggregating cians responsible for the health of the subject. One or more of 10 the bone cages described above can be selected for delivery of compound includes, but is not limited to, fibrin, fibrinogen the one or more immunogens and one or more adjuvants. The and/or thrombin. For example, fibrinogen is known to play a one or more immunogens and one or more adjuvants can be role in platelet aggregation. Coll. Anthropol. 29:341-9, 2005, provided with or added to the bone cages, and/or released which is incorporated herein by reference. from one or more cells or tissues provided with or added to the The device including one or more bone cages can comprise 15 bone cages, and/or released from the cells comprising the one or more immunogens and one or more adjuvants. The one semi-permeable component provided with or added to the or more immunogens and one or more adjuvants can be bone cages. The device including one or more bone cages Surrounded by the semi-permeable component. In an aspect, including the one or more immunogens and one or more the one or more immunogens and one or more adjuvants can adjuvants optionally in combination with cells or tissues and/ be adsorbed to the bone cage. The bone cage binds one or or semi-permeable component can be implanted in the Sub more immunogens and one or more adjuvants. The bone cage ject to allow delivery of the one or more immunogens and one can bind covalently or ionically to one or more immunogens or more adjuvants. and one or more adjuvants. The bone cage can bind these Bone Cage Device Including Immunogen and Adjuvant as a molecules following their release from the bone cage and/or Vaccine living cells and/or tissues. The one or more immunogens and 25 The device including one or more bone cages includes one one or more adjuvants comprise part of the bone wall. The one or more immunogens provided in combination with one or or more immunogens and one or more adjuvants can be bound more adjuvants, for active immunization of a Subject against to the semi-permeable component and/or one or more cells or a pathological condition, e.g., infectious disease or neoplastic tissues. The one or more immunogens and one or more adju disease. An immunogen is a Substance that, as a result of 30 coming in contact with appropriate tissues of an animal body, vants can be released from, provided by, secreted from, and/or can induce an immune response, for example formation of diffuse from cells of the bone wall, the semi-permeable com antibodies and/or cell-mediated immunity. Information and ponent, and/or one or more cells or tissues. examples of immune responses can be found in Delves et al. Biologically active molecules, e.g., one or more immuno Roitt's Essential Immunology, 11th edition, Wiley-Black gens and one or more adjuvants, include any molecule that 35 well, 2006, which is incorporated herein by reference. An has a measurable biological action in a subject. For example, immunogen includes any type of biological or synthetic com biologically active molecules would include, but not be lim pound including, but not limited to, a compound from a ited to, any molecules described in this disclosure including, pathogenic organism, a Substance endogenous to the Subject but not limited to, molecules that enhance or reduce bone causing a pathological condition in the Subject, or a synthetic restructuring including bone resorption and deposition, and/ 40 Substance. or that enhance or reduce an immune response. These bio Immunogens from pathogens. The device including one or logically active molecules would include, but not be limited more bone cages can be used to administer one or more to, pharmaceutically acceptable compounds including immunogens and one or more adjuvants that constitute a parenteral drugs, nutrients, and vitamins including, but not Vaccine against a pathogen, e.g., viruses, bacteria, fungi, and limited to, those described in this disclosure for the treatment 45 parasites. Examples of pathogens include, but are not limited of particular diseases or disorders. to, viruses, e.g., herpes simplex virus (HSV), hepatitis A The device including one or more bone cages includes one virus, hepatitis B virus (HBV), hepatitis C virus (HCV), or more immunogens and one or more adjuvants optionally cytomegalovirus (CMV), dengue virus, flavivirus, Epstein wherein one or more cells or tissues produce the one or more Barr virus (EBV), influenza virus, measles virus, human immunogens and/or the one or more adjuvants. A semi-per 50 immunodeficiency virus (HIV), human papilloma virus meable component can Surround the one or more immuno (HPV), Japanese encephalitis virus, norovirus, polio virus, gens and one or more adjuvants optionally in combination rotavirus, respiratory syncytial virus (RSV), ebola virus, with one or more cells or tissues. The cells can be autologous, rabies virus, Sendai virus, severe acute respiratory syndrome allogeneic, or Xenogeneic with respect to a subject within (SARS) coronavirus, smallpox virus, West Nile virus, yellow whom they may be implanted. The cells can be cultured in 55 fever virus; bacteria, e.g., Mycobacterium tuberculosis (tu vitro. In an aspect, the cells can be non-immunogenic and/or berculosis), Chlamydia trachomatis (trachoma), Haemophi are configured to be recognized as self by a subject within lus influenzae (otitis media), Neisseria meningitidis (menin whom they is implanted. In an aspect, the one or more cells or gitis), Streptococcus pneumoniae (pneumonia), Escherichia tissues can be genetically engineered. The one or more cells coli (intestimal disorders) Staphylococcus aureus, Bacillus or tissues can be genetically engineered to release, provide, 60 anthracic (anthrax), Borrelia burgdorferi (Lyme’s disease); diffuse and/or extrude the one or more immunogens and/or and parasites, e.g., Plasmodium (malaria), Leishmania, Try one or more adjuvants. panosoma Cruzi, Trypanosoma brucei, Ascaris lumbricoides The one or more living cells and/or tissues can include, but (ascariasis), hookworm, Onchocerca volvulus (river blind are not limited to, cells and/or tissues that produce, express ness), Schistosoma (Schistosomasis), Trichuris trichiura (tri and/or secrete immune/inflammation-related, biochemical 65 churasis). function-related, metabolism-related, and/or hormone-re Immunogens used with the device including one or more lated biologically active molecules. The one or more living bone cages as a vaccine against a pathogen can be derived US 8,682,619 B2 71 72 from any of a number of sources including, but not limited to, chanical methods, or a combination thereof. Examples of living microorganisms that are naturally avirulent or that have mechanical methods for generating a cell lysate include, but been modified to attenuate their virulence while retaining are not limited to, Sonication, homogenization by dounce or adequate immunogenic properties; heat and/or chemically glass beads, and osmotic lysis. Examples of non-mechanical inactivated/killed virulent microorganisms; immunogens methods include, but are not limited to, detergents, alkali, and extracted from or secreted by an infectious agent; immuno enzymatic degradation (e.g., lysozyme, lysostaphin, Zymol gens produced by recombinant DNA technology; a live, yase, lyticase). The immunogen can be a whole cell lysate. recombinant vector producing immunogens in vivo in the Alternatively, the immunogen can be a subfraction of the vaccinated host; plasmid DNA; immunogens produced by whole cell lysate, e.g., the cell wall subfraction. A whole cell chemical synthesis in vitro. Examples of commonly admin 10 lysate can be subfractionated by differential centrifugation to istered vaccines containing live, attenuated microorganisms generate a cell wall/membrane fraction. Alternatively, a include those for yellow fever, measles, oral polio, varicella, whole cell lysate can be subfractionated using beads or other rubella, mumps, and rotavirus. Examples of commonly particles that include one or more antibody directed to a administered vaccines containing inactivated/killed microor biomolecule associated with one or more cell subfraction. ganisms include those for influenza, cholera, bubonic plague, 15 Immunogens can include all or parts of viruses, bacteria, polio, rabies and hepatitis A. Examples of commonly admin parasites, and other microorganisms, e.g., coats, capsules, istered vaccines derived from toxins include those for tetanus cells walls, flagella, fimbrae, and toxins. and diphtheria. Examples of vaccines using one or more The immunogen can be one or more specific biomolecules isolated immunogens from a microorganism include the associated with the pathogen, particularly biomolecules hepatitis B vaccine that is composed of only the Surface expressed on the Surface of the pathogen. Pathogen associ proteins of the virus and the virus-like particle (VLP) vaccine ated biomolecules can be purified from a natural source. against human papillomavirus (HPV). Alternatively, pathogen associated biomolecules, for The immunogen can be a live, attenuated pathogen, e.g., a example, proteins, can be generated by recombinant DNA live, attenuated virus, bacteria, or parasite. In some instances, technology using standard methodologies. Recombinant the live attenuated pathogen can be naturally-occurring. In an 25 DNA technology can also be used to produce enzymes aspect, the live attenuated pathogen is generated by environ required for modification to endogenous proteins, lipids, mental and/or genetic manipulation. Examples of environ polysaccharides or lipopolysaccharides. For example, cDNA mental manipulation include, but are not limited to, chemical encoding all or part of an immunogen or one or more enzyme or radiation induced attenuation of the virulent strain or required for modification or production thereof, can be iso extensive passage of the virulent strain at Suboptimal tem 30 lated using polymerase chain reaction (PCR) amplification in peratures. For example, cold adapted Strains of influenza A combination with primers based on DNA sequence available and influenza B have been developed by multiple passages at in the Genbank Database. Benson, et al., Nucleic Acids Res. progressively lower temperatures in primary chicken kidney 36:D25-30, 2008, which is incorporated herein by reference. cells, resulting in attenuated influenza strains that only repli Production or modification of an immunogen can include, cate at 25°C. or well below normal mammalian body tem 35 e.g., cDNA encoding glycosylation enzymes for glycosyla perature. Murphy & Coelingh, Viral Immunol. 15:295-323, tion of a protein, glycoprotein, or lipopolysaccharide. cDNA 2002, which is incorporated herein by reference. As another encoding glycosylation enzymes can be configured to correct example, non-replicating, but metabolically active Plasmo abberant glycosylation in protein, glycoprotein, or dium falciparum sporozoites for malaria vaccination have lipopolysaccharide associated with tumor cells. The modifi been generated by irradiation. Luke & Hoffman, J. Exp. Biol. 40 cation may only be needed for Surface expression or may be 206:3803-3808, 2003, which is incorporated herein by refer part of the immunogen itself. In some instances, the recom ence. Alternatively, live, attenuated pathogen can be gener binant expression of one or more pathogen protein can result ated by reverse genetics, whereby the infectious pathogen is in formation of viral-like particles in which expressed capsid produced entirely from recombinant cDNA and predeter proteins, for example, spontaneously assemble into particles mined changes in the nucleotide sequence are introduced by 45 that are structurally similar to authentic virus. Roy & Noad site-directed mutagenesis to generate a new, attenuated vac Human Vaccines 4:5-8, 2008, which is incorporated herein by cine strain. See, e.g., Murphy & Collins, J. Clin. Invest. 110: reference. 21-27, 2002; Neumann, et al., Proc. Natl. Acad. Sci. USA. The immunogen can be produced by cells encapsulated in 96:9345-9350, 1999, which are incorporated herein by refer the bone cage. The cells encapsulated in the bone cage may CCC. 50 naturally express the immunogen, or the cells may be geneti The immunogen can be an inactivated pathogen, e.g., an cally engineered to express the immunogen. The genetically inactivated virus, bacteria, or parasite, in which the ability of engineered cells can be bacteria, yeast, parasites, insect cells, the pathogen to replicate has been eliminated, but the ability or mammalian cells. The mammaliancells can be autologous, to mount an immune response remains intact. An inactivated allogeneic or Xenogeneic relative to the Subject. In an aspect, virus, bacteria or parasite can be generated using heat, UV 55 the immunogen can be a live, attenuated pathogen, e.g., virus irradiation, formalin or other chemicals such as B-propiolac or bacteria that is propagated in the encapsulated cells. Alter tone or a photoinducible agent. For example, the Ebola virus natively, the immunogen can be one or more specific proteins can be inactivated with the photoinducible alkylating agent generated in the cells using standard recombinant DNA tech 1.5-iodonaphthylazide without compromising immunoge niques. For example, the cDNA sequence corresponding to nicity and structural integrity. See, e.g., Warfield, et al., J. 60 the immunogen can be generated using standard polymerase Infect. Dis. 196 Suppl2:S276-S283, 2007, which is incorpo chain reaction (PCR) amplification and an appropriate cDNA rated herein by reference. library or reverse-transcribed mRNA with primers designed In an aspect, the immunogen can be all or part of a cell based on the known cDNA sequence of the immunogen from, lysate generated, for example, from a microbial antigen, viral e.g., the GenBank Database. Benson, et al., Nucleic Acids antigen, parasite antigen, fungal antigen, plant antigen, ani 65 Res. 36:D25-30, 2008, which is incorporated herein by ref mal antigen, endogenous antigen, or synthetic antigen. A cell erence. In an aspect, site-directed mutations can be made to lysate can be generated by mechanical methods, non-me nucleotide sequence of codons within the immunogen coding US 8,682,619 B2 73 74 sequence to align the sequence with mammalian codon usage mercially available peptide synthesizer (e.g., ABI 433A Pep to enable more efficient expression in mammalian cells. See, tide Synthesizer from Applied Biosystems, Inc., Foster City, e.g., Garmory, et al., Genetic Vaccines Ther: 1:2, 2003, which Calif.). is incorporated herein by reference. The cDNA encoding the The device including one or more bone cages including an immunogen is cloned into an expression vector, transfected immunogen and an adjuvant can further include one or more into a cell line, and cloned cells expressing the immunogen circular plasmid DNAS that include genes encoding all or part biomolecule, which may or may not be modified by glycosy of one or more target immunogens, or one or more enzyme lation and/or lipidation, are identified using standard meth required for modification or production thereof, e.g., protein ods. The cells can transiently or stably express the immuno glycosylation or production of lipopolysaccharide, under the gen. The relative expression of the immunogen by the cells 10 transcriptional control of a promoter region active in host cells. Promoters include, but are not limited to, SV40 pro can be assessed using any of a number of assay systems. The moter, rous sarcoma virus (RSV) promoter, adenovirus pro expression of messenger RNA (mRNA) corresponding to the moter, cytomegalovirus (CMV) immediate early promoter. In immunogen can be assessed by quantitative PCR, Northern an aspect, site-directed mutations can be made to codons analysis, in situ hybridization, or other methods designed to 15 within the sequence encoding the immunogen or enzyme to assess the presence and/or quantity of a specific mRNA in a align the sequence with mammalian codon usage to enable cell. The expression of actual immunogen in the cells can be more efficient in vivo expression from the plasmid DNA. See, assessed by Western analysis, immunocytochemistry, or e.g., Garmory, et al., Genetic Vaccines Ther: 1:2, 2003, which other methods designed to assess the presence and/or quantity is incorporated herein by reference. The bone cage including of a specific protein in a cell. Secretion of the immunogen out the plasmid DNA encoding an immunogen and/or an adju of the genetically engineered cell can be assayed, for vant can be administered to a Subject, taken up by the cells in example, by analysis of in vitro culture medium using, e.g., an proximity to the site of administration, and the host cells immunoassay system such as an enzyme-linked immunosor expressing the immunogen and/or the adjuvant in the Subject bent assay (ELISA) with immunoreagents specific for the produce the pathogen-related immunogen. The plasmid DNA immunogen. Secretion of the immunogen out of the geneti 25 can be incorporated as a hydrogel within the bone cage. One cally engineered cell can further be assayed, for example, by or more DNA vaccines can be used within the bone cage. For analysis in vivo for presence of the immunogen in serum or example, the HIV DNA vaccine pGA2/JS2 (from GeoVax, tissue of the Subject. In an aspect, the genetically engineered Atlanta, Ga.) expresses several HIV proteins including those cells expressing the immunogen are encapsulated in alginate encoded by the genes gag, pro, RT, env, tat, Vpu, and rev. DNA or other physiologically compatible encapsulation medium 30 based vaccines, e.g., for HIV. avian influenza H5N1, SARS, hepatitis C virus, West Nile virus, tuberculosis, and malaria, prior to incorporation into the bone cage. See, e.g., Read, et have been or are currently undergoing clinical trials and a al., Nature Biotechnol. 19:29-34, 2001: Orive, et al., Nature DNA vaccine for West Nile virus has been approved for Medicine. 9:104-107, 2003: U.S. Patent Application Nos. Veterinary use. 2004/0005302; 2007/0258901; 2008/0107686 which are 35 Tumor Immunogens. incorporated herein by reference. The device including one or more bone cages can be used The immunogen can include one or more polypeptides, to administer one or more immunogens and one or more which may or may not be modified by glycosylation and/or adjuvants that constitute a vaccine to treat neoplastic disease, lipidation. In some instances, the one or more polypeptides e.g., tumor cells or cancer cells. Cancer vaccines are designed can represent one or more immunogenic portions of a protein 40 to stimulate a Subject’s immune system to recognize and associated with a pathogen. The immunogenic polypeptides eliminate cancer cells. Cancer vaccine strategies can include, can be predicted using computational modeling. See, e.g., for example, whole cell vaccines, antigen therapy vaccines, Florea, et al., Proceedings 2003 IEEE Bioinformatics Con antigen-presenting cell vaccines, and non-specific therapy ference, Aug. 11-14, 2003, p. 17-26: Toussaint, et al., PLOS and cytokine therapy. Cancer vaccines can be used against Comput. Biol. 4:e1000246, 2008, which are incorporated 45 neoplastic diseases that include, but are not limited to, pan herein by reference. Alternatively, the one or more peptides creatic cancer, prostate cancer, cervical cancer, breast cancer, can be mimotopes that act as Surrogate immunogens for bio ovarian cancer, bladder cancer, kidney cancer, multiple molecules that are otherwise not very immunogenic Such as, myeloma, non-Small cell lung cancer, colorectal cancer, leu for example, the carbohydrates found on the surface of some kemia, melanoma, glioma, gastric cancer, esophageal cancer, pathogens. See, e.g., Monzavi-Karbassi, et al., Trends Bio 50 head and neck cancer, hepatic cancer, renal cell carcinoma, technol. 20:207-214, 2002, which is incorporated herein by testicular cancer, or uterine cancer. reference. A mimotope is a macromolecule that mimics the The immunogen included in a device including one or structure of an epitope or that portion of the immunogen more bone cages for use as a cancer Vaccine can be all or part recognized by an antibody or other receptor, and is able to of a tumor cell. The immunogen can be a multivalent cell induce an antibody response identical to that elicited by the 55 culture of different tumor cells lines established ex vivo prior authentic epitope. Mimotopes can be synthetic compounds, to use in the bone cage. The cell culture can include non e.g., shaped gels. Alternatively, mimotopes can be synthetic autologous immunogens and the use of multiple cell lines to peptides screened from combinatorial Solid-phase peptide help ensure that at least Some of the antigens in the vaccine are phage libraries using an antibody or other receptor that nor shared by the subjects own tumor. Alternatively or in addi mally binds to the immunogen of interest. For example, stud 60 tion, the immunogen can include an autologous cell line ies describe screening a peptide library with a neutralizing, prepared by harvesting tumor cells from the subject to be protective antibody against respiratory syncytial virus (RSV) treated. See, e.g., Berger, et al., J. Pharm. Pharmaceut. Sci. to identify peptides that mimic the epitope of the fusion (F) 10: 144-152, 2007, which is incorporated herein by reference. protein of the virus. Steward. Biologicals 29:215-219, 2001, The autologous and/or non-autologous tumor cells can be which is incorporated herein by reference. Once identified, 65 killed prior to (re)introduction into the subject. An example of the one or more peptides can be generated using either recom a tumor killing agent is dinitrophylate. Alternatively, the binant DNA techniques or by chemical synthesis with a com autologous tumor cells can be otherwise alive but inactivated US 8,682,619 B2 75 76 by lethal irradiation. The autologous or non-autologous libraries), or combinations thereof. See, e.g., Carter, et al., tumor cells can be genetically modified by recombinant DNA Endocrine-Related Cancer 11:659-687, 2004, which is incor techniques to boost the immune response. For example, the porated herein by reference. autologous or non-autologous tumor cells can be genetically One or more immunogens in a device including one or engineered to ectopically express IL-2, IL-12, IL-21, GM more bone cages for use as a cancer vaccine can be produced CSF, or other cytokine. The device including one or more by cells encapsulated in the bone cage. The cells encapsulated bone cages including tumor cells can be administered alone in the bone cage can naturally express the immunogen. Alter or in combination with autologous antigen presenting cells, natively, the cells can be genetically engineered to express the e.g., dendritic cells. The immunogen can be one or more immunogen. The genetically engineered cells can be bacteria, 10 yeast, parasites, insect cells, or mammalian cells. The mam tumor-associated antigens. A tumor-associated antigen can malian cells can be autologous, allogeneic or Xenogeneic be, for example, a endogenous protein or other molecule, e.g., relative to the Subject. The immunogen can be one or more lipid, lipoprotein, polysaccharide or lipopolysaccharide, that specific tumor antigens generated in the cells using standard is otherwise well sequestered from the immune system, that is recombinant DNA techniques as described herein. The cDNA normally produced in extremely small quantities, that is nor 15 encoding the immunogen, or one or more enzymes required mally produced only in certain stages of development, that for modification or production thereof, is cloned into an carries inappropriate modification such as glycosylation, or expression vector, transfected into a cell line, and cells whose structure is modified due to one or more mutation, or a expressing the immunogen biomolecule are identified using combination thereof. See, e.g., Hakomori S. Proc Natl Acad standard methods. The cells can transiently or stably express Sci USA. 99:10231-10233, 2002, which is incorporated the immunogen. The relative expression of the immunogen herein by reference. Alternatively, the tumor-associated anti by the cells can be assessed using any of a number of assay gen can be a viral protein derived from a virally induced systems as described herein. In an aspect, the genetically tumor. Examples of tumor associated antigens that might be engineered cells expressing the immunogen are encapsulated used as immunogens include, but are not limited to, prostatic in alginate or other physiologically compatible encapsulation acid phosphatase (PAP), carcinoembryonic antigen (CEA), C. 25 medium prior to incorporation into the bone cage. See, e.g., fetoprotein, prostate specific antigen (PSA), CA-125, 32-mi Read, et al., Nat. Biotechnol. 19:29-34, 2001; U.S. Patent croglobulin, B-hCG, bombesin, CA19-9, CA 15-3, chromog Application Nos. 2008/0107686 and 2007/0258901 which ranin A, thyroglobulin, TA 90, MART-1/Melan-A, MART-2, are incorporated herein by reference. tyrosinase, Gp 100, MAGE-1, MAGE-3, BAGE, GAGE, One or more immunogens can be one or more peptide URLC10, Her2/neu, GM2, MUC1, MUC2G, globo H, 30 epitopes derived from a tumor antigen. See, e.g., Sangha & LAGE-1, TTK-567, neuron-specific enolase (NSE), prostate Butts, Clin Cancer Res. 13:4652s-4654s, 2007, which is specific membrane antigen (PSMA). An extensive list of incorporated herein by reference. In an aspect, the one or tumor antigens is provided in Novellino, et al., Abstract, more peptides can represent one or more immunogenic por Cancer Immunol. Immunother. 54:187-207, 2005, which is tions of a protein associated with a tumor. The immunogenic incorporated herein by reference. All or part of a tumor anti 35 peptides can be predicted using computational modeling. gen can be produced as recombinant protein using standard See, e.g., Florea, et al., Proceedings 2003 IEEE Bioinformat techniques. For example, cDNA encoding all or part of a ics Conference, Aug. 11-14, 2003, p. 17-26: Toussaint, et al., tumor antigen, or one or more enzyme required for modifi PLOS Comput. Biol. 4:e 1000246, 2008, which are incorpo cation or production thereof, can be isolated using poly rated herein by reference. The immunogen can be one or more merase chain reaction (PCR) amplification in combination 40 peptide mimotopes that mimic epitopes associated with with primers based on DNA sequence available in the Gen tumor-associated carbohydrate antigens or other tumor asso bank Database. Benson, et al., Nucleic Acids Res. 36:1525 ciated antigens. See, e.g., Bramswig, et al., Clin. Cancer Res. 30, 2008, which is incorporated herein by reference. Produc 13:6501-6508, 2007, which is incorporated herein by refer tion or modification of an immunogen can include, e.g., ence. Alternatively, the immunogen can be a synthetic peptide cDNA encoding glycosylation enzymes for glycosylation of 45 that combines a tumor associated carbohydrate antigen a protein, glycoprotein, or lipopolysaccharide. cDNA encod covalently linked to a T-cell epitope and a B-cell epitope, ing glycosylation enzymes can be configured to correct enabling the immunogen to elicit both a humoral response abberant glycosylation in protein, glycoprotein, or and a cellular immune response. U.S. Patent Application No. lipopolysaccharide associated with tumor cells. Alterna 20090041836, which is incorporated herein by reference. In tively, the cDNA encoding all or part of a tumor antigen can 50 another aspect, a melanoma vaccine can include a device be from a commercial source (from, e.g., Origene, Rockville, including one or more bone cages including a peptide derived Md.). from the tumor antigen gp 100 linked to the T-helper epitope One or more immunogens in a device including one or from tetanus toxoid. Slinghuff, et al., Clin. Cancer Res. more bone cages useful as a tumor antigen in a cancer vaccine 7:3012-3024, 2001, which is incorporated herein by refer can be identified using a number of analytical methods 55 ence. The peptides for use as immunogens in the bone cage including, but not limited to, genomics (e.g., comparative can be generated using chemical synthesis or can be incorpo genomic hybridization (CGH) array, spectral karyotyping rated into a plasmid DNA. See, e.g., Fest, et al., Cancer Res. (SKY)), transcriptomics (e.g., microarrays, representational 66:10567-10575, 2006, which is incorporated herein by ref. difference analysis (RDA), serial analysis of gene expression CCC. (SAGE), suppression subtractive hybridization (SSH), cancer 60 One or more immunogens can be one or more circular profiling array (CPA), quantitative real time PCR (QPCR), plasmid DNAS that include genes encoding all or part of one RNA in situ hybridization), proteomics (2-dimensional gel or more target immunogens, or one or more enzyme required electrophoresis, mass spectrometry, glycomics, immunohis for modification or production thereof, under the transcrip tochemistry (IHC), tissue arrays, fluorescence activated cell tional control of a promoter region active in host cells. Pro sorting (FACS), serological identification of antigens by 65 moters include, but are not limited to, SV40 promoter, rous expression cloning (SEREX)), antibody technologies (e.g., sarcoma virus (RSV) promoter, adenovirus promoter, murine or human monoclonal hybridomas, phage display cytomegalovirus (CMV) immediate early promoter. The US 8,682,619 B2 77 78 bone cage including the plasmid DNA encoding an immuno haemocyanin, or a conjugate of angiotensin II linked to virus gen and/oran adjuvant can be administered to a Subject, taken particles, as an immunogen to treat disease in the Subject. up by the cells in proximity to the site of administration, and A method for treating a pathological condition includes the host cells in the Subject produce the pathogen-related treatment for drug or substance addiction. The device includ immunogen. The plasmid DNA can be incorporated as a ing one or more bone cages can include norcocaine with hydrogel within the bone cage. One or more DNA vaccines inactivated cholera toxin as an immunogen. A large protein can be used within the bone cage. DNA vaccines for a number molecule attaches to cocaine, which stimulates response from of cancers are currently undergoing clinical evaluation and antibodies which destroy the molecule. This prevents the include e.g., vaccines for prostate cancer, breast cancer, kid cocaine from crossing the blood-brain barrier negating the ney cancer, liver cancer, cervical cancer, lymphoma, and 10 euphoric high and rewarding effect of cocaine caused from melanoma. stimulation of dopamine release in the mesolimbic reward One or more immunogens can be an antigen presenting pathway. Martell et al., Biol. Psychiatry 58: 158-164, 2006, cell, for example, a dendritic cell that has been genetically Ann NYAcadSci, 1141: 257-269, 2008, which are incorpo modified to express one or more tumor antigens. Autologous rated herein by reference. dendritic cells can be isolated from a subject by leukopher 15 A method for treating obesity can includes providing a esis, transfected with an expression vector that includes, for device including one or more bone cages wherein GIP (gas example, DNA sequence encoding all or part of one or more tric inhibitory peptide; or glucose-dependent insulinotropic tumor antigens, and Subsequently used for immunization of polypeptide) is an immunogen to treat disease in the Subject. the Subject. See, e.g., Nakamura, et al., Clin. Cancer Res. A method for treating autoimmune disease, e.g., type 1 dia 8:2742-2749, 2002, which is incorporated herein by refer betes, can include providing self-antigen as an immunogen to ence. Alternatively, the autologous dendritic cells can be treat disease in the Subject. A method for treating Substance stimulated or activated ex vivo in the presence of a tumor cell abuse can include providing an antibody to bind the drug in lysate or mixture of tumor antigens and Subsequently used for the bloodstream, thereby blocking entry and/or reducing the immunization of a Subject. See, e.g., Hirschowitz, et al., J. rate of entry of the drug into the central nervous system. Clin. Oncol. 22:2808-2815, 2004, which is incorporated 25 The one or more immunogens includes, for example, herein by reference. B-amyloid associated with Alzheimer's disease. See, e.g., Other Immunogens. Wilcock & Colton, J. Alzheimers Dis. 15:555-569, 2008, The device including one or more bone cages can be used which is incorporated herein by reference. to administer one or more immunogens and one or more In an aspect, the device including one or more bone cages adjuvants that constitute a vaccine to treat a pathological 30 can be used to administer one or more immunogens derived condition in the Subject, e.g., an allergic response, wherein from drugs of abuse such as, for example, nicotine, cocaine, the immunogen can be an allergen. Examples of allergens methamphetamine, phencyclidine, and morphine. See, e.g., include, but are not limited to, inhaled allergens (e.g., grass, Orson, et al., Ann. N.Y. Acad. Sci. 1141:257-269, 2008, which weed, and tree pollens, mold spores, chemicals, cockroach is incorporated herein by reference. calyx, dust mite excretions, animal dander, Saliva), ingested 35 Adjuvants. allergens (e.g., food, food Supplements, home remedies, The device including one or more bone cages include one medications), contact allergens (e.g., cosmetics, fragrances, or more immunogens in combination with one or more adju plants, detergents, chemicals, metals, latex), and injected vants. Adjuvants are used as immune potentiators or immu allergens (e.g., medications, insect venom). For example, a nomodulators to improve the immune response to the vaccine number of clinical trials are underway to develop a vaccine 40 immunogens. An adjuvant can be incorporated into a vaccine against peanut allergies. In this latter instance, the immuno formulation to enhance, accelerate and/or prolong the spe gen can be peanut powder or one or more isolated peanut cific immune response towards the desired response to vac proteins such as, for example, Ara hl. Ar2 hw, and Arah?. cine immunogens. Adjuvants potentially enhance the immu See, e.g., Li, et al., J. Immunol. 170:3289-3295, 2003, which nogenicity of immunogens, modify the nature of the immune is incorporated herein by reference. 45 response, reduce the amount of immunogen needed for a The device including one or more bone cages includes one Successful immunization, reduce the frequency of booster or more immunogens that can be one or more biomolecules immunizations needed and improve the immune response, for associated with a pathogenic state in the Subject including, example in elderly and immunocompromised vaccinees. In but not limited to, neoplastic disease, atherosclerosis, hyper addition, adjuvants can aid in presentation of the antigen, tension, autoimmune disease, diabetes, or Substance addic 50 defined by the physical appearance of the antigen in the tion. A method for treating a pathological condition in the vaccine; antigen/adjuvant uptake; distribution (targeting to Subject includes a device including one or more bone cages specific cells); immune potentiation/modulation that includes and one or more immunogens and one or more adjuvants that activities that regulate both quantitative and qualitative constitute a vaccine to treat a condition in the Subject, wherein aspects of the ensuing immune responses; the protection of the pathological conditions includes, but is not limited to, 55 the antigen from degradation and elimination. Selectively, atherosclerosis (Cardiol Rev. 16: 288-300, 2008), hyperten adjuvants can be used to optimize a desired immune response, sion (Drugs, Abstract: 68: 2557-2560, 2008), obesity (PLoS e.g., with respect to immunoglobulin classes and induction of ONE 3: e3163, 2008), autoimmune diseases including dia cytotoxic or helper T lymphocyte responses. In addition, cer betes (Immunol Cell Biol, Abstract: 86: 139-145, 2008), drug tain adjuvants can be used to promote antibody responses at or substance addiction (Ann NY Acad Sci, 1141: 257-269, 60 mucosal Surfaces. 2008), which are incorporated herein by reference. Adjuvants can be classified according to their source (natu A method for treating atherosclerotic disease can include ral, synthetic or endogenous), mechanism of action, or physi providing a device including one or more bone cages wherein cal or chemical properties. The current most common oxidized LDL is an immunogen to treat atherosclerotic dis described adjuvant classes include mineral salts, e.g., alumi ease. A method for treating hypertension can include provid 65 num hydroxide and aluminum or calcium phosphate gels; oil ing an immunogen to inhibit renin-angiotensin System emulsions and surfactant based formulations, e.g., MF59 (RAS), e.g., modified angiotensin I coupled to keyhole limpet (sorbitan trioleate, microfluidized detergent stabilized oil-in US 8,682,619 B2 79 80 water emulsion), QS21 (purified saponin), AS02 (SBAS2, interleukin receptors sIL-1r and sIL-2r; C-reactive protein; oil-in-water emulsion, MPL, and QS-21), Montanide ISA-51, CD11b; histamine; serotonin; apolipoprotein A1; f32-micro and ISA-720 (stabilized water-in-oil emulsion); particular globulin; bradykinin; D-dimer; endothelin-1; eotaxin; factor adjuvants, e.g., viroSomes (unilamellar liposome vehicles, VII: fibrinogen; GST haptoglobin: IgA; insulin; IP-10; lep e.g., those incorporating influenza hemagglutinin), AS04 tin: LIF; lymphotactin; myoglobin: OSM; SGOT, TIMP-1: (SBAS4A1 salt with MPL), ISCOMS (structured complex tissue factor; VCAM-1: VWF; thromboxane; platelet activat of Saponins and lipids), polylactide co-glycolide (PLG), ing factor (PAF); immunoglobulins. See, e.g., Tomai, et al., archaeosomes (liposomes comprised of glycerolipids of Expert Rev. Vaccines 6:835-847, 2007, which is incorporated Archaea); microbial derivatives (natural and synthetic), e.g., herein by reference. monophosphoryl lipid A (MLP), Detox (MLP. M. phlei cell 10 wall skeleton), AGP (RC-529, synthetic acylated monosac In an aspect, the adjuvant can be a molecule, for example a charide), DC-Chol (self-assembling lipoidal immunostimu pathogen-associated molecular patterns (PAMPs), that is rec lators), OM-174 (lipid A derivative), CpG oligonucleotides ognized by a component of the innate immune system, called (synthetic oligonucleotides containing immunostimulatory a pattern recognition receptor (PRRs). PRRs can, for CpG motifs), modified LT and CT (genetically modified bac 15 example, include secreted molecules that circulate in blood terial toxins); endogenous human immunomodulators, e.g., and lymph and can trigger responses such as the complement GM-CSF, IL-2 (cytokines that can be administered either as cascade and Subsequent accelerated phagocytosis; Surface protein or plasmid-encoded), TRICOM (B7-1, ICAM-1, and receptors on phagocytic cells like macrophages that bind the LFA-3), Immudaptin (C3d tandem arrays); inert vehicles, pathogen for engulfment, for example mannose-binding pro e.g., gold particles. tein (MBP); and receptors that bind the pathogen initiating a In general, adsorbents and particulate adjuvants aid in pre signal leading to the release of effector molecules Such as sentation of the antigen to the immune system, whereas cytokines. In a further aspect, the adjuvant can be a molecule microbial, synthetic and endogenous adjuvants can directly that binds to and/or induces a response in a PRR that is one or stimulate or modulate the immune system. Adjuvant emul more Toll-like receptor (TLRs). TLRs, which can exist as sions can be used to present the immunogen to the immune 25 heterodimers, play an important role in immune response by system, promote slow immunogen release and protect the recognizing molecular patterns associated with pathogens. immunogen from rapid elimination. Mineral salt adjuvants Ligands that are or mimic PAMPs and activate immune cells can be used to induce an inflammatory response at the site of via TLRs can be used as vaccine adjuvants. See, e.g., injection, promote synthesis of pro-inflammatory cytokines, Pulendranand Ahmed, Cell, 124:849-63, 2006; Kanzleret al., and stimulate innate immunity important for the initial steps 30 Nat. Med. 13: 552-559, 2007: Graham PLoS Med. 3:e57, of the immune response. 2006, Celis Cancer Res. 67: 7945-7947, 2007, Tomai, et al., In an aspect, the adjuvants are one or more immunomodu Expert Rev. Vaccines 6:835-847, 2007, which are incorpo lators, such as but not limited to, interleukin 2 (IL-2), IL-12, rated herein by reference. Molecules recognized by TLR2 IL-21, GM-CSF, or other cytokines, alone or in combination, include a wide array of microbial molecules representing that are capable of vigorously driving immune responses 35 broad groups of species such as gram-positive and gram stimulated by vaccines. As an example, a multivalent mela negative bacteria, as well as mycoplasma and yeast (e.g., noma cell vaccine appears more effective when encapsulated Pam3CSK4; HKLM). TLR2 can be a heterodimer with TLR1 into liposomes containing IL-2 or GM-CSF. See, e.g., van or TLR6. TLR3 ligands include double stranded RNA, a Slootenet al., IntJPharm. 183:33-36, 1999: Koppenhagenet molecular pattern associated with viral infection. Polyi al., Clin Cancer Res. 4: 1881-1886, 1998; van Slooten et al., 40 nosine-polycytidylic acid (poly(I:C)), a synthetic analog of Pharm Res. 17:42-48, 2000; Ben-Yehuda et al., Vaccine 21: dsRNA, is the ligand of choice for TLR3. Molecules recog 3.169-3178, 2003; Reynolds, et al., Clin. Cancer Res. 9: 657 nized by TLR4 include lipopolysaccharide (LPS) and lipid A. 662, 2003, which are incorporated herein by reference. Low toxicity versions of LPS, monophosphoryl lipid A Examples of other immunomodulatory adjuvants include, but (MPL) and a chemical mimetic, RC529 are efficient adju are not limited to, interferons (IFN) IFN-O, IFN-?3, and IFN-y: 45 vants for CD4-positive T-cells. Thompson, et al., J. Leukoc. other interleukins (IL) IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, Biol. 78: 1273-1280, 2005, which is incorporated herein by IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, reference. One TLR5 ligand is flagellin, the major component IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-27, IL-28, of the bacterial flagellar filament. TLR7/TLR8 ligands IL-29, IL-30, IL-31, and IL-32; tumor necrosis factor (TNF) include GU-rich short single-stranded RNA as well as small TNF-C. and TNF-B; granulocyte colony stimulating factor 50 synthetic molecules Such as imidazoquinolines and nucleo (G-CSF); granulocyte-macrophage colony stimulating factor side analogues (e.g., Imiquimod, Residuimod (R-848)). (GM-CSF); macrophage colony-stimulating factor (M-CSF); TLR9 ligands include specific unmethylated CpG oligode erythropoietin (EPO); and thrombopoietin (TPO); any of a oxynucleotides (CpG-ODN) sequences that distinguish number of chemotactic cytokines (chemokines) including, microbial DNA from mammalian DNA, and synthetic CpG but not limited to, CC chemokines CCL1 through CCL28 55 ODN are used in vaccine therapies. See, e.g., Kanzler et al., exemplified by RANTES (CCL5), MCP-1 (CCL2), LARC Nat. Med. 13:552-559, 2007, which is incorporated herein by (CCL20), MIP-1C. (CCL3), and MDC(CCL22); CXC reference. Additional natural and synthetic TLR agonists chemokines CXCL1 through CXCL17 exemplified by LIX include, but are not limited to, IMO-2055, IMO-2125, (CXCL5), GCP-2 (CXCL6) and BCA-1 (CXCL13): C QAX935, monophosphoryl lipid A, loxoribine, isatoribine, chemokines XCL1 and XCL2: CX3C chemokine C3CL1 60 3M-001, 3M-002, 3M-003, SD-101, and CPG 7909. (fractalkine); and chemokine like molecules exemplified by The adjuvant can be one or more biomolecules that stimu MIF; and other immunodulators anaphylatoxin fragments late activation, proliferation, and/or differentiation of T cell C3a, C4a, and C5a from the complement pathway; leukot lymphocytes. Examples of T cell stimulators include, but are rienes LTA4, LTB4, LTC4, LTD4, LTE4, and LTF4; prostag not limited to, enterotoxins, MHC-peptide complexes, CD80 landins; growth factors EGF, FGF-9, FGF-basic, growth hor 65 (B7-1), B7-2, antibodies to CD2, CD28, CD3; phorbolesters, mone, stem cell factor (SCF), TGF-3- and VEGF; soluble IL-2, protein kinase C activators such as phorbol myristate receptors to tumor necrosis factor receptor (STNFr); soluble acetate, calcium ionophores such as inonmycin, agents that US 8,682,619 B2 81 82 trigger T cell receptor (TCR/CD3) activation, CD86, tumor Temporal Release of Immunogen and Adjuvant from Bone necrosis factor (ligand) superfamily member 14 (TNFSF14), Cage Device CD5, and ICOS. The release of the one or more immunogen and the one or The adjuvant can be one or more biomolecules that stimu more adjuvant from a device including one or more bone late activation, proliferation, and/or differentiation of B cell cages can be temporally controlled. Temporal release can be lymphocytes. Examples of B cell stimulators include, but are controlled by the properties of the bone cage, the formulation not limited to, antigen dependent lipopolysaccharide, of the immunogen and/or adjuvant placed in the bone cage, or CD98hc, phorbol esters, interleukin 4 (IL-4), interleukin 15 a combination thereof. Temporally controlled release of the (IL-15), tumor necrosis factor (ligand) Super family member one or more immunogen and the one or more adjuvant from 10 the bone cage is useful for primary immunization and sec 13.b (TNFSF13B), TNFSF13C (B cell-activating factor; ondary immunization to establish memory cells responsive to BAFF), TLR7 and TLR9 agonists (e.g., 852A, 3M-003, the pathogenic organism or pathogenic condition. For CpG2006), IFN-O. or IFN-B. example, vaccines for diphtheria-tetanus-pertussis (DTaP) is In an aspect, the one or more adjuvants incorporated into recommended to be given in 5 doses between 2 months and 18 the one or more bone cages can be a purified protein, for 15 months and a final dose at 4-6 years, with a tetanus and example, one or more cytokine immunomodulator. The adju diphtheria vaccine boosted every 10 years. As a further vant can be purified from a natural source Such as plasma, example, vaccination with Gardasil Rhuman papillomavirus cells or tissue. Alternatively, the adjuvant can be purified from quadrivalent (types 6, 11, 16, 18) vaccine is recommended in a genetically engineered cell line in which the adjuvant has three doses over approximately six months. The method for been expressed using standard recombinant DNA techniques. treating an infectious disease in a subject can include provid The one or more adjuvants can be admixed with the one or ing a device including one or more bone cages as a single more immunogens and loaded into the bone cage. Alterna administered dose to deliver the one or more immunogen and tively, the adjuvant and the immunogen can be placed in the one or more adjuvant over an extended period of time separate bone cages or in separate compartments within a from 2 months up to 24 months. bone cage. 25 The temporal release of immunogen and adjuvant from the The one or more adjuvants can be produced in cells encap device including one or more bone cages can be controlled Sulated in the bone cage, wherein the encapsulated cells can based on the size of the pores in the bone cage relative to the naturally express the adjuvant. Alternatively, the encapsu size of the immunogen and/or adjuvant. The Smaller the lated cells can be genetically engineered to express the adju pores, the slower the release of immunogen and/or adjuvant vant. The genetically engineered cells can be bacteria, yeast, 30 from the bone cage. In addition, release of immunogen and adjuvant can be controlled by the number of pores. The fewer insect cells or mammalian cells. The mammalian cells can be the pores, the slower the release of immunogen and adjuvant autologous, allogeneic, or Xenogeneic. In an aspect, the adju from the bone cage. The pores can range in size from about 1 vant is a protein or peptide Such as, for example, a cytokine nanometer to about 20 micrometers and can be dependent immunomodulator, which may or may not be modified, for 35 upon the size of the immunogen and adjuvant. For example, example by glycosylation or lipidation. A protein or peptide, proteins and peptides range in size from Sub-nanometer to and/or one or more enzymes required for modification or greater than 16 nanometers (e.g., diameter of immunoglobu production thereof, can be expressed in a genetically engi lin molecule); plasmid DNA ranges in size from Sub-nanom neered cell using standard recombinant DNA techniques. In eter to greater than 25 nanometer; live or killed whole viral an aspect, the cDNA corresponding to the adjuvant and the 40 particles range in size from about 10 nanometers to about 300 immunogen, or one or more enzymes required for modifica nanometers; live or killed bacteria range in size from 100 tion or production thereof, can be incorporated into the same nanometers to 6 micrometers; and attenuated Plasmodium expression vector and transfected into a mammalian cell line. falciparum sporozoites are about 10 micrometers in length. Alternatively, the cDNA corresponding to the adjuvant and The pores may be uniform in size or may vary in size. Dif the immunogen can be incorporated into distinct expression 45 ferent compartments of the bone cage can have pores of vectors. The two or more expression vectors can be trans different sizes allowing for differential release rates from fected simultaneously into the same cells, creating a single each of the compartments. genetically engineered cell line that expresses both the adju The temporal release of the one or more immunogens vant and the immunogen. Alternatively, the two or more and/or one or more adjuvants from the device including one or expression vectors can be transfected into separate cultures of 50 more bone cages can be controlled by the formulation of the the same or differing cells and the two genetically engineered immunogen and adjuvant. The formulation can allow for cell lines incorporated into the bone cage. In an aspect, the immediate release, extended release, delayed release, or a genetically engineered cells expressing the adjuvant and/or combination thereof. An immediate release formulation can the immunogen are encapsulated in alginate or other physi be a simple aqueous solution, e.g., Saline, into which the ologically compatible encapsulation medium prior to incor 55 immunogen and/or adjuvant have been dissolved. An poration into the bone cage. extended or delayed release formulation can include a matrix The adjuvant can be administered as part of a plasmid DNA through which the immunogen and/or adjuvant must diffuse into which DNA sequence encoding all or part of the adjuvant to escape the bone cage. Examples of matrix material for use has been incorporated. Methods for generating a plasmid in extended and/or delayed release formulation include, but DNA for this purpose have been described herein. In this 60 are not limited to, insoluble plastics (e.g., methyl acrylate instance, the plasmid DNA encoding the adjuvant is loaded methylmethacrylate, polyvinyl chloride, and polyethylene), into the bone cage and the adjuvant protein is expressed by the hydrophilic polymers (e.g., methylcellulose, hydroxypropy host cells following implantation of the bone cage. The plas lmethylcellulose, Sodium carboxymethylcellulose, and car mid DNA can also include DNA encoding one or more immu bopol 934), and fatty compounds (e.g., carnauba wax, glyc nogen. Alternatively, the DNA encoding one or more immu 65 eryl tristearate). In an aspect, the immunogen and/or adjuvant nogen and the one or more adjuvants can be incorporated into can be encapsulated in Small particles Surrounded by a mem separate plasmid DNAs. brane barrier coat through which the immunogen and/or adju US 8,682,619 B2 83 84 vant diffuse over time. Examples of material for use in gen out of the bone cage. Examples of stimuli-responsive gels erating a membrane barrier coat include, but are not limited include pH/electro-responsive gels (e.g., hyaluronic acid, to, hardened gelatin, methyl- or ethylcelluloses, polyhy chondroitin Sulphate, agarose, carbomer, Xanthan gum, cal droxymethacrylate, hydroxypropylcellulose, polyvinylac cium alginate, acrylate and methacrylate derivatives Such as tate, and various waxes. Alternatively, the immunogen and 5 partially hydrolyzed polyacrylamide, polydimethylamino adjuvant can be formulated in a hydrogel material including propyl acrylamide, and poly(methacrylic acid) PMAA, poly natural polymers (e.g., chitosan, alginate, fibrin, collagen, ethyloxazoline), thermo-responsive gels (e.g., poly(N-iso gelatin, hyaluronic acid, and dextran), synthetic monomers propylacrylamide), poly(N-vinylcaprolactam), polyethylene (e.g., hydroxyethyl methacrylate, N-(2-hydroxypropyl) glycol, poly(propylene oxide) and combinations thereof), and methacrylate, N-vinyl-2-pyrrolidone, N-isopropyl acryla 10 magnetically responsive gels (e.g., incorporation of magnetic mide, vinyl acetate, acrylic acid, methacrylic acid, polyeth ylene glycol acrylate/methacrylate, and polyethylene glycol carriers such as magnetite, iron, nickel and/or cobalt into the diacrylate/dimethacrylate), or a combination thereof. See, polymer matrix). See, e.g., Anal. Rec. Pat. Endo. Metab. e.g., Lin & Metters. Adv. Drug Deliv Rev. 58:1379-1408, Immune Drug Dis. 1:83-90, 2007; Liu, et al., Nano Today 2006, which is incorporated herein by reference. General 15 4:52-65, 2009 which are incorporated herein by reference. information regarding formulation can be found in Reming The stimuli-responsive gel can be constructed Such that it ton: The Science and Practice of Pharmacy; 20" Edition; releases the one or more immunogens and/or the one or more Cover page (3 pgs.); printed on Mar. 19, 2010; Lippincott adjuvants is response to a specific analyte. For example, Yang Williams & Wilkins, Baltimore, Md. which is incorporated et al. describe a hydrogel system that incorporates two or herein by reference. See also Heit, et al., Eur: J. Immunol. more overlappingaptamers that serve as recognition elements 37:2063-2074, 2007; U.S. Pat. No. 5,762,965; U.S. Pat. No. for an analyte. Yang, et al., J. Am. Chem. Soc. 130:6320-6321, 5,820,873; U.S. Pat. Appl. No. 2007/0026005, which are 2008, which is incorporated herein by reference. The two or incorporated herein by reference. more partially overlappingaptamers are copolymerized into a The device including one or more bone cages can be con polyacrylamide-based hydrogel. At least one of the aptamers structed utilizing cells cultured in vitro including, but not 25 is a recognition element that binds to an analyte. The interac limited to, stem cells, fibroblasts, endothelial cells, osteo tion of the analyte with the aptamer recognition element blasts and/or osteoclasts. The in vitro cultured cells can be causes the two partially overlapping aptamers to separate configured to form the bone cage structure or configured to from one another and to change the properties of the hydro restructure the bone cage wallorinner compartment. Restruc gel, releasing the contents of the hydrogel. The one or more turing the device including one or more bone cages is useful 30 aptamers can interact with an analyte that is all or part of a to design compartments that can provide temporal release of pathogen or tumor associated biomolecule. In response to the one or more immunogens and one or more adjuvants from interacting with the analyte, the hydrogel can release one or the bone cage. Temporal release can depend upon the size of more immunogens and/or adjuvants incorporated in the the bone cage compartment, the thickness of the bone cage hydrogel. wall, and the size and configuration of pores in the bone cage 35 wall. As discussed in more detail herein, the bone cage wall The synthesis of the one or more immunogens and/or one can be slowly degraded to release one or more of the one or or more adjuvants by genetically engineered cells incorpo more immunogens and one or more adjuvants. rated into the device including one or more bone cages can be Triggered Release of Immunogen and/Adjuvant from Bone controlled by a trigger, for example, one derived from a Cage Device 40 pathogen or tumor. The cells can be genetically engineered to The release of one or more immunogens and/or one or include a receptor that is responsive to the trigger and is linked more adjuvants from a device including one or more the bone to the expression of the immunogen and/or adjuvant. cage can be controlled by a trigger, for example, a biomol Examples of expression systems that are linked to receptor ecule, a specific analyte, a pathogen or tumor cell, or an activation by a trigger include those linked to signaling externally-administered compound. The trigger can stimulate 45 through the Toll-like receptors (TRL) such as, for example, a immediate or Sustained release of the immunogen and/or TLR-signaling reporter plasmid. See, e.g., pNIFTY from adjuvant from the bone cage. Alternatively, the trigger can InvivoGen, San Diego, Calif.; Roger, et al., Biochem.J. 387: stimulate the synthesis of the immunogen and/or adjuvant by 355-365, 2005, which are incorporated herein by reference. cells incorporated in the bone cage. The trigger can be a TLRs are activated by pathogen-associated molecular pat biomolecule. Examples of biomolecules include, but are not 50 terns (PAMPs) such as, for example, bacterial lipopolysac limited to, pathogen associated biomolecules (e.g., toxins, charide (LPS), flagellin, and lipoteichoic acid and viral pep polysaccharides, double stranded RNA, CpG polynucle tidoglycan, double-stranded RNA, and unmethylated CpG otides), tumor-associated biomolecules (e.g., tumor antigens, motifs. tumor markers), other disease-associated biomolecules (e.g., Alternatively, the synthesis of the immunogen and/or adju B-amyloid), allergens (e.g., food allergen), or other biomol 55 vant by genetically engineered cells incorporated into the ecules (e.g., drugs of abuse). Alternatively, the trigger can be device including one or more bone cages can be controlled by a physiological change induced by the pathogen, tumor, dis an external trigger administered to the Subject. For example, ease, or allergic response. Examples of a physiological cells can be engineered to Switch on genes in response to change include, but are not limited to, changes in pH, tem administration of the dietary supplement vitamin H (biotin: perature, osmolarity, hypoxia, ion concentrations, and endog 60 Weber, et al., Metabolic Eng. 11:117-124, 2009, which is enous biomolecule concentrations (e.g., cytokines). incorporated herein by reference). The expression system can The immunogen and the adjuvant can be incorporated into include an inducible promoter that is activated in the presence a stimuli-responsive gel that is further incorporated into the of an inducing agent. Examples of expression systems with bone cage. The stimuli-responsive gel, for example, may inducible promoters include, but are not limited to, lactose undergo Swelling and deswelling in response to environmen 65 inducible expression systems, tetracycline-inducible expres tal conditions. The Swelling and deswelling of the gel alters sion systems, doxycycline-inducible expression system, the ability of the immunogen and/or the adjuvant to diffuse cumate-inducible expression system, metal (e.g., Zinc)-in US 8,682,619 B2 85 86 ducible expression system, ethanol-inducible expression sys more adjuvants, optionally in combination with one or more tem, rapamycin-inducible expression system, and ecdysone cells or tissues that produce the one or more immunogen inducible expression system. and/or the one or more adjuvant is shown in FIG. 2. FIG. 2A The synthesis of the immunogen and/or adjuvant by cells shows a buckeyball shaped cage 201 in which the pentagonal incorporated into the bone cage can be controlled by a trigger and hexagonal shapes are comprised of bone 210. FIG. 2B that is a physiological trigger. For example, studies describe shows a barrel-like shape 202, in which the vertical and an inducible expression system that is activated by a hypoxic horizontal members are comprised of bone 210 with pores in environment. Lee, et al., Abstract, JControl Release 115:113 between 220. FIG. 2C shows a rectangular structure 203, 119, 2006, which is incorporated herein by reference. Other comprised of a bone wall 210 containing large openings as examples might include expression systems that incorporate 10 pores 220. a heat shock protein promoter. In an aspect, the external wall has one or more openings, Configuration and Structure of Bone Cage Device Including and the openings are closable. Closable refers to the opening One or More Immunogens and One or More Adjuvants configured to be completely or partially filled, such that the The device including one or more bone cages can partially opening can be made no longer larger than the pore size of the Surround or can completely Surround the one or more immu 15 bone. The closure has a width sufficiently greater than the nogens and one or more adjuvants, optionally in combination width of the opening to allow attachment to the external wall with one or more cells or tissues that produce the one or more completely Surrounding the opening, and can be secured by immunogen and/or the one or more adjuvant. Examples of any method known in the art. In an aspect, the closure spans bone cages that completely Surround the one or more immu the entire width of the opening, and/or the entire length. The nogens and one or more adjuvants, optionally in combination plug or closure can be comprised of bone, including, but not with one or more cells or tissues are shown in FIG.1. In FIG. limited to, anorganic, artificial, demineralized, cultured in 1A, a rectangular cage 100 is depicted, showing the bone wall vitro, autologous, allogeneic or Xenogeneic bone, or any com 110 with pores 120 partially surrounded by a semi-permeable bination thereof. component 130 optionally comprised of cells 140 (e.g., at Aspects of a device including one or more bone cages with least one altered microorganism). FIG. 1B shows a cross 25 closable openings are shown in FIG. 3. FIG. 3A shows a section of the rectangular cage 100, showing the optional rectangular cage 301 comprised of bone 310 containing pores exterior semi-permeable component 130 optionally com 320 containing an opening 360 that connects with the internal prised of cells 140, and the optional interior semi-permeable cavity 350. The opening 360 is closable by the insertion of a component 130, as well as the bone structure 110 with pores plug370 made of bone 310 of a size to approximately entirely 120, and the internal cavity 150 with optional living cells 140. 30 fill the opening. FIG. 3B shows the two open halves of a petri The device including one or more bone cages partially dish-shaped cage 302 made of bone 310 containing pores 320 surrounds the one or more immunogens and one or more in which one half 304 has a uniformly slightly smaller diam adjuvants, optionally in combination with one or more cells eter than the other half306 so that the sides of 306 overlap the or tissues that produce the one or more immunogen and/or the sides of 304 on closure such that an internal cavity 350 one or more adjuvant. “Partially surrounds’ refers to the 35 remains. The two halves are optionally secured by sliding a external wall of the bone cage surrounding less than 100% of partially internally protruding edge 385 under a partially the one or more immunogens and one or more adjuvants externally protruding edge 380. On closing, 304 and 306 are optionally in combination with one or more cells or tissues in positioned such that 380 and 385 can slide past each other. the internal cavity. “Less than 100% includes any integer Once 385 is past 380,304 and 306 are twisted such that 380 percentage from 1% to 99%, for example, 10%, 25%, 50%, 40 and 385 align. FIG. 3C shows the two open halves of an egg 75%, and 95%. shell-shaped structure 303 made of bone 310 comprising Examples of devices including one or more bone cages pores 320, where the edges 390 and 395 of the two halves 305 with external walls that partially surround the internal cavity and 307, respectively, optionally mate to allow a screw-type include, but are not limited to, those where the external wall seal, forming an internal cavity 350. is a lattice, and/or where there are openings in the wall that are 45 In an embodiment, as shown in FIG.4, a blood brainbarrier larger than the pore size of the bone. Examples of lattice work device includes one or more cells. FIG. 4A shows a cross external walls include, but are not limited to, those patterned section of one orientation of an ovoid blood brain barrier after buckeyballs. endothelium device 401 comprised of endothelial cells 410 Examples of external walls with openings include, but are optionally containing interendothelial tight junctions 420, P not limited to, those with openings designed to facilitate the 50 glycoprotein efflux pump 430 at interior plasma membrane placement of the semi-permeable membrane, and the one or surfaces 490, ion transporters 440 and glucose receptor 450 at more immunogens and one or more adjuvants optionally in exterior plasma membrane surfaces 495, pinocytic vesicles combination with the one or more cells or tissues, for 460, mitochondria 470, and nuclei 480. FIG. 4B shows a example, within the internal cavity. In an aspect, the width of cross-section of another orientation of an ovoid blood brain the one or more openings in the external wall can be any 55 barrier endothelium device 402 comprised of endothelial integer um from approximately 1 to approximately 1,000 cells 410 optionally containing interendothelial tight junc including, but not limited to, approximately 2 um, 3 um, 4 um, tions 420, the P glycoprotein efflux pump 430 at exterior 5um, 8 um, 10um, 12 Jum, 15um, 20um, 25um, 50 um, 100 plasma membrane surfaces 495, ion transporters 440 and um, 200 um, 300 um, 500 um, 600 um, 800 um and 1,000 um. glucose receptor 450 at interior plasma membrane Surfaces In an aspect, the width can be approximately 1 um to 1,000 60 490, pinocytic vesicles 460, mitochondria 470, and nuclei um, 2 um to 800 um, 5um to 750 um, 10um to 500 um, 20um 480. to 250 um, 10um to 100 um, 5um to 50 um, 1 um to 10 um, In an embodiment, as shown in FIG. 5, a blood cerebrospi 2 um to 20 lum, 1 um to 50 um, 50 um to 500 um, or 250 um nal fluid barrier device is described. FIG. 5A shows a cross to 1,000 um in width, and the length is the width of the section of one orientation of an ovoid choroid plexus epithe external wall as described above. 65 lium device 501 comprised of epithelial cells 510 optionally Examples of devices including one or more bone cages that containing exterior plasma membrane microvilli520, interior partially Surround the one or more immunogens and one or plasma membrane interdigitations 530, interepithelial tight US 8,682,619 B2 87 88 junctions 540 near the external Surface, ion transport systems cessors, a networked computer, a tablet personal computer, a 550 on interior and exterior plasma membranes, organic laptop computer, a mobile device, a mobile telephone, or a transport systems on interior and exterior plasma membranes personal digital assistant computer. 560, mitochondria 570 and nuclei 580. FIG. 5B shows a In an embodiment 1340, the system further comprises one cross-section of another orientation of an ovoid choroid or more instructions on a recordable medium that when plexus epithelium device 502 comprised of epithelial cells executed on the at least one computing device cause the at 510 optionally containing interior plasma membrane least one computing device to generate at least one output to microVilli 520, exterior plasma membrane interdigitations a user readable display. 530, interepithelial tight junctions 540 near the internal sur As depicted in FIG. 14, in an embodiment 1400, the at least face, ion transport systems 550 on interior and exterior 10 one output includes at least one graphical illustration of the at plasma membranes, organic transport systems 560 on interior least one auxotrophic microorganism, at least one component and exterior plasma membranes, mitochondria 570 and nuclei thereof, or at least one product thereof; at least one property of 580. the treatment device; or at least one property of dispensing the Referring to FIG. 12, a logic flowchart is depicted for a at least one therapeutic agent from the treatment device. In an method 1101 for modulating a pathological condition in a 15 embodiment 1410, the at least one output includes at least one subject. The method 1101 includes providing 1102 a device protocol for administering the at least one treatment device to comprising one or more bone cages including one or more at least one biological tissue. In an embodiment 1420, the user immunogens and one or more adjuvants. In an aspect 1103, includes at least one entity. In an embodiment 1430, the entity the one or more bone cages is configured to be non-weight includes at least one person, or computer. In an embodiment bearing when implanted into a soft tissue of a Subject. In a 1440, the user readable display includes a human readable further aspect 1104, the pathological condition in the subject display. In an embodiment 1450, the user readable display includes infectious disease, neoplastic disease, atherosclero includes one or more active displays. In an embodiment 1460, sis, hypertension, autoimmune disease, diabetes, or Substance the user readable display includes one or more passive dis addiction. The method further includes providing 1105 one or plays. In an embodiment 1470, the user readable display more cells or tissues encapsulated in the bone cage and con 25 includes one or more of a numeric format, graphical format, figured to produce the one or more immunogens. The method or audio format. In an embodiment 1480, the system further further includes providing 1106 one or more cells or tissues comprises one or more instructions on a recordable medium encapsulated in the bone cage and configured to produce the that when executed on the computing device cause the com one or more adjuvants. In an aspect 1107, the one or more puting device to evaluate the at least one biological tissue for adjuvants includes a biologically derived agent. In an aspect 30 one or more indicators prior to, during, or Subsequent to 1108, the one or more adjuvants includes a synthetically administering the at least one treatment device to the at least derived agent. In an aspect 1109, the one or more immuno one biological tissue. gens include a microbial antigen, viral antigen, parasite anti As depicted in FIG. 15, in an embodiment 1500, to evaluate gen, plant antigen, animal antigen, endogenous antigen, or at least one biological tissue for one or more indicators synthetic antigen. In an aspect 1110, the one or more immu 35 includes to evaluate at least one of an assay, image, or gross nogens include protein, lipid, lipoprotein, glycolipid, glyco assessment of the at least one biological tissue prior to, dur protein, proteoglycan, polysaccharide, or lipopolysaccha ing, or Subsequent to administering the at least one treatment ride. device to the at least one biological tissue. In an embodiment Bone encompasses all types of bone, including, but not 1510, the assay includes at least one technique including limited to, organic, anorganic, demineralized, freeze-dried, 40 spectroscopy, microscopy, electrochemical detection, poly and artificial bone. The bone can be cultured in vitro, and/or nucleotide detection, histological examination, biopsy analy genetically engineered. The bone can be living or dead. The sis, fluorescence resonance energy transfer, electron transfer, bone can be autologous, allogeneic, or Xenogeneic with enzyme assay, electrical conductivity, isoelectric focusing, respect to a subject within whom the bone is implanted. The chromatography, immunoprecipitation, immunoseparation, bone can be a combination of one or more of the types of bone 45 aptamer binding, filtration, electrophoresis, immunoassay, or described above. radioactive assay. As depicted in FIG. 13, a system 1300 comprises 1310 at In an embodiment 1520, the at least one image includes one least one computing device; at least one treatment device or more images acquired by at least one of laser, holography, including at least one semi-permeable barrier Substantially X-ray crystallography, optical coherence tomography, com enclosing at least one auxotrophic microorganism, the at least 50 puter-assisted tomography scan, computed tomography, one auxotrophic microorganism including at least one nucleic magnetic resonance imaging, positron-emission tomography acid construct encoding at least one therapeutic agent, the Scan, ultrasound, X-ray, electrical-impedance monitoring, barrier defining an interior region and exterior region; microscopy, spectrometry, flow cytommetry, radioisotope wherein the device includes at least one metabolite required imaging, thermal imaging, infrared visualization, multipho by the at least one auxotrophic microorganism; the treatment 55 ton calcium-imaging, photography, or in silico generation. In device further comprising at least one pump for dispensing at an embodiment 1530, the system further comprises one or least one therapeutic agent, the pump including electronic more instructions on a recordable medium that when circuitry configured to send or receive signals from the com executed on the computing device cause the computing puting device; and one or more instructions on a recordable device to alter at least one microorganism isolated from at medium that when executed on the computing device cause 60 least one biological tissue. the computing device to regulate dispensing of the at least one As depicted in FIG. 16, the system 1600 further comprises therapeutic agent from the at least one treatment device. one or more instructions on a recordable medium that when In an embodiment 1320, the at least one computing device executed on the computing device cause the computing includes one or more of a desktop computer, phone, personal device to amplify the at least one microorganism isolated digital assistant, remote controller, workstation computer, or 65 from the at least one biological tissue. In an embodiment computing system. In an embodiment 1330, the at least one 1610, the system further comprises one or more instructions computing system includes one or more of a cluster of pro ona recordable medium that when executed on the computing US 8,682,619 B2 89 90 device cause the computing device to reinstate the at least one more instructions on a recordable medium that when microorganism to the biological tissue from which it was executed on the at least one computing device cause the isolated subsequent to alteration. In an embodiment 1620, the computing device to generate an output to a user readable system further comprises one or more instructions on a display. In an embodiment 1915, the system further com recordable medium that when executed on the computing prises one or more instructions on a recordable medium that device cause the computing device to predetermine at least when executed on the at least one computing device cause the one microorganism strain or type for altering in order to at least one computing device to compare a value associated produce at least one therapeutic agent based on at least one with the first possible dataset with a second dataset including feature of at least one biological tissue. In an embodiment values of at least one predictive parameter. In an embodiment 1630, the at least one feature of the at least one biological 10 1920, the first input includes one or more values derived from tissue includes at least one property of one or more microor at least one property of the at least one treatment device. ganism populations associated with the at least one biological As depicted in FIG. 20, in an embodiment 2000, the first tissue. In an embodiment 1640, the system further comprises input includes at least one parameter for generating the at one or more instructions for delivering at least one agent least one auxotrophic microorganism. In an embodiment Sufficient to induce death in the at least one auxotrophic 15 2010, the first input includes at least one parameter for mak microorganism. In an embodiment 1650, the one or more ing the at least one treatment device. In an embodiment 2020. instructions for delivering at least one agent Sufficient to the at least one parameter for making the at least one treat induce death in the at least one auxotrophic microorganism ment device includes one or more of constitution of the at includes one or more instructions for delivering the at least least one treatment device, configuration of the at least one one agent at least one of locally or systemically. treatment device, formulation of the at least one therapeutic As depicted in FIG. 17, in an embodiment 1700, the system agent, type of auxotrophic microorganism, strain of aux further comprises a cryptographic logic component. In an otrophic microorganism, configuration of the at least one embodiment 1710, the cryptographic logic component is con inducible genetic element of the auxotrophic microorganism, figured to implement at least one cryptographic process or or alteration of the at least one auxotrophic microorganism. cryptographic logic. In an embodiment 1720, the crypto 25 In an embodiment 2030, the second input includes one or graphic logic component is configured to implement one or more values related to the at least one parameter for admin more processes associated with at least one of a cryptographic istering at least one treatment device to the at least one bio protocol, decryption protocol, or encryption protocol. In an logical tissue. In an embodiment 2040, the at least one param embodiment 1730, the cryptographic logic component is con eter for administering the at least one treatment device figured to implement one or more processes associated with 30 includes one or more of biological tissue type; biological at least one of a regulatory compliance protocol, regulatory tissue function; biological tissue size; biological tissue con protocol, or authentication protocol. In an embodiment 1740. stitution; biological tissue architecture: biological tissue the cryptographic logic component is configured to imple durability; biological tissue temperature; temperature of ment one or more processes associated with at least one of an administration conditions; depth of administration of the at authorization protocol, activation protocol, or treatment regi 35 least one treatment device; biological tissue source; one or men protocol. In an embodiment 1750, the cryptographic more temporal coordinates; one or more spatial coordinates; logic component includes one or more of a crypto-algorithm, angle of administration of the at least one treatment device; signal-bearing media, crypto controller, or cryptographic force of administration of the at least one treatment device; module. velocity of administration of the at least one treatment device: As depicted in FIG. 18, in an embodiment 1800 the cryp 40 quantity of treatment devices administered; rate of adminis tographic logic component is configured to generate informa tration of more than one treatment device; method of admin tion associated with at least one of an authentication protocol, istration of the at least one treatment device; timing of admin authorization protocol, anti-microbial agent reservoir deliv istration of the at least one treatment device; rate of ery protocol, activation protocol, encryption protocol, production of the at least one therapeutic agent of the at least decryption protocol authorization instruction, prescription 45 one treatment device, or rate of dispensing of the at least one dosing instruction, or prescribed regimen instruction. In an therapeutic agent from the at least one treatment device. embodiment 1810, the cryptographic logic component is con As depicted in FIG. 21, the system 2100 further comprises figured to generate information associated with at least one of one or more instructions on a recordable medium that when an activation code, error code, command code, or authoriza executed on the at least one computing device cause the at tion code. In an embodiment 1820, the cryptographic logic 50 least one computing device to determine a graphical illustra component is configured to generate information associated tion of the second possible dataset. In an embodiment 2120. with at least one of a cryptographic protocol, decryption the system further comprises one or more instructions on a protocol, encryption protocol, regulatory compliance proto recordable medium that when executed on the at least one col, or regulatory use protocol. computing device cause the at least one computing device to As depicted in FIG. 19, a system 1900 comprises 1910 at 55 determine from the comparison at least one parameter for least one computing device; one or more instructions on a making or administering the at least one treatment device. In recordable medium that when executed on the at least one an embodiment 2130, the system further comprises one or computing device cause the at least one computing device to more instructions on a recordable medium that when receive a first input associated with a first possible dataset, the executed on the at least one computing device cause the at first possible dataset including data representative of at least 60 least one computing device to access the first possible dataset one parameter for making or administering at least one treat in response to the first input. In an embodiment 2140, the ment device to at least one biological tissue, wherein the at system further comprises one or more instructions on a least one treatment device includes at least one semi-perme recordable medium that when executed on the at least one able barrier structured to substantially enclose at least one computing device cause the at least one computing device to auxotrophic microorganism; wherein the at least one aux 65 generate the first possible dataset in response to the first input. otrophic microorganism includes at least one nucleic acid In an embodiment 2150, the system further comprises one or construct encoding at least one therapeutic agent; and one or more instructions on a recordable medium that when US 8,682,619 B2 91 92 executed on the at least one computing device cause the at microorganism. In an embodiment 2370, the system further least one computing device to determine a graphical illustra comprises means for administering the at least one treatment tion of the first possible dataset. In an embodiment 2160, the device to at least one biological tissue. at least one computing device includes one or more of a In an embodiment 2400, the system further comprises desktop computer, workstation computer, phone, personal means for evaluating the at least one biological tissue for one digital assistant, remote controller, or computing system. In or more indicators prior to, during, or Subsequent to admin an embodiment 2170, the at least one computing system istering the at least one treatment device to at least one bio includes one or more of a cluster of processors, a networked logical tissue. In an embodiment 2410, the one or more indi computer, a tablet personal computer, a laptop computer, a cators include at least one of an assay, image, or gross assessment of the at least one biological tissue prior to, dur mobile device, a mobile telephone, or a personal digital assis 10 ing, or Subsequent to at least one administration of the at least tant computer. one treatment device. In an embodiment 2420, the assay As depicted in FIG. 22, in an embodiment 2200, the output includes at least one technique including spectroscopy, includes at least one graphical description of the at least one microscopy, electrochemical detection, polynucleotide treatment device, at least one component thereof, or at least detection, histological examination, biopsy analysis, fluores one cell or Substance associated with at least one biological 15 cence resonance energy transfer, electron transfer, enzyme tissue. In an embodiment 2210, the user includes at least one assay, electrical conductivity, isoelectric focusing, chroma entity. In an embodiment 2220, the entity includes at least one tography, immunoprecipitation, immunoseparation, aptamer person, or computer. In an embodiment 2230, the user read binding, filtration, electrophoresis, immunoassay, or radioac able display includes a human readable display. In an embodi tive assay. In an embodiment 2430, the at least one image ment 2240, the user readable display includes one or more includes one or more images acquired by at least one of laser, active displays. In an embodiment 2250, the user readable holography, X-ray crystallography, optical coherence tomog display includes one or more passive displays. In an embodi raphy, computer-assisted tomography scan, computed ment 2260, the user readable display includes one or more of tomography, magnetic resonance imaging, positron-emission a numeric format, graphical format, or audio format. In an tomography scan, ultrasound, X-ray, electrical-impedance embodiment 2270, the system further comprises one or more 25 monitoring, microscopy, spectrometry, flow cytommetry, instructions for receiving a third input associated with at least radioisotope imaging, thermal imaging, infrared visualiza one feature of at least one subject. In an embodiment 2280, tion, multiphoton calcium-imaging, photography, or in silico the at least one feature of the at least one subject includes one generation. In an embodiment 2440, the system further com or more of a physiological condition; genotype; phenotype; prises one or more instructions on a recordable medium that genetic profile; proteomic profile; lipidomic profile; gly 30 when executed on the computing device cause the computing comic profile; system biology profile; circulatory condition; device to evaluate the at least one biological tissue for one or respiratory condition; lymph condition; anatomic, genetic or more indicators relating to one or more of: administering the proteomic characteristic; response to previous treatment; at least one device, at least one component thereof, or at least weight; height; medical diagnosis; familial background; one product thereof; cell or tissue formation; cell or tissue results of one or more medical tests; ethnic background; body 35 growth; cell or tissue apoptosis; cell or tissue necrosis; cell mass index; age; presence or absence of at least one disease or division; cellular cytoskeletal rearrangement; cell or tissue condition; species; ethnicity; race; allergies; gender, presence secretion; cell or tissue differentiation; status of the at least or absence of at least one biological, chemical, or therapeutic one auxotrophic microorganism of the at least one treatment agent in the Subject; pregnancy status; lactation status; medi device; status of the at least one treatment device; or status of cal history; or blood condition. 40 the at least one therapeutic agent. As depicted in FIG. 23, in an embodiment 2300, the user As depicted in FIG.25, in an embodiment 2500, the system readable display includes one or more of a display of one or further comprises means for transmitting one or more signals more differences in the comparison of at least one value that include information relating to the accepting a first input related to the first input and at least one value related to at least or a second input and information related to the evaluating the one property of the at least one treatment device. In an 45 at least one biological tissue. In an embodiment 2510, the embodiment 2310, the user readable display includes one or means for transmitting one or more signals includes means more of a display of one or more differences in the compari for transmitting one or more signals associated with selection Son of at least one value related to the second input and at least of at least one parameter for making the at least one treatment one value related to at least one parameter for administration device. In an embodiment 2520, the means for transmitting of the at least one treatment device to the at least one biologi 50 one or more signals includes means for transmitting one or cal tissue. In an embodiment 2320, the system further com more signals associated with selection of at least one param prises means for transmitting one or more signals that include eter for administering the at least one treatment device. In an information related to the processing of the first input and the embodiment 2530, the system further comprises means for second input. In an embodiment 2330, the means for trans obtaining genetic sequence information from at least one mitting one or more signals includes means for transmitting 55 microorganism isolated from at least one biological tissue. In one or more signals associated with selection of at least one an embodiment 2540, the system further comprises means for parameter for making the at least one treatment device. In an altering the at least one microorganism isolated from the at embodiment 2340, the means for transmitting one or more least one biological tissue. In an embodiment 2550, the sys signals includes means for transmitting one or more signals tem further comprises means for amplifying the at least one associated with selection of at least one parameter for admin 60 microorganism isolated from the at least one biological tis istering the at least one treatment device to the at least one sue. In an embodiment 2560, the system further comprises biological tissue. In an embodiment 2350, the means for means for reinstating the at least one microorganism isolated transmitting one or more signals includes means for transmit from the at least one biological tissue Subsequent to alter ting one or more signals associated with comparing the infor ation. In an embodiment 2570, the system further comprises mation related to the processing of the first input and the 65 means for predetermining at least one microorganism strain second input. In an embodiment 2360, the system further or type for altering to produce at least one therapeutic agent comprises means for generating the at least one auxotrophic based on at least one feature of at least one biological tissue. US 8,682,619 B2 93 94 In an embodiment 2580, the at least one feature of the at least As depicted in FIG. 28, in an embodiment 2800, the com one biological tissue includes at least one property of one or puter-implemented method further comprises executing one more microorganism populations associated with the at least or more instructions for evaluating the at least one biological one biological tissue. tissue for one or more indicators relating to one or more of As depicted in FIG. 26, a computer-implemented method administering the at least one treatment device, at least one 2600 comprises 2610 executing one or more instructions component thereof, or at least one product thereof; cell or located on a recordable medium for regulating dispensing of tissue formation; cell or tissue growth; cell or tissue apopto at least one treatment device including at least one semi sis; cell or tissue necrosis; cell division; cellular cytoskeletal permeable barrier Substantially enclosing at least one aux rearrangement; cell or tissue secretion; cell or tissue differ otrophic microorganism, the at least one auxotrophic micro 10 entiation; status of the at least one auxotrophic microorgan ism of the at least one treatment device; status of the at least organism including at least one nucleic acid construct one treatment device; or status of the at least one therapeutic encoding at least one therapeutic agent, the barrier defining an agent. In an embodiment 2810, the computer-implemented interior region and exterior region; wherein the device method further comprises executing one or more instructions includes at least one metabolite required by the at least one 15 for obtaining genetic sequence information from at least one auxotrophic microorganism. In an embodiment 2620, the microorganism isolated from at least one biological tissue. In computer-implemented method further comprises generating an embodiment 2820, the computer-implemented method at least one output to a user readable display. In an embodi further comprises executing one or more instructions for ment 2630, the generating the at least one output includes altering the at least one microorganism isolated from the at generating at least one graphical illustration of one or more of least one biological tissue. In an embodiment 2830, the com the at least one auxotrophic microorganism, at least one com puter-implemented method further comprises executing one ponent thereof, or at least one product thereof, at least one or more instructions for amplifying the at least one microor property of the at least one treatment device; or at least one ganism isolated from the at least one biological tissue. In an property of dispensing the at least one treatment device. In an embodiment 2840, the computer-implemented method fur embodiment 2640, the generating the at least one output 25 ther comprises executing instructions for replacing the at least includes generating at least one protocol for generating the at one microorganism isolated from the at least one biological least one auxotrophic microorganism. In an embodiment tissue subsequent to alteration. In an embodiment 2850, the 2650, the at least one output includes at least one protocol for computer-implemented method further comprises executing administering the at least one treatment device to at least one one or more instructions for predetermining at least one 30 microorganism Strain or type for altering to produce at least biological tissue. In an embodiment 2660, the user includes at one therapeutic agent based on at least one feature of at least least one entity. In an embodiment 2670, the entity includes at one biological tissue. In an embodiment 2860, the at least one least one person, or computer. In an embodiment 2680, the feature of the at least one biological tissue includes at least user readable display includes a human readable display. one property of one or more microorganism populations asso As depicted in FIG. 27, in an embodiment 2700, the user 35 ciated with the at least one biological tissue. readable display includes one or more active displays. In an As depicted in FIG. 29, a computer program product 2900, embodiment 2710, the user readable display includes one or in an embodiment 2910 comprises a recordable medium bear more passive displays. In an embodiment 2720, the user read ing one or more instructions for regulating dispensing of at able display includes one or more of a numeric format, least one treatment device including an semi-permeable bar graphical format, or audio format. In an embodiment 2730, 40 rier structured to Substantially enclose at least one aux the computer-implemented method further comprises execut otrophic microorganism, and wherein the at least one aux ing one or more instructions for evaluating at least one bio otrophic microorganism includes at least one nucleic acid logical tissue for one or more indicators prior to, during, or construct encoding at least one therapeutic agent. In an Subsequent to administering the at least one treatment device embodiment 2920, the recordable medium includes a com to the at least one biological tissue. In an embodiment 2740, 45 puter-readable medium. In an embodiment 2930, the record the evaluating at least one biological tissue for one or more able medium includes a communications medium. In an indicators includes evaluating at least one of an assay, image, embodiment 2940, the computer program product further or gross assessment of the at least one biological tissue prior comprises one or more instructions for evaluating the at least to, during, or Subsequent to administering the at least one one biological tissue for one or more indicators prior to, treatment device to the at least one biological tissue. In an 50 during, or Subsequent to administering the at least one treat embodiment 2750, the assay includes at least one technique ment device. In an embodiment 2950, the computer program including spectroscopy, microscopy, electrochemical detec product further comprises one or more instructions for evalu tion, polynucleotide detection, histological examination, ating the at least one biological tissue for one or more indi biopsy analysis, fluorescence resonance energy transfer, elec cators relating to one or more of administering the at least one tron transfer, enzyme assay, electrical conductivity, isoelec 55 treatment device, at least one component thereof, or at least tric focusing, chromatography, immunoprecipitation, immu one product thereof; cell or tissue formation; cell or tissue noseparation, aptamer binding, filtration, electrophoresis, growth; cell or tissue apoptosis; cell or tissue necrosis; cell immunoassay, or radioactive assay. In an embodiment 2760. division; cellular cytoskeletal rearrangement; cell or tissue the at least one image includes one or more images acquired secretion; cell or tissue differentiation; status of the at least by at least one of laser, holography, X-ray crystallography, 60 one auxotrophic microorganism of the at least one treatment optical coherence tomography, computer-assisted tomogra device; status of the at least one treatment device; or status of phy Scan, computed tomography, magnetic resonance imag the at least one therapeutic agent. In an embodiment 2960, the ing, positron-emission tomography scan, ultrasound, X-ray, first input includes at least one parameter for generating the at electrical-impedance monitoring, microscopy, spectrometry, least one auxotrophic microorganism. In an embodiment flow cytommetry, radioisotope imaging, thermal imaging, 65 2970, the second input includes at least one parameter for infrared visualization, multiphoton calcium-imaging, pho administering the at least one treatment device to at least one tography, or in silico generation. biological tissue. US 8,682,619 B2 95 96 As depicted in FIG. 30, in an embodiment 3000, the output obtained from the calvarial, rib and/or iliac bone of the sub includes at least one protocol for generating the at least one ject. To obtain autologous bone from the iliac crest, a small auxotrophic microorganism. In an embodiment 3010, the incision (4-5 cm) is made just below the anterior iliac wing output includes at least one protocol for administering the at and a 2x2 centimeter area of bone is exposed on top of the least one treatment device to the at least one biological tissue. crest. A rongeur, osteotome or chisel is used to remove a In an embodiment 3020 the at least one output includes at portion of cortical and/or cancellous bone from the top of the least one graphical illustration of one or more of the at least crest. Alternatively, the sample of bone is excised from the one auxotrophic microorganism, at least one component subject using a microsaw (e.g., FRIOS(R) MicroSaw, FRIA thereof, or at least one product thereof; at least one property of DENT GmbH, Mannhein, Germany). The excised bone is the at least one treatment device; or at least one property of 10 optionally cleaned of any associated tissue, fat, and/or blood dispensing the at least one treatment device. In an embodi components. ment 3030, the user includes at least one entity. In an embodi The autologous bone used to form the one or more bone ment 3040, the entity includes at least one person, or com cages is further cut with a bone saw into Smaller pieces (e.g., puter. In an embodiment 3050, the user readable display 5x5x5 millimeter) or to even smaller pieces using a saw includes a human readable display. In an embodiment 3060, 15 microtome, e.g., Leica SP2600 (Leica Microsystems Nuss the user readable display includes one or more active dis loch GmbH, Postfach 1120, Heidelberger Strasse 17-19, plays. In an embodiment 3070, the user readable display D-69226 Nussloch, Germany). The bone is optionally ground includes one or more passive displays. In an embodiment to the desired dimensions using a grinding table and a series 3080, the user readable display includes one or more of a of graded abrasive sand papers (from, e.g., Buehler, Ldt. numeric format, graphical format, or audio format. In an Lake Bluff, Ill.) or using an automated grinding instrument embodiment 3090, the computer program product further (e.g., Exakt Apparatebau GMBH, Norderstedt, Germany). comprises one or more instructions for obtaining genetic Calipers are used to assess thickness (e.g., Mitutoyo digital, sequence information from at least one microorganism iso Kanagawa, Japan). The shape is rectangular, or smoothed to lated from the at least one biological tissue. an oblong, although other shapes may be implemented. An In an embodiment 3100, the computer program product 25 interior cavity is hollowed into a bone cage using a microma further comprises one or more instructions for altering the at chining laser. For example, a Nd:YAG laser rod is used to cut least one microorganism isolated from the at least one bio the interior of the bone leaving an approximately 10 logical tissue. In an embodiment 3110, the computer program micrometer thick bone wall. The bone wall is further perfo product further comprises one or more instructions for ampli rated with multiple pores 1 to 2 micrometer in diameter using fying the at least one microorganism isolated from the at least 30 the laser. A second piece of bone is micromachined and one biological tissue. In an embodiment 3120, the computer shaped to form a bone cap or plug. program product further comprises one or more instructions Alternatively, the bone cage is manufactured from bone for reinstating the at least one microorganism isolated from powder derived from autologous, allogeneic, or Xenogeneic the at least one biological tissue Subsequent to modification. bone obtained by biopsy, Surgery, autopsy, or necropsy. For In an embodiment 3130, the computer program product fur 35 example, an allogeneic bone sample is obtained from a donor ther comprises one or more instructions for predetermining at undergoing hip replacement. In this instance, the head of the least one microorganism type for altering to produce at least femur is removed, Substantially cleaned of associated tissue, one therapeutic agent based on at least one feature of the at fat, and blood components and cut into Smaller pieces (e.g., least one biological tissue. In an embodiment 3140, the at 1x1 x1 centimeter) using a bone saw. Any residual blood least one feature of the at least one biological tissue includes 40 components are removed from the bone pieces by soaking in at least one property of one or more microorganism popula purified water for 12 to 24 hours. Fat and protein material are tions associated with the at least one biological tissue. substantially removed from the bone pieces by boiling in As one of skill in the art would appreciate, a computing purified water for 12 to 72 hours. The bone pieces are pulver device as described herein includes any instrument contain ized into a powder using a bone mill (from, e.g., Aesculap Inc. ing a processor to execute instructions, for example a phone, 45 USA, Center Valley, Pa.; Medtronic, Minneapolis, Minn.). personal digital assistant, remote-controller, desktop com Residual fatty material is removed from the bone powder by puter, workstation computer, or computing system. extracting the powder in an organic solvent mixture, e.g., chloroform and methanol (1:1 V/v), for 24 hours with or Prophetic Example 1 without agitation. The bone powder is rinsed repeatedly with 50 purified water to remove the organic solvent. The washed Device Including Bone Cage Formed from Cortical bone powder is dried in an oven at 60° C. Residual protein Bone and/or Cancellous Bone material is removed by soaking the bone powder in a 4% (w/v) solution of sodium hypochlorite for 12 to 72 hours with A device including one or more bone cages configured to or without agitation. The bone powder is rinsed repeatedly include one or more immunogens and one or more adjuvants 55 with purified water to remove the sodium hypochlorite and is delivered into a soft tissue of a subject for use as a vaccine dried in an oven at 60° C. The bone powder is further milled againstapathogen, cancer, or tumor in the Subject. The device to generate a finer bone powder. The bone powder is sieved includes a bone cage formed from autologous, allogeneic, or through a series of sieves of various mesh size openings, e.g., Xenogeneic cortical bone and/or cancellous bone. The one or 10-200 micrometers, 200-425 micrometers, 425-1000 more immunogens is delivered as a live attenuated or an 60 micrometers, 1000-2000 micrometers, etc. The bone powder inactivated viral particle. The one or more adjuvants include is heated at 600° C. for 3-5 hours to remove any residual complete Freund's adjuvant. organic material and to sterilize the powder. A similar process The device including one or more bone cages utilizes bone is used to generate milled bone from a Xenogeneic donor. obtained from the Subject (autologous) or from a donor Sub The bone powder is compressed using compression mold ject (allogeneic) by biopsy, Surgery, or autopsy. Alternatively, 65 ing techniques into appropriate building blocks for forming bone is obtained from a non-human Subject (Xenogeneic), for the bone cages. Alternatively, the bone powder is admixed example, from bovine orporcine Subjects. Autologous bone is with a biocompatible substance to form a paste that is molded US 8,682,619 B2 97 98 into appropriate cage forms. Examples of appropriate bio phate as described by Barberi, et al., PLoS Med. 2:e161, 2005, compatible Substances for this purpose include fibrinogen, which is incorporated herein by reference. Various assays are MATRIGELTTM basement membrane matrix, gelatin, algi used to assess osteoblast differentiation, e.g., expression of nate, polyglycolide, polylactide, glycolide-lactide copoly collagen, osteopontin, osteonectin, osteocalcin, and bonesia mer, etc. In some instances, heating the formed cages poly loprotein, alkaline phosphatase assay, and mineralization merizes the biocompatible material and forms a rigid cage assay. like structure. Scaffolds for use in bone tissue engineering to form one or The bone cage is molded to include pores sufficiently large more bone cages include synthetic polymers (e.g., polylactic enough for passive diffusion of live/attenuated or inactivated acid and polyglycolic acid and copolymers, thereof, polyca viral particles including the one or more immunogens and/or 10 prolactone, polyanhydrides, and polypohsphaZenes; polym the one or more adjuvants, e.g., complete Freund's adjuvant. ethylmethacrylate, polytetrafluoroethylene, polyhydroxyeth In general, viral particles range in size from 10 to 300 nanom eters. The complete Freund's adjuvant is bound ionically or ylmethacrylate); ceramics (e.g., alumina, hydroxyapatite); covalently to the Surface of the bone cage structure. native polymers (e.g., Type I collagen); and composites The live/attenuated or inactivated viral particles including 15 thereof. Growth conditions include nutrients, growth factors, the immunogen and adjuvant, e.g., complete Freund's adju appropriate temperature, and oxygenation. vant, are injected into the one or more cavities of the bone Bone tissue configured to form one or more bone cages is cage. The one or more cavities include micro-holes large engineered using bone marrow-derived stem cells grown in enough to allow diffusion of the viral particles. The number of three-dimensional Superporous poly(ethylene glycol)diacry viral particles added to each bone cage is dependent upon the late (PEGDA) hydrogels. See, e.g., Keskar, et al., Abstract, size of the one or more bone cage cavities, the number of bone Tissue Eng. Part A 15, 1695-707 (2009), which is incorpo cages to be used for immunization, and the expression effi rated herein by reference. PEGDA is combined with a foam ciency of the genetically engineered cells. For example, a stabilizer and initiators N.N.N',N'-tetramethylethylene-di bone cage with an internal cavity measuring 1x1 x0.3 centi amine and ammonium persulfate and the pH adjusted to pH meters accommodates a volume of 300 microliters of viral 25 3.7 with citric acid. The mixture is heated at 37° C. for 2 particles. Assuming a maximum packing density of 0.524 and minutes. Sodium bicarbonate is added to the mixture with a particle diameter of 300 nanometers, as many as 4x10 constant stirring to evenly distribute the salt and evolving gas. densely packed viral particles are incorporated into the bone Polymerization is allowed to proceed for 30 minutes. cage cavity. The viral particles are Suspended within a hydro Residual monomers are removed with rinsing in distilled gel at this density or at a lower density and incorporated into 30 water. The hydrated hydrogel is cut into appropriate sized the bone cage cavity. The cavity is plugged with an additional pieces (e.g., 5x3 millimeter) and seeded with mesencymal bone fragment or with another material to immunoisolate the cells derived from bone marrow aspirates. The cells seeded on viral particles. One or more of the bone cages are implanted the hydrogel are grown in an osteogenic medium containing into a subject. The viral particles diffuse out of the bone cage Dulbecco's modified Eagle's medium (DMEM), fetal bovine and into the Surrounding tissue of the Subject. 35 serum, penicillin/streptomycin, dexamethasone, (3-glycero Prophetic Example 2 phosphate, and ascorbic acid 2-phosphate. After 3-7 weeks in culture, gross calcification is observed in the form of a white Device Including Bone Cage Formed by Growing deposit on and within the hydrogel cell culture system. The Bone on a Scaffold 40 bone-like material is further processed using machining tech niques as described above to form a cage-like structure. In a A device including one or more bone cages configured to further aspect, the hydrated hydrogel is shaped into a cage include one or more immunogens and one or more adjuvants like structure and seeded with mesenchymal cells to form the is delivered into a soft tissue of a subject for use as a vaccine one or more bone cages. againstapathogen, cancer, or tumor in the Subject. The device 45 Alternatively, the bone cage is derived from bone tissue includes a bone cage formed from growing bone progenitor generated in the absence of an underlying exogenous scaf cells on an appropriate scaffold. Bone is formed by growing fold. Bone marrow cells are collected from the pelvic bone bone progenitor cells on a scaffold in the presence of appro from the Subject or from an allogeneic donor using standard priate growth conditions. Bone progenitor cells are differen medical procedures. The collected cells are grown in Dulbec tiated into osteoblasts for formation of bone and osteoclasts 50 co's modified eagle medium (DMEM) supplemented with for resorption of bone. Bone-forming osteoblasts are differentiated from preo 20% fetal bovine serum, basic fibroblast growth factor steoblastic cells such as, for example, MC3T3 cell line, pri (bFGF), dexamethasone, and antibiotics/antimycotics. After mary cells isolated from bone, mesenchymal stem cells iso 48 hours, non-adherent cells are removed. The cells are trans lated from bone marrow, adipose tissue, placenta, cartilage, 55 ferred to dishes containing SYLGARDR polymer coated and other tissues. Duplomb, et al., Stem Cell 25:544-552, with laminin and grown in the Supplemented medium 2007, which is incorporated herein by reference. Mesenchy described above. After the cells reach confluence, two pins mal stem cells, for example, are isolated from bone marrow are placed in the SYLGARDR polymer about 1.5 centimeters by Percoll density gradient centrifugation and differentiated apart and ascorbic acid and organic phosphate are added to into osteoblasts using a basal culture medium Supplemented 60 the medium. Over the course of 6 weeks, cells lay down with fetal bovine serum (FBS), L-glutamine, penicillin/strep extracellular matrix, differentiate and begin to form a tube tomycin, dexamethasone, ascorbate, and B-glycerophos like solid structure that exhibits bone-like qualities including phate. Ogura, et al., J. Oral Sci. 46:207-213, 2004, which is calcification and increased stiffness, see e.g., Syed-Picard, et incorporated herein by reference. Alternatively, mesenchy al., Tissue Eng. Part A 15:187-195, 2009, which is incorpo mal stem cells are derived from embryonic stem cells and 65 rated herein by reference. Bone-like structures formed in this differentiated into osteoblast-like cells using medium Supple manner are further processed as described above to form one mented with dexamethasone, ascorbate, and B-glycerophos or more bone cages. US 8,682,619 B2 99 100 Prophetic Example 3 Aldrich, St. Louis, Mo.). Alternatively, a vector using an adenovirus expression system or other promoter or viral Genetically Engineered Cells Generating expression systems is used (see, e.g., Promega, Madison, Immunogen/Adjuvant for Device Including Bone Wis.; Clontech Laboratories, Inc., Mountain View, Calif.; Cage Invitrogen, Carlsbad, Calif.). Alternatively, the cDNA for CEA and/or IL-2 is obtained from a commercial source, in a A device including one or more bone cages and configured mammalian expression vector expressed under control of a to include one or more immunogens and one or more adju CMV promoter (see, e.g., Origene, Rockville, Md.). vants is delivered into a soft tissue of a subject for use as a vaccine against cancer or a tumor in the Subject. The device 10 In some instances, the cDNA corresponding to CEA and to includes genetically engineered cells expressing one or more IL-2 are incorporated into the same expression vector and immunogens and/or one or more adjuvants for use as a vac transfected into the mammalian cell line. Alternatively, the cine against a cancer or tumor. The device includes a bone cDNA corresponding to CEA and to IL-2 are incorporated cage formed from synthetic or artificial materials, from into distinct expression vectors. The two expression vectors excised bone, or from in vitro tissue engineering. In this 15 are transfected simultaneously into the same cells. In this instance, the genetically engineered cells are encapsulated by instance, a single genetically engineered cell line is generated the bone cage. The bone cage itself has pores that allow that expresses both CEA and IL-2. Alternatively, the two passage of the immunogen and the adjuvant produced by the expression vectors are transfected into separate cultures of the encapsulated cells, but the pores are Small enough to prevent same or differing mammalian cells. For example, the CEA passage of the genetically engineered cells. expression vector is transfected into one culture of Chinese The immunogen and the adjuvant include any of a number hamster ovary (CHO) cells while the IL-2 expression vector is of tumor specific antigens. For example, the tumor is colorec transfected into another culture of CHO cells, creating two tal carcinoma. The device including one or more bone cages distinct genetically engineered cell lines. includes the immunogen, carcinoembryonic antigen (CEA) or polypeptide epitopes thereof, and the adjuvant, the immu 25 Prior to incorporating the genetically engineered cells into nomodulator interleukin 2 (IL-2), or interleukin 21 (IL-21). the bone cages, the relative expression of CEA and IL-2 by the Shievely, et al., Crit. Rev. Oncol Hematol. 2: 355-399, 1985; cells is assessed using any of a number of assay systems. The Frederiksen et al., Cancer Immunol. Immunother. 57: 1439 expression of messenger RNA (mRNA) corresponding to 1449, 2008; Parrish-Novak et al., Journal of Leukocyte Biol CEA and IL-2 is assessed by quantitative PCR, Northern ogy; 72:856-863, 2002: Lamprecht et al., Blood 112: 3339 30 analysis, in situ hybridization, or other methods designed to 3347, 2008, which are incorporated herein by reference. In a assess the presence and/or quantity of a specific mRNA in a further example, the cancer is metastatic melanoma. The cell. The expression of CEA and IL-2 in the cells is assessed device including one or more bone cages includes the immu by Western analysis, immunocytochemistry, or other meth nogen, one or more of the tumor antigens or polypeptide ods designed to assess the presence and/or quantity of a epitopes thereof: Melan-A/MART-1, MAGE-3, Gp 100/pmel 35 specific protein in a cell. The secretion of CEA and IL-2 out 17, Tyrosinase, TRP-1/-2, P. polypeptide, MC1R, B-catenin, of the genetically engineered cell and into the culture medium or MART-2. Kawakami, et al., J Exp Med 180:347-52, 1994: is assayed using an immunoassay system with immu Bakker, et al., J Exp Med 179: 1005-9, 1994; Brichard, et al., noreagents specific for CEA and IL-2. In an example assay, J Exp Med 178: 489-95, 1993; Kawakami, et al., J Immu the Supernatant from the genetically engineered mammalian nother 21:237-46, 1998: Wang, et al., J Exp Med 184: 2207 40 cells is collected and Subjected to analysis by enzyme-linked 16, 1996; Touloukian, et al., Cancer Res 61: 8100-4, 2001; Salazar-Onfray, et al., Cancer Res 57: 4348-55, 1997: Alex immunosorbent assays (ELISAs) specific for CEA and IL-2 ander, et al., Abstract, Urology 51: 150-7, 1998; Robbins, et (from, e.g., Signosis, Inc., Sunnyvale, Calif.). al., J Exp Med 183: 1185-92, 1996, which are incorporated The genetically engineered cells expressing the CEA herein by reference. The adjuvant in combination with the 45 immunogen and IL-2, or the metastatic melanoma immuno melanoma tumor antigen is the immunomodulator interleu gen and IL-21, are injected into the one or more cavities of the kin 21 (IL-21), interleukin 12 (IL-12), or incomplete Fre bone cage. The one or more cavities include micro-holes large unds adjuvant. Frederiksen et al., Cancer Immunol. Immu enough to allow diffusion of the immunogen and adjuvant but nother. 57: 1439-1449, 2008; Gajewski, et al., Clin Cancer Small enough to prevent release of the genetically engineered Res. 7(3 Suppl): 895s-901s, 2001, which are incorporated 50 cells. The number of cells added to each bone cage is depen herein by reference dent upon the size of the one or more bone cage cavities, the Immunization with CEA and IL-2 is used to induce an number of bone cages to be used for immunization, and the immune response against CEA-expressing colorectal carci expression efficiency of the genetically engineered cells. For noma cells. Immunization with metastatic melanoma tumor example, a bone cage with an internal cavity measuring 1x1 x antigen and IL-21 is used to induce an immune response 55 against metastatic melanoma cells. Standard molecular biol 0.3 centimeters accommodates a volume of 300 microliters of ogy techniques are used to express the CEA and IL-2 in a cells. Assuming a maximum packing density of 0.524 and a mammalian cell line. The cDNA sequences corresponding to cell diameter of 15 micrometers, as many as 80 million CEA and IL-2 are generated using standard polymerase chain densely packed cells are incorporated into the bone cage reaction (PCR) amplification and an appropriate cDNA 60 cavity. The cavity is plugged with an additional bone frag library or reverse-transcribed mRNA with primers designed ment or with another material to immunoisolate the geneti based on the known cDNA sequence of CEA and IL-2 from, cally engineered cells. One or more of the bone cages are e.g., the GenBank Database. Benson, et al., Nucleic Acids implanted into a subject. The genetically engineered cells are Res. 36:D25-30, 2008, which is incorporated herein by ref retained within the confines of the implanted bone cage, while erence. All or a portion of the cDNA sequences corresponding 65 the expressed CEA and IL-2 are secreted from the genetically to CEA and IL-2 are cloned into expression vectors contain engineered cells and diffuse out of the bone cage and into the ing the cytomegalovirus (CMV) promoter (see, e.g., Sigma Surrounding tissue of the Subject.