(19) &   

(11) EP 2 377 401 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 19.10.2011 Bulletin 2011/42 A01N 63/00 (2006.01) A01N 65/00 (2009.01) C12N 5/00 (2006.01) C12N 5/02 (2006.01) (2010.01) (21) Application number: 11174205.2 C12N 5/071

(22) Date of filing: 29.04.2004

(84) Designated Contracting States: • Bukhman, Mordechay AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 21891 Carmiel (IL) HU IE IT LI LU MC NL PL PT RO SE SI SK TR • Stern, Baruch, S. Designated Extension States: 34366 Haifa (IL) AL HR LT LV MK • Shalhevet, David 36090 Kiryat Tivon (IL) (30) Priority: 01.05.2003 US 466793 P • Shavitt, Menachem, D. 06.08.2003 US 492754 P 20142 D.N. Misgav (IL) • Pearlman, Andrew, L. (62) Document number(s) of the earlier application(s) in 20164 D.N. Miscav (IL) accordance with Art. 76 EPC: • Noam, Shani 04760621.5 / 1 653 807 30900 Zichron Yaakov (IL) • Almon, Einat (71) Applicant: Medgenics, Inc. 23840 Timrat (IL) Palo Alto, CA 94303 (US) (74) Representative: Modiano, Micaela Nadia (72) Inventors: Modiano & Partners • Bellomo, Stephen, F. Thierschstrasse 11 30900 Zichron Yaakov (IL) 80538 München (DE) • Lippin, Itzhak 42920 Moshav Beit Yitzhak (IL) Remarks: • Piva, Guillermo, Alberto This application was filed on 15-07-2011 as a Winston Salem, NC North Carolina 27104 (US) divisional application to the application mentioned • Rosenberg, Lior under INID code 62. 84965 Omer (IL)

(54) A genetically modified dermal micro-organ expressing erythropoietin

(57) The present invention is directed to a genetically cro-organ and diffusion of cellular waste out of said cells modified dermal micro-organ expressing at least one re- so as to minimize cellular toxicity and concomitant death combinant gene product, wherein said dermal micro-or- due to insufficient nutrition and accumulation of waste in gan consists essentially of a plurality of dermal compo- said dermal micro organ, wherein at least some of said nents and does not include epidermal layers, which main- cells of said genetically modified dermal micro-organ ex- tains the micro- architecture and three dimensional struc- press at least a portion of at least one recombinant gene ture of the dermal tissue from which it is derived, having product, wherein said recombinant gene product is eryth- dimensions selected so as to enable passive diffusion of ropoietin. adequate nutrients and gases to cells of said dermal mi- EP 2 377 401 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 377 401 A1 2

Description augmentation procedures. To operate this device, which is not commonly used, a sharpened cutting tube, which FIELD OF THE INVENTION includes a reusable thick walled tube with an inner diam- eter of approximately 4.5 mm, is manually rotated at a [0001] The invention relates to the field of tissue based 5 very slow speed. Using this type of device generally re- micro organs, therapeutic tissue based micro organs and quires applying pressure to the skin surface directly methods and apparatuses for harvesting, processing, im- above the harvest site and installing sutures with active planting and manipulating dermal tissue. tugging as the cutting tube is pushed forward. Further- more, the resulting harvested dermis is generally not uni- BACKGROUND OF THE INVENTION 10 form in dimensions and includes "plugs" of epidermis at either end of the dermal core. [0002] Various methods for delivering therapeutic agents are known. For example, therapeutic agents can SUMMARY OF THE INVENTION be delivered orally, transdermally, by inhalation, by in- jection and by depot with slow release. In each of these 15 [0007] Embodiments of some aspects of the present cases the method of delivery is limited by the body proc- invention provide a DMO/DTMO with the ability to be esses that the agent is subjected to, by the requirement maintained ex-vivo in a generally viable state, which may for frequent administration, and limitations on the size of allow various manipulations to be performedon the DMO, molecules that can be utilized. For some of the methods, while keeping a high production and secretion level of the amount of therapeutic agent varies between admin- 20 the desired therapeutic agent. In addition, embodiments istrations. of some aspects of the present invention provide a meth- [0003] A dermal micro organ (DMO), which can be sus- od of harvesting a DMO and subsequently implanting a tained outside the body ("ex-vivo" or "in-vitro") in an au- DTMO without forming keratin cysts or keratin micro- tonomously functional state for an extended periods of cysts, e.g., upon implantation of the DTMO subcutane- time, and to which various manipulations can be applied, 25 ously or deeper in the body. Furthermore, it will be ap- may then be implanted subcutaneously or within the body preciated by persons skilled in the art that the methods for the purpose of treating diseases, or disorders, or for and devices according to some embodiments of the plastic surgical purposes. The DMO can be modified to present invention may be relatively uncomplicated and, express a gene product of interest. These modified der- therefore, the level of skill required from a professional mal micro organs are generally referred to as Dermal 30 to carry out the methods and/or to use the devices of the Therapeutic Micro Organs (DTMOs). present invention may not be as demanding as those [0004] Skin micro organs, including layers of epider- required in conventional procedures. mal and dermal tissues, for example; as outlined in [0008] Some exemplary embodiments of the invention PCT/IL02/0880, have been observed to be associated provide a dermal micro organ (DMO) having a plurality with a number of clinical challenges. Harvesting of a skin 35 of dermal components, which may include cells of the sample leaves a superficial wound on the patient that dermal tissue and a surrounding matrix. The DMO ac- may last several weeks and may leave scars. The har- cording to embodiments of the invention may generally vested skin sample requires significant processing to retain a micro-architecture and three dimensional struc- generate micro organs from this sample. Also, implanta- ture of the dermal organ from which it is obtained and tion of skin micro organs subcutaneously or deeper in 40 the dimensions of the DMO may allow passive diffusion the body have been found to result in the development of adequate nutrients and gases to the cells and diffusion of keratin cysts or keratin micro-cysts. Additionally, im- of cellular waste out of the cells so as to minimize cellular plantation of skin micro organs as a graft onto the skin toxicity and concomitant death-due to insufficient nutri- surface in "slits" requires significant technical expertise tion and accumulation of waste. in order to handle the MO while maintaining its proper 45 [0009] In some exemplary embodiments of the inven- orientation. tion, the dermal micro organ of the invention does not [0005] Harvesting of dermis, e.g., to be used as a "filler produce keratin or produces negligible amounts of ker- material" in a plastic surgical or cosmetic procedure, is atin. known in the art. Conventional harvesting techniques in- [0010] In some embodiments of the invention, the der- clude using a dermatome or scalpel to peel away a layer 50 mal micro organ does not produce keratin and/or keratin of epidermis in order to expose a section of dermis. The cysts following subcutaneous or deeper implantation in dermatome or scalpel may then be used again to man- a body. ually harvest the exposed section of dermis. [0011] Inanother embodiment of the invention, the der- [0006] Another conventional apparatus for harvesting mal micro organ of the invention produces micro keratin dermis, albeit not commonly used, is the Martin Dermal 55 cysts following that will atrophy within a relatively short Harvester marketed by Padgett (Part No. P-225) for the period of time, e.g., days or weeks after subcutaneous indication of harvesting dermal cores from the back for implantation. subsequent implantation into the lips during cosmetic lip [0012] Inanother embodiment of the invention, the der-

2 3 EP 2 377 401 A1 4 mal micro organ of the invention contains hair follicles netically modified dermal micro organ of the invention and sebaceous glands, which will atrophy after a short produces substantially no keratin. period of time, e.g., days or weeks. [0018] In some embodiments, the invention provides [0013] Inanother embodiment of the invention, the der- a method of delivering to a recipient a recombinant gene mal micro organ of the invention contains glands that will 5 product produced by the dermal micro organ. connect to the skin surface after a short period of time, [0019] In some embodiments, the invention provides e,g., days or weeks. a method of inducing a local or systemic physiological [0014] Further exemplary embodiments of the inven- effect by implanting a dermal micro organ in a recipient. tion provide a method and apparatus of harvesting a der- [0020] In another embodiment the invention provides mal micro organ. The method may include stabilizing 10 a method of delivering a protein of interest to a subject. and/or supporting a skin-related tissue structure from The method includes implanting the genetically modified which a dermal micro organ is to be harvested, e.g., such dermal micro organ into the skin, under the skin or at that the skin-related tissue structure is maintained at a other locations in the body. desired shape and/or position, separating at least a por- [0021] In another embodiment, the invention provides tion of the dermal micro organ from the skin- related tissue 15 a method of implanting a dermal micro organ so as to structure, and isolating the separated dermal micro organ avoid or to reduce keratin cyst formation. from the body. According to some of these exemplary embodiments, the support configuration may include a BRIEF DESCRIPTION OF DRAWINGS first tubular element, and the cutting tool may include a second tubular element adapted to be inserted along and 20 [0022] Non-limiting embodiments of the invention are substantially coaxially with the first element. According described in the following description, to be read with to other exemplary embodiments, the support configura- reference to the figures attached hereto. In the figures, tion may include a vacuum chamber having an inner sup- identical and similar structures, elements or parts thereof port surface able to maintain the skin- related tissue struc- that appear in more than one figure are generally labeled ture at a desired shape and/or position to enable the cut- 25 with the same or similar references in the figures in which ting tool to separate the DMO from the skin- related tissue they appear. Dimensions of components and features structure. shown in the figures arechosen primarilyfor convenience [0015] Further exemplary embodiments of the inven- and clarity of presentation and are not necessarily to tion provide a genetically modified dermal micro organ scale. expressing at least one recombinant gene product the 30 [0023] Figure 1 is a schematic block diagram of an dermal micro organ having a plurality of dermal compo- exemplary method of producing and utilizing dermal ther- nents, including cells and matrix of the dermal tissue, apeutic micro organs (DTMOs), in accordance with an which retain the micro- architecture and three dimension- exemplary embodiment of the invention; al structure of the dermal tissue from which they are ob- [0024] Figures 2A and 2B show, respectively, a corre- tained, and having dimensions selected so as to allow 35 lation analysis between in-vitro secretion of pre- implant- passive diffusion of adequate nutrients and gases to the ed mIFNα-TMOs and hEPO-TMOs and the serum in- cells and diffusion of cellular waste out of the cells so as vivo levels following their implantation, in accordance to minimize cellular toxicity and concomitant death due with an embodiment of the invention, toinsufficient nutrition and accumulation of waste, where- [0025] Figures 3A and 3B show, respectively, elevated in at least some of the cells of the dermal micro organ 40 serum hEPO levels determined by an ELISA assay and express at least one recombinant gene product or at least reticulocyte count elevation after autologous TMO im- a portion of said at least one recombinant gene product. plantation in a miniature swine, in accordance with an [0016] Yet further exemplary embodiments the inven- embodiment of the invention; tion provide a genetically modified dermal micro organ [0026] Figure 4 is a schematic illustration of a graph expressing at least one recombinant protein, the dermal 45 showing Secretion levels of human erythropoietin micro organ having a plurality of dermal components, (hEPO) by DTMO-hEPO prepared from six different hu- including cells and matrix of the dermal tissue, which re- man skins; tain the micro-architecture and three dimensional struc- [0027] Figure 5: shows histology of DTMO and split ture of the dermal tissue from which they are obtained, thickness skin TMO; and having dimensions selected so as to allow passive 50 [0028] Figure 6 shows ihnmunohistochemistry (IHC) diffusion of adequate nutrients and gases to the cells and and Hematoxylin & Eosin (H&E) staining of DTMO; diffusion of cellular waste out of then cells so as to min- [0029] Figure 7 demonstartes In vivo hEPO serum lev- imize cellular toxicity and concomitant death due to in- els and physiological effect on hematocrit levels following sufficient nutrition and accumulation of waste, wherein subcutaneous implantation of DTMO-hEPO and split at least some of the cells of the dermal micro organ ex- 55 thickness skin TMO-hEPO in SCID mice; press at least a portion of at least one recombinant pro- [0030] Figure 8 demonstrates clinical and histological tein. analysis of DTMO- hEPO and split thickness skin TMO- [0017] In some embodiments of the invention, the ge- hEPO implanted subcutaneously in SCID mice;

3 5 EP 2 377 401 A1 6

[0031] Figure 9 shows hHistological analysis of skin in the art, and the general principles defined herein may MOs grafted in skin slits (split thickness skin MO, right) be applied to other embodiments. Therefore, the present or implanted S.C. (DMO, Left) 17 days post implantation invention is not intended to be limited to the particular in healthy volunteers; embodiments shown and described, but is to be accord- [0032] Fig. 10 is a schematic flowchart illustrating a 5 ed the widest scope consistent with the principles and method of harvesting a DMO according to some exem- novel features herein disclosed. In other instances, well- plary embodiments of the invention; known methods, procedures, and components have not [0033] Figs. 11a-11c are schematic illustrations of ex- been described in detail so as not to obscure the present emplary stages of harvesting a DM0 in accordance with invention. the method of Fig. 10; 10 [0046] In the following detailed description, numerous [0034] Fig. 12 is a schematic illustration of a clamping specific details are set forth in order to provide a thorough tool that may be used by a dermal harvesting apparatus understanding of the present invention. However, it will in accordance with some exemplary embodiments of the be understood by those skilled in the art that the present invention; invention may be practiced without these specific details. [0035] Fig. 13 is a schematic illustration of a dermal 15 harvesting apparatus including a coring tube inserted into EXEMPLARY DEFINITIONS OF TERMS USED HERE- source tissue for a DMO, and harvesting coaxially with IN an inner guide needle in accordance with some exem- plary embodiments of the invention; [0047] The term "explant" as used herein, refers in [0036] Figs, 14a-14c are schematic illustrations of a 20 some embodiments of the invention, to a removed sec- front view, a side view, and top view, respectively, of a tion of living tissue or organ from one or more tissues or dermal vacuum harvesting apparatus according to an ex- organs of a subject. emplary embodiment of the invention; [0048] The term "dermal micro organ" or "DMO" as [0037] Fig. 15 is a schematic illustration of a cross- used herein, refers in some embodiments of the inven- sectional side view of the apparatus of Figs 14a- 14c sup- 25 tion, to an isolated tissue or organ structure derived from porting a dermal micro organ at a desired position ac- or identical to an explant that has been prepared in a cording to one exemplary embodiment of the invention; manner conducive to cell viability and function, while [0038] Fig. 16 is a schematic illustration of across- sec- maintaining at least some in vivo interaction similar to tional view of the apparatus of Fig. 15 externally support- the tissues or organ from which it is obtained. Dermal ing a dermal micro organ to be harvested at a desired 30 micro organs may include plurality of dermal components position; that retain the micro-architecture of the tissue or organ [0039] Fig. 17 is a schematic illustration of a dermal from which they were derived, and three dimensional harvesting apparatus according to another exemplary structure of the dermal tissue from which they are de- embodiment of the invention; rived, heaving dimensions selected so as to allow pas- [0040] Fig. 18 is a schematic illustration of a harvesting 35 sive diffusion of adequate nutrients and gases to cells apparatus according to yet another exemplary embodi- within the MO and diffusion of cellular waste out of the ment of the invention; cells of the MO so as to minimize cellular toxicity and [0041] Fig. 19 is a schematic illustration of implement- concomitant death due to insufficient nutrition and accu- ing the harvesting apparatus of Fig. 18 for harvesting a mulation, of waste. Dermal micro organs may consist es- DMO; 40 sentially of a plurality of dermis components (tissue com- [0042] Fig. 20 is a flow chart illustrating a DTMO im- ponents of the skin located below the epidermis). These planting method, according to some embodiments of the components may contain skin fibroblast, epithelial cells, present invention; other cell types, bases of hair follicles, nerve endings, [0043] Fig. 21 is a flow chart illustrating a DTMO ab- sweat and sebaceous glands, and blood and lymph ves- lating method, according to some embodiments of the 45 sels. Wherever used hereinbelow, the description of the present invention; and embodiments related to MO relates also to dermal MO [0044] Fig. 22 is a schematic illustration of a system Whenever the term "dermal tissue" is used, it also relates for processing a harvested DMO according to exemplary to "dermal organ". embodiments of the invention. [0049] As used herein, the term "microarchitecture" re- 50 fers, in some embodiments of the invention, to the char- DETAILED DESCRIPTION OF EXEMPLARY EMBOD- acteristic of the explant in which, in one embodiment at IMENTS least about 50%, in another embodiment, at least about 60%, in another embodiment at least about 70%, in an- [0045] The following description is presented to enable other embodiment, at least about 80 %, and in another one of ordinary skill in the art to make and use the inven- 55 embodiment, at least about 90% or more of the cells of tion as provided in the context of a particular application the population, maintain, in vitro, their physical and/or and its requirements. Various modifications to the de- functional contact with at least one cell or non-cellular scribed embodiments will be apparent to those with skill substance with which they were in physical and/or func-

4 7 EP 2 377 401 A1 8 tional contact in vivo. Preferably, the cells of the explant sweat glands and/or sebaceous glands and/or blood and maintain at least one biological activity of the organ or lymph vessels and/or elastin fibers and/or collagen fibers tissue from which they are isolated. and/or endothelial components and/or immune system [0050] The term "donor" as used herein, refers in some derived cells and/or extra-cellular matrix. As shown by embodiments of the invention to a subject, from which 5 the test results summarised in the Examples section be- the explant is removed and used to form, or which is low (Example 5, Figure 8), conventional subcutaneous already in the form of, one or more micro organs. implantation of a micro organ including epidermal layers [0051] The term "therapeutic micro organ (TMO)" as ("split thickness skin MO") in mice and pigs (data in pigs used herein, refers in some embodiments of the invention is not shown), may result in formation of keratin cysts or to a micro organ (IVID) that can be used to facilitate a 10 macro-keratin cysts. In contrast, when skin tissue is sam- therapeutic objective, such as, for example, an MO that pled to obtain a DMO according to exemplary embodi- has been genetically altered or modified to produce a ments of the invention, no cysts or macro cysts are ob- therapeutic agent, such as a protein or and RNA mole- served in mice, pigs or in humans. It should be noted that cule. The therapeutic agent may or may not be a naturally the biological activity (for example secretion of a thera- occurring body substance. Wherever used hereinbelow, 15 peutic protein, e.g., erythropoietin and elevation of he- the description of the embodiments related to TMO re- matocrit as a result) of a DTMO according to embodi- lates also to DTMO which is a therapeutic Dermal MO ments of the invention may be comparable to or even which may be in some embodiments of the invention ge- higher than split thickness skin derived TMO (see Exam- netically modified. ple 4). Namely, both types of preparation may release [0052] The term "implantation" as used herein, refers 20 the same amount of erythropoietin; however, the DTMO in some embodiments of the invention, to introduction of may produce and secrete higher protein levels per unit one or more TMOs or DTMOs into a recipient, wherein than those of split thickness derived TMO. said TMOs or DTMOs may be derived from tissues of [0059] In general, production of DTMOs may include the recipient or from tissues of another individual or an- DMO harvesting, maintaining the DMO and/or modifying imal. The TMOs or DTMOs can be implanted in a slit25 the DMO and/or genetically altering them and, in some within the skin, by subcutaneous implantation, or by embodiments, verifying the production of a desired agent placement at other desired sites within the recipient body. (for example proteins) by the DMO. Utilization of the DT- [0053] The term "recipient" as used herein refers, in MO may include production, within a patient’s or animal’s some embodiments of the invention, to a subject, into own body, of therapeutic substance, such as proteins, which one or more TMOs or DTMOs are implanted. 30 for treatment of a subject. For example, the DTMO can [0054] The term "clamping" (e.g., the skin) as used be implanted into or under the skin or within the body of herein may refer to any similar action or any action with the subject to produce the agent/protein in vivo. In the a similar purpose, for example, "pinching" (e.g., the skin). case of tissue from another subject, the implant is op- [0055] The term "in vitro" as used herein should be tionally protected from reaction by the recipient’s immune understood to include "ex-vivo". 35 system, for example, by housing the DTMO in an immu- [0056] The term "coring tube" as used herein may re- noprotective capsule or sheath. For example, a mem- late, individually or collectively, to the terms "cutting tool", brane can be positioned to surround the DTMO, either "cutting tube" and "coring needle", as well as to any other by placing the DTMO in a capsule prior to implantation elements with similar functionalities. or otherwise. The membrane should have a pore size [0057] While, for clarity and completeness of presen- 40 that is sufficiently large to allow for the passage of nutri- tation, all aspects of the production and utilization of DT- ents, waste and the therapeutic agent yet sufficiently MOs are described in this document, and embodiments small to prevent passage of cells of the immune system. of the invention are described from the start of the proc- [0060] In some embodiments of the invention, the der- esses to their ends, it should be understood that each of mal micro organ may contain tissue of a basal epidermal the aspects described herein can be used with other45 layer and, optionally, other epidermal layers of the skin. methodologies and/or equipment for the carrying out of In other embodiments, the dermal micro organ does not other aspects and can be used for other purposes, some include basal layer tissue. of which are described herein. The present invention in- [0061] In some embodiments of the invention, the cludes portions devoted to the preparation and mainte- DMO does not include epidermal layers. In other embod- nance of dermal micro organs for transformation into DT- 50 iments, the DMO may contain a few layers of epidermal MO. It should be understood that the dermal micro organs tissue. produced according to these aspects of the invention can [0062] In one embodiment of the invention, the DMO be used for purposes other than for transformation into includes the entire cross-section of the dermis. In another DTMOs embodiment of the invention, the dermal micro organ in- [0058] In some embodiments of the invention, the mi- 55 cludes part of the cross- section of the dermis. In a further cro organ is a dermal micro organ including a plurality of embodiment, the DMO includes most of the cross section dermis components, for example, fibroblasts and/or ep- ofthe dermis,namely, most of thelayers andcomponents ithelial components containing nerve endings and/or of the dermis including the papillary and reticular dermis.

5 9 EP 2 377 401 A1 10

In a further embodiment, the DMO includes primarily der- be, for example, about 60-100 mm in length. In another mal tissue, but may also include fat tissue. In some em- embodiment, the dimensions of the tissue being harvest- bodiments of the invention the DMO does not produce ed may be, for example, about 70 -100 mm in length. In keratin or produces a negligible amount of keratin, there- another embodiment, the dimensions of the tissue being by preventing the formation of keratin cysts following sub- 5 harvested may be, for example, about 80 - 100 mm in cutaneous implantation in a recipient. length, In another embodiment, the dimensions of the [0063] The DMO to be harvested can be removed from tissue being harvested may be, for example, about 90  the body by any means of removing tissue known in the 100 mm with an aspect of some embodiments of the in- art, such as biopsy procedures. The harvesting proce- vention, a closed, sterile, bioreactor apparatus may be durekeeps intact the micro- architecture of the tissue from 10 used to carry, support and/or alter the DMO or DTMO which it is removed. In one embodiment the DMO may throughout a harvesting, mm in length. In another em- be obtained by direct biopsy and be then cut to the re- bodiment the length may be around 20 mm. In another quired size or have non-desired tissue cut from it. In an- embodiment, the length may be about 30 mm. In another other embodiment, a tissue sample may be obtained by embodiment, the length may be about 40 mm. direct biopsy, in which the desired size of the dermal mi- 15 [0066] When a dermal MO has the above listed dimen- cro organ is obtained and no further processing is re- sions, it maybe maintained in vitro, e.g., in a growth me- quired. dium under proper tissue culture conditions for extended [0064] In some embodiments of the invention, the der- periods of time, for example, several days, several weeks mal micro organ is directly harvested from the body, and or several months. The DMO may be maintained, for ex- the dimensions of a cutting tool used to harvest the der- 20 ample, in-vitro in defined growth media. In one exemplary mal micro organ may be, for example, about 1  4 mm embodiment the growth media may include growth fac- in diameter. In another embodiment, the dimension may tors, fetal calf serum (FCS), or human serum, e.g., Syn- be, for example, 1.71 mm in diameter. In another em- thetic Serum Substitute (SSS). In another exemplary em- bodiment the dimension may be, for example, 1-3 mm in bodiment the growth media may include serum either diameter. In another embodiment, the dimension may 25 from the donor or the recipient subject. In yet another be, for example, 2-4 mm in diameter, In another embod- embodiment the growth media may include autologous iment the dimension may be, for example, 1-2 mm in serum. diameter. In another embodiment the dimension may be, [0067] In accordance with an aspect of some embod- for example, about 1.5 mm in diameter. In another em- iments of the invention, a closed, sterile, bioreactor ap- bodiment, the dimension may be, for example, about 2 30 paratus may be used to carry, support and/or alter the mm in diameter. In some embodiments, the harvested DMO or DTMO throughout a harvesting, alteration and dermal micro organ may not retain its cylindrical shape implantation process, e.g., from harvesting to implanta- after harvesting, i.e., at least one dimension of its cross tion, as described in detail below, e.g., with reference to section may expand while at least another dimension of Fig. 22. According to some exemplary embodiments, at its cross section may contract. In one embodiment, for 35 least part of the bioreactor apparatus may be formed of example, at least one dimension may be 0.5 - 3.5 mm disposable material. and at least one dimension may be 1.5  10 mm. [0068] In accordance with an aspect of some embod- [0065] In another embodiment, the dimensions of the iments of the invention, the bioreactor apparatus may be tissue being harvested may be, for example, about 5-100 loaded into a docking station, which may be used to carry mm in length. In another embodiment, the dimensions of 40 out various processes and/or to maintain the DMO/DT- the tissue being harvested may be, for example, about MO under desired conditions. The apparatus may be op- 10 - 60 mm in length. In another embodiment, the dimen- tionally computer controlled according to a protocol. sions of the tissue being harvested may be, for example, [0069] In accordance with an aspect of some embod- about 20 - 60 mm in length. In another embodiment, the iments of the invention, only a portion of the DTMO gen- dimensions of the tissue being harvested may be, for45 erated may be used in a given treatment session. The example, about 20  50 mm in length. In another em- remaining DTMO tissue may be returned for mainte- bodiment, the dimensions of the tissue being harvested nance and/or may be stored (e.g., cryogenically or oth- may be, for example, about 20 - 40 mm in length. In an- erwise) for later use. other embodiment, the dimensions of the tissue being [0070] It is a feature of some embodiments of the in- harvested may be, for example, about 20  100 mm in 50 vention that a large number of dermal micro organs may length. In another embodiment, the dimensions of the be processed together in a batch process into DTMOs, tissue being harvested may be, for example, about 30  as described below. This may allow for more convenient 100 mm in length. In another embodiment, the dimen- processing, but will not allow for determination of the se- sions of the tissue being harvested may be, for example, cretion level of each DTMO separately. about 40 - 100 mm in length. In another embodiment, the 55 [0071] In some exemplary embodiments of the inven- dimensions of the tissue being harvested may be, for tion a potency assay may be performed for the therapeu- example, about 50-100 mm in length. In another embod- tic agent, which may be produced and/or secreted by iment, the dimensions of the tissue being harvested may either a single DTMO or a batch of DTMOs, The potency

6 11 EP 2 377 401 A1 12 assay may include, for example, a cell proliferation assay bioreactor, as will be described below) in which it can be in which the proliferation response of the cells is mainly genetically altered. A suitable genetic modification agent dependent on the presence of the therapeutic agent in is prepared (block 208). Alternative exemplary methods the growth media of the cells. of preparing the agent include creation of aliquots with a [0072] The term "skin-related tissue structure", as 5 desired amount, using a predefined dilution buffer of used herein, refers to a structure of tissue components modifying agent such as for example a viral vector, pos- that may be stabilize and/or supported by apparatuses sible cryogenic storage and thawing of the modifying defined herein to enable the harvesting of a dermal micro agent, under controlled temperature (0-4°C), and validat- organ therefrom. A skin-related tissue structure may in- ing the activity of the modifying agent. All of these proc- clude components of the epidermal tissue, and compo- 10 esses are well known in the art. At this point the DMO nents of the dermal tissue. Optionally, the skin-related can be stored cryogenically, for later introduction at the tissue structure may include fat tissue and/or muscle tis- same place in the process. This can be performed using sue in the vicinity of the dermal tissue. known protocols for gradual freezing of tissues and cells, [0073] According to some embodiments of the inven- using for example, DMEM medium containing 10% DM- tion, a method of harvesting the dermal micro organ may 15 SO. include stabilizing and supporting a skin-related tissue [0077] At block 210 the DMO is genetically altered. As structure from which a dermal micro organ is to be har- described above, many methods of genetic alteration are vested, e.g., such that at least the dermal micro organ known and may be used in conjunction with the present and/or one or more other tissue segments in its vicinity invention. As an example, the following description is are maintained at a desired shape and/or position, sep- 20 based on using a viral vector to insert a gene into the arating at least a portion of the dermal micro organ from cells of the DMO. This process is well known and will not surrounding tissue, and extracting the separated dermal be further described, except as to the particular method- micro organ, as described in detail below. ology and apparatus for introducing the virus to the DMO. [0074] Figure 1 shows an overview of a methodology [0078] At block 212 the genetically altered DTMO is 200 for producing and utilizing DMOs and DTMOs, in 25 optionally tested for production and secretion rates of the block diagram form, in accordance with an exemplary therapeutic agent. There are various methods of deter- embodiment of the invention. At block 202 a DMO is har- mining the quantity of secretion, for example, ELISA, oth- vested from a subject. In some embodiments of the in- er immunoassays, spectral analysis, etc. In addition the vention, the DMO is harvested from the same subject to quality of the secretion is optionally tested, for example which therapy will later be applied. In an embodiment of 30 for sterility and activity of the secreted protein. This may the invention, the DMO is from dermal tissue. Optionally, be performed periodically or continuously on-line. At this other tissues are harvested and used in a manner similar point the DTMO can be cryogenically stored for later use. to that described below with reference to dermal tissue. [0079] At blocks 214 and 216, the amount of DTMO While the method described below is exemplary, other required for producing a desired therapeutic effect is de- methods of harvesting tissue samples can be used in 35 termined. As indicated below, the therapeutic dose re- some embodiments of the invention. If desired, the DMO quirements can be estimated from measured secretion can be cryogenically stored for later use (i.e., introduction rates, patient parameters and population statistics on the at the same stage of the process). Alternatively, for cer- estimated or known relationship between in vitro secre- tain embodiments, the DMO can be implanted directly tion and in vivo serum levels. back into the patient from which it was harvested to pro- 40 [0080] At block 218 the selected number of the DTMOs duce a therapeutic, cosmetic, or other physiological af- are loaded into implantation tools. Exemplary implemen- fect. tation tools have been described above. If needed, for [0075] In order for a DMO to be a viable micro organ, allografts or xenografts or for other reasons, the DTMO it must have at least one dimension that is small enough can be encapsulated. If the DTMO must be transported that nutrients can diffuse to all the cells of the DMO from 45 prior to being transported to the implantation tools, it is a nutrient medium which contacts the DMO and that optionally held (220) in a maintenance station, in which waste products can diffuse out of the DMO, and into the the temperature, humidity, etc. are held at levels that al- medium. This enables the DMO to be viable in, vitro long low the DTMO to stay viable during transport. The re- enough for the further processing described below and maining DTMO material is optionally maintained in vitro for the optional further utilization of the DMO as a source 50 for future use. This can be at warm incubator conditions for a therapeutic agent, such as a protein. The method (30-37 °C), in conditions as described above or at cool of harvesting a DMO as described above, generally re- incubator conditions (4°C), which mayprolong its viability sults in a DMO having an in vitro life of several months. in vitro. [0076] After the DMO is harvested, it is optionally vis- [0081] At block 224, a subset of the DTMOs is implant- ually inspected to determine that it is properly formed and 55 ed into the subject. An exemplary embodiment of a meth- that it has the desired dimensions. Inspection can also od of implantation is described above. Other methods of be performed optically. It is then optionally mounted on doing so will occur to persons of skill in the art and are a holder and transported (block 206) to an apparatus (the primarily dependent on the specific geometry of the mi-

7 13 EP 2 377 401 A1 14 cro-organ being used. Animal studies have shown that generally correspond to the length of the DMO to be har- the DMOs and DTMOs remain viable in vivo, in the sense vested. For example, inner guide 1110 may be inserted that the DTMO continues to produce and secrete the ther- manually, and hand guided within the dermis at a proc- apeutic agent for a period of weeks and months following ess. Alternatively, inner guide 1110 may be inserted into implantation (Figure 7). In animal studies, therapeutic 5 and along the subcutaneous space, by manually sensing amounts are produced for periods up to 160 days (or the boundary between the fibrous dermis and an under- longer). While the tissue of the DMO or DTMO appears lying smooth fatty layer as the inner guide is inserted. to be integrated or well taken into the tissue of the subject [0087] As indicated at block 1006, the method may op- into which it is implanted (especially if the tissue is im- tionally include guiding inner guide 1110 to exit the skin, planted in a tissue of the same kind from which it was 10 e.g., at incision 1130. According to some exemplary em- harvested), the cells including the DMO or the DTMO bodiments, the distance between incisions 1190 and continue to produce and secrete the therapeutic agent. 1130 may be approximately equal to or larger than a re- [0082] In either case, the in vivo performance of the quired length of the DMO to be harvested. DTMO is optionally determined (block 228). Based on [0088] As indicated at block 1008, the method may al- this evaluation for example, and/or on past patient data 15 so include inserting a tubular cutting tool coaxially with (block 226), patient dosage may then be adjusted (block and around inner guide 1110, such that the DMO may 230) by increasing the amount of the implant or removing betrapped, i.e., positioned,between the inner guide 1110 some of the implant, as described below. As the efficacy and the cutting tool. This may be achieved, for example, of the implant changes, additional DTMO can be implant- by using a tubular cutting tool having an inner diameter ed. 20 larger than the outer diameter of inner guide 1110. The [0083] Genetic alteration may generally include genet- cutting tool may include any suitable cutting tool, for ex- ically engineering a selected gene or genes into cells that ample, a coring tube 1150. Coring tube 1150 may include causes the cells to produce and optionally to secrete a a generally symmetrically sharpened tubular tool, e.g., a desired therapeutic agent such as a protein. In an em- hypo tube processed to have sharpened cutting edge bodiment of the invention, at least part of the process of 25 with a desired shape. Coring tube 1150 may include, for sustaining the DMO during the genetic alteration, as well example, a standard medical grade tube, having a thin as the genetic alteration itself, may be performed in a wall, e.g., having a thickness of between 0.05mm and bioreactor, as described below. 0.3mm. Coring tube 1150 may have a diameter, for ex- [0084] Reference is now made to Fig. 10, which sche- ample, between 1 mm and 10 mm. The dimensions, e.g., matically illustrates a flowchart of a method of harvesting 30 the diameter, of coring tube 1150 and/or the dimensions a dermal micro organ according to some exemplary em- of inner guide 1110 may be predetermined based on the bodiments of the invention, and to Figs. 11a-11c, which volume and/or dimensions of the DMO intended to be schematically illustrate exemplary stages of harvesting harvested. Coring tube 1150 may have a sharpened end as dermal micro organ 1160 located under a skin tissue ("tip") 1140 adapted to serve as a cutting edge. Coring portion 1120 in accordance with the method of Fig. 10. 35 tube 1150 may be inserted through tissue portion 1120, [0085] As indicated at block 1002, the method may op- preferably after creating initial incisions, E.G., INCISION tionally include locally administering an anesthetic, e.g., 1130, on the outer surface of the skin in order to prevent as is known in the art, to the vicinity of the DMO to be harvesting of epidermal tissue. harvested. [0089] According to one exemplary embodiment of the [0086] As indicated at block 1004, the method may fur- 40 invention, e.g., as illustrated in Fig. 11b, the method may ther include inserting an inner guide 1110 into tissue por- include initially positioning end 1140 of coring tube 1150 tion 1120. Thin incisions ("lance cuts") 1190 and 1130 over a distal end of inner guide 1110, e.g., at incision may be formed in the outer skin, preferably using a sur- 1130, and sliding coring tube 1150 along the length of gical lance, scalpel, or other sharp probe, in order to allow inner guide 1110, e.g., towards incision 1190, to harvest easier insertion of inner guide 1110, and also to prevent 45 the dermal DMO. or minimize the harvesting of epidermal tissue. Inner [0090] As indicated at block 1010, in one embodiment guide 1110 may be inserted into portion 1120 via incision the method may include rotating the cutting tool while 1190, e.g., generally parallel to the skin surface and/or advancing the cutting tool, e.g., towards the proximal end at a desired depth within the dermis or just under the of the inner guide. For example, a medical drill or other skin. Inner guide 1110 may include a thin needle, rod, or 50 suitable tool or rotation mechanism may be used to rotate any other suitable thin, generally straight, object able to coring tube 1150 while it is advanced manually or auto- be placed inside the dermis or in a subcutaneous space. matically, thereby more smoothy harvesting DMO 1160. For example, inner guide 1110 may include a needle of For example, a proximal end 1180 of coring tube 1150 size 20-25G, for example, about 22G, as is known in the may be connected to a medical drill 1170, such as, for art. Inner guide 1110 may be inserted into the dermis or 55 example, the Aesculap Micro Speed drill manufactured subcutaneous space and/or pushed generally horizon- by Aesculap AG & Co. KG, Am Aesculap Platz, D- 78532 tally, i.e., generally in parallel with the skin surface. The Tuttlingen, Germany, which may include a control unit, length of penetration of guide 1110 within the dermis may a motor, a connection cord, a hand piece and/or a foot

8 15 EP 2 377 401 A1 16 switch, catalogue numbers GD650, GD658, GB661, that any combination of the above actions may be imple- GB166 andGB660, respectively. Such adrill, orany other mented to perform harvesting according to embodiments suitable drill or rotation mechanism, may be used to rotate of the invention. Further, other actions or series of actions the cutting edge of the cutting tool at a rotational speed may be used. appropriate for cutting of the dermal tissue, for example, 5 [0096] According to some embodiments of the inven- a relatively high rotational speed, for example, a speed tion, the harvesting method may additionally include ex- higher than 1,000 RPM, e.g., between 1,000 RPM and ternally stabilizing and/or supporting the DMO to be har- 10,000 RPM. For example, tube 1150 may be rotated at vested and/or tissue in the vicinity of the DMO to be har- a rotational speed higher than 2,000 RPM, e.g., approx- vested e.g., using an external support device and/or imately 7,000 RPM. Alternatively, a relatively low rota- 10 mechanism, for example, in addition to internally stabi- tional speed of less than 1000 RPM may be used, or no lizing and/or supporting the dermis, e.g., by the inner rotation at all, as described below, Optionally, the rota- guide, as described below. tional speed of the drill may vary in an oscillatory manner, [0097] Reference is also made to Fig. 12, which sche- i.e., the direction of rotation may vary periodically be- matically illustrates a stabilizing clamping tool 1200, tween "clockwise" and "counterclockwise" directions.15 which may be used in conjunction with a dermal harvest- While rotated by drill 1170, coring tube 1150 may be man- ing apparatus in accordance with some exemplary em- ually or automatically advanced, e.g., towards the prox- bodiments of the invention. imal end of inner guide 1110, e.g., towards incision 1190. [0098] According to exemplary embodiments of the in- The method may also include stopping the forward mo- vention, tool 1200 may include a clamping mechanism tion of coring tube 1150, for example, when tip 1140 has 20 having clamping edges 1210. For example, tool 1200 been advanced just beyond incision 1190. According to may include a pinching clamp or forceps, e.g., as are some exemplary embodiments of the invention, at least known in the art. Tool 1200 may include a spring clamp part of an inner surface and/or an outer surface of tube having a constant clamping force, or a controllably vari- 1150 may be coated with a low motion material, e.g., able clamping force. Tool 1200 may be closed, clamping Teflon, Parylene or any other suitable coating material, 25 edges 1210may be positioned beneathinner guide 1110. e.g., to ease the separation of the harvested tissue from Clamping edges 1210, when brought close together, may the inner surface of the cutting tool in a subsequent action function to stabilize and/or support inner guide 1110 and/or to reduce any forces acting on the tissue during and/or a skin portion 1240 associated with the DMO to the cutting action, as described below. be harvested, such that the DMO may be stabilized while [0091] In another embodiment, a fast-acting, e.g., 30 being out by tube 1150. Coring tube 1150, in this case, spring-loaded, insertion mechanism may be used to as- may be pushed through clamping edges 1210 concentric sist coring tube 1150 in penetrating the harvesting target or non-concentric to inner guide 1110, while force is ap- and cutting the dermis, e,g., with substantially no rota- plied. According to some exemplary embodiments of the tional motion of the coring tube. invention, clamping edges 1210 may include at least one [0092] As indicated at block 1012, the method may in- 35 or two rows of serrated teeth 1260 in order to provide clude withdrawing inner guide 1110, e.g., having DMO improved clamping of portion 1240 and reduce, e.g., min- 1160 impaled thereon, from within coring tube 1150, imize,lateral movement of the skin during the coring proc- thereby to extract DMO 1160 from portion 1120. ess. [0093] According to some embodiments. DMO 1160 [0099] Other tools and/or mechanisms may be used may be left impaled on inner guide 1110. In such a case, 40 to apply force to the outer skin in order to cause similar inner guide 1110 may be used to handle, transport, compression of the dermis surrounding the inner guide. and/or manipulate the DMO 1160. Alternatively DMO Alternatively, other devices and/or methods for stabiliz- 1160 may be, for example, carefully removed from inner ing the dermis to be harvested may be used, such as guide 1160 into a bioreactor processing chamber, e.g., twisting the inner guide and holding it at a substantially as described in detail below with reference to Fig. 22, or 45 fixed position with respect to the rotation of the coring onto various transfer devices (not shown) adapted for tube. transferring the DMO to a different mount or into a cham- [0100] Reference is also made to Fig. 13, which sche- ber for further processing. Such transfer devices may matically illustrates a cross sectional view of coring tube include, for example, forceps, vacuum grippers or any 1150 inserted coaxially over and along inner guide 1110 other mechanical devices able to grip DMO 1160 and/or 50 in accordance with some exemplary embodiments of the push DMO 1160 off inner guide 1110. In addition, suitable invention. fluids, such as sterile fluids, may be used, either alone [0101] According to some embodiments of the present or in conjunction with the means listed above, to assist invention, inner guide 1110 may be placed in skin portion in removing the DMO from inner guide 1160. 1120 at a position such that an axis 1125 of guide 1110 [0094] As indicated at block 1014, the method may al- 55 is positioned substantially at the center of DMO 1160. In so include withdrawing the cutting tool, e.g., coring tube such a case, coring tube 1150 may be substantially co- 1150, from skin portion 1120. axially aligned with inner guide 1110, such that DMO [0095] It will be appreciated by those skilled in the art 1160 is impaled on inner guide 1110 in an approximately

9 17 EP 2 377 401 A1 18 symmetrical manner. 1406, having a top support surface 1430 fluidically con- [0102] However, accordingto other exemplary embod- nected via a plurality of channels 1404 to a vacuum inlet iments of the invention, the inner guide and the coring 1402. Vacuum inlet 1402 may be fluidically connected to tube may be positioned in any other suitable arrange- at least on vacuum source, e.g., a vacuum pump (not ment. For example, the inner guide may be positioned in 5 shown), to provide a vacuum condition to chamber 1406. the subcutaneous space, such that the desired DMO to Surface 1430 and/or channels 1404 may be configured be harvested may be primarily located above the inner to enable attaching to surface 1430 at least part of an guide and wrapped around it. Accordingly, the coring epidermal layer 1508 associated with DMO 1510, e.g., tube may be inserted over the inner guide and/or guided located generally above DMO 1510, when a vacuum con- such that the inner guide is positioned close to or touches 10 dition is applied to chamber 1406, e.g., by the vacuum the lower inner surface of the coring tube as it cuts the source. DMO. In such a case, the inner guide may hold the DMO, [0107] Apparatus 1400 may also include a guiding which may rest, for example, along the upper surface of channel 1416 for guiding a cutting tool, e.g., a coring tube the inner guide when being removed. 1520, and maintaining the cutting tool at a predetermined [0103] According to some embodiments of the present 15 location, e.g. a predetermined distance from upper sur- invention, the above described manual procedures may face 1430. For example, the upper surface of cutting tool befacilitated by an integratedapparatus (not shown)con- 1520 may be located at a distance, for example, of ap- figured to perform some or all of the above procedures proximately 1mm from upper surface 1430. In other em- for harvesting the DMO. For example, in regard to one bodiment, other ranges, such as for example, 0.3-2.0 harvesting method embodiment, the integrated appara- 20 mm, may also be used, Channel 1416 may include, for tus may be configured to enable positioning and guiding example, a generally cylindrical channel having a diam- the insertion of inner guide 1110, attaching clamping tool eter slightly larger than the outer diameter of coring tube 1200, guiding the insertion of coring tube 1150 and con- 1520. Coring tube 1520 may include a coring needle hav- trolling its movement during the cutting process, and/or ing a size of, e.g., between 1 mm and 10 mm, for example, removing DMO 1160 being attached to inner guide 1110. 25 14G (corresponding to an outer diameter of approximate- Such an apparatus may enable relatively simple opera- ly 2.11mm) and having a symmetrically sharpened cut- tion when performing a harvesting procedure. ting edge. [0104] According to some exemplary embodiments of [0108] According to exemplary embodiments of the in- the invention, a method of harvesting a DMO from a sub- vention, surface 1430 may be flat, generally curved, or ject may include generating and/or maintaining a skin- 30 may have any other suitable shape. For example, in one related tissue structure associated with the DMO, e.g., embodiment, surface 1430 may have a radius of curva- located generally at a targeted harvest site for harvesting ture of about 3.5mm. In one embodiment, chamber 1406 the DMO, at a desired shape and position such that the may have a width of, for example, about 4mm. . Further- cutting tool may be able to separate at least part of the more, in some embodiments, chamber 1406 may have DMO from tissue in the vicinity of the DMO. For example, 35 a height of, for example, about 5mm, In other embodi- an epidermis portion in the vicinity of the targeted harvest ments, other ranges, such as for example, 3-25 mm, may site may be lifted, e.g., by attaching at least part of the also be used for the radius of curvature of surface 1430 epidermis portion to a predefined, e.g., substantially flat, and/or the width and/or height of chamber 1406, for ex- surface area such that at least part of the skin-related ample, any desired dimensions in the range of 3-25 mm tissue structure may be lifted and maintained at the de- 40 may be used in some embodiments of the invention. The sired shape and/or position. According to some exem- length of chamber 1406 may be generally similar to the plary embodiments, attaching the epidermis to the pre- length of the DMO being harvested, far example, approx- defined surface may include applying a vacuum condi- imately 30 mm in length; however, other ranges, for ex- tion, e.g., as described below. Alternatively or addition- ample, in the range of 5 - 100 mm, may be used for the ally, attaching the epidermis to the predefined surface 45 chamber length. may include applying an adhesive to the surface. [0109] According to some exemplary embodiments, [0105] Reference is now made to Figs. 14a- 14c, which apparatus 1400 may include two channels 1408 located schematically illustrate a front view, a side view, and a at least partially along two sides of chamber 1406, re- top view, respectively, of a dermal harvesting apparatus spectively, to allow clamping epidermis layer 1508, as 1400 for harvesting a DMO according to one exemplary 50 described below. Channels 1408 may be positioned, e.g., embodiment of the invention, and to Fig. 15, which sche- centered, at a desired height, for example, at approxi- matically illustrates a cross-section side view of appara- mately the same height as where the center of the DMO tus 1400 being implemented for externally supporting a is to be harvested. In one embodiment, the center of skin-related tissue structure including DMO 1510 at a channels 1408 may be positioned at a height of about 2 desired position according to one exemplary embodi- 55 mm below upper surface 1430. so that the clamping may ment of the invention. stabilize and/or support the tissue being out. According [0106] Apparatus 1400 may include a vacuum cham- to these exemplary embodiments, apparatus 1400 may ber, e.g., a generally cylindrical longitudinal chamber also include two flexible membrane elements 1412, on

10 19 EP 2 377 401 A1 20 eitherthe inner surface or outer surface of channels 1408, chamber 1406. so as to allow external clamping of the tissue without [0113] The method may further include rotating coring substantially affecting the vacuum condition applied to tube 1520, e.g., using drill 1170 (Fig. 11) at a relatively chamber 1406. According to other embodiments of the high rotational speed, e, g., higher than 1,000 RPM, e.g., invention, apparatus 1400 may not include elements5 between 1,000 RPM and 10,000 RPM. For example, cor- 1412 and/or channels 1408. ing tube 1520 may be rotated at a rotational speed higher [0110] According to exemplary embodiments of the in- than 2,000 RPM, e.g., approximately 7,000 RPM. Alter- vention, a method of harvesting DMO 1510 using appa- natively, a relatively low rotational speed of less than ratus 1400 may include forming two incisions (not 1000 RPM may be used, or no rotation at all, as described shown), e.g., forming two lance cuts using a scalpel, in 10 above. The method may also include advancing coring a skin portion associated with DMO 1510 at a predeter- tube 1520 along vacuum chamber 1406, e.g., at least mined distance, e.g., approximately 30mm, which may along the entire length of chamber 1406. Coring tube correspond to the points at which coring tube 1520 is 1520 may be guided through channel 1416 in order to intended to enter and exit epidermis 1508 ("the entry and ensure that dermal micro organ 1510 is harvested from exit penetration sites"). The incisions may be formed in 15 approximately the same depth in the skin-related tissue order to ensure that there will be substantially no epider- structure along chamber 1406. Coring tube 1520 may be mal component at the two ends of harvested DMO 1510, advanced manually, or using a motorized actuator (not and/or to maintain a desired shape of the penetration shown), e.g., to control the speed at which coring tube sites such that they may heal efficiently, i.e., quickly 1520 may advance. and/or leaving relatively small scars. The method may 20 [0114] The method may also include detaching DMO also include placing apparatus 1400 in contact with epi- 1510 from tissue surrounding DMO 1510. For example, dermis layer 1508 ("the harvest site") such that the inci- apparatus 1400 may include an extension 1418, e.g., sions are positioned underneath chamber 1406, i,e., in having a length of between 1mm and 5 mm and a radius between points 1410 and 1414. The incisions may be substantially equal to the radius of channel 1416, located positioned at points 1410 and/or 1414, respectively, or 25 substantially opposite channel 1416 suchthat coringtube may be positioned between points 1410 and 1414 to help 1520 may advance into extension 1418 after going force the lance cuts to "open" once the vacuum condition through chamber 1406. Alternatively, a cutting surface is applied to chamber 1406. According to some exem- 1440, e.g., formed of Silicone or other suitable material, plary embodiments, apparatus 1400 may optionally in- may be positioned in extension 1418 such that the coring clude a mechanism configured for creating the lance30 tube may cut into surface 1440 to detach the harvested cuts, for example, spring loaded lancets that produce the DMO. Additionally, a vacuum condition may be applied lance cuts, e.g., after apparatus 1400 is placed on the within coring tube 1520, e.g., from its back end, such that harvest site and before the vacuum condition is applied DMO 1510 may be actively drawn into coring tube 1520, to chamber 1406. thus urging final detachment of the DMO from the sur- [0111] The method may also include inserting coring 35 rounding tissue. tube 1520 into channel 1416. Coring tube 1520 may be [0115] The method may further include with drawing connected, for example, via a connector, e.g., a Jacobs coring tube 1520, including therein DMO 1510, from ap- Chuck or a friction holder, to a medical drill or any other paratus 1400. suitable tool and/or mechanism, e.g., drill 1170 (Fig. 11), [0116] Reference is made to Fig. 16, which schemat- able to rotate coring tube 15211, Optionally, the rotational 40 ically illustrates a cross- sectional side view of apparatus speed of the drill may vary in an oscillatory manner, i.e., 1400 being implemented for externally supporting a skin- the direction of rotation may vary periodically between related tissue structure at a desired position according "clockwise" and "counterclockwise" directions. to another exemplary embodiment of the invention. [0112] The method may also include applying a vacu- [0117] According to the exemplary embodiment of Fig. um condition to chamber 1406, e.g., by activating the 45 16, improved stabilization of dermis 1506 and/or im- vacuum source, Consequently, the skin-related tissue proved prevention of recruitment of fat 1504 into vacuum structure may be drawn into chamber 1406 and epider- chamber 1406 may be accomplished by external clamp- mis 1508, e.g., between the lance cuts, may be firmly ing of the skin-related tissue structure supported within held against surface 1430. Epidermis 1508, dermis 1506, the vacuum chamber. Forexample, a clamping tool 1600, and/or fatty tissue components 1504 may additionally be 50 e,g., analogous to the clamping tool described above with drawn into chamber 1406, depending on the thickness reference to Fig. 12, may be implemented to "pinch" the of each of these tissue layers and the dimensions of skin-related tissue structure supported inside vacuum chamber 1406. Thus, the dimensions of chamber 1406 chamber 1406, e.g., symmetrically. Two clamping ends may be designed in accordance with the anticipated 1502 of clamping tool 1600 may be inserted into channels thickness of one or more of the tissue layers and/or ex- 55 1408, respectively. Tool 1600 may be closed such that terior clamping, e.g., as described herein, may be applied clamping ends 1502 may press down against flexible el- such that fat tissue 1504 drawn into vacuum chamber ements 1412. Thus, the skin-related tissue structure in 1406 may be forced downwards and substantially out of chamber 1406 may be clamped from the sides without

11 21 EP 2 377 401 A1 22 substantially affecting the vacuum condition in chamber 1 mm or more, and a suitable length, e.g., substantially 1406. A clamping force applied by clamping ends 1502 equal to the desired length of the DMO to be harvested. may correspond, for example, to a constant or variable [0124] According to the exemplary embodiments of force of a spring 1512 or other suitable device. Fig. 18, a single incision, e.g., lance cut, may be formed, [0118] Although the above description may refer to a 5 e.g., using a scalpel blade, through which HST 1806 may vacuum chamber having a generally constant shape be inserted together with cutting tool 1808, e.g., as a and/or size along its longitudinal axis, it will be appreci- single unit, at the desired position underneath or in the ated by those skilled in the art that, according to other skin, preferably in the subcutaneous space with notch embodiments of the invention, the vacuum chamber may 1802 oriented upwards towards dermis layer 1840. Cut- have any other predetermined size and/or shape, e.g., 10 ting tool 1801 may be positioned within HST 1806 during as described below. penetration such that window cutout 1802 may be [0119] Reference is now made to Fig. 17, which sche- "closed" to allow a generally smooth penetration of HST matically illustrates a dermal harvesting apparatus 1700 1806. Tool 1808 and HST 1806 inserted therein may run according to another exemplary embodiment of the in- along the subcutaneous interface for the length of notch vention. 15 1802,and end 1804 maynot exitthrough the skin surface. [0120] Apparatus 1700 may include a vacuum cham- Once appropriately positioned, tool 1808 may be retract- ber 1701 including an elevated protrusion 1706. Elevated ed to expose notch 1802 and allow for dermal tissue to protrusion 1706 may have a predetermined size and/or substantially fill the notch. Appropriate pressure on the shape adapted, for example, to enable the creation of a skin surface may be applied, e.g., using a suitable clamp- "plateau" of a single layer of skin tissue in a generally flat 20 ing tool, for example, as described above with reference orientation, elevated above the trajectory of a coring tube to Fig 12, and/or a vacuum condition may be applied from 1716. For example, section 1706 may be higher than within HST 1802 by a vacuum manifold (not shown), e.g., other sections of chamber 1701, such that a fat layer located under notch cutout 1802, to assist the dermis to 1718 may be drawn into section 1706 and supported substantially fill notch 1802. Tool . 1808 may be connect- along the trajectory of coring tube 1716. As a result, after 25 ed to a motor, e.g., as described above, to rotate tool harvesting a DMO of a predetermined length, coring tube 1808 at a rotational speed appropriate for cutting of the 1716 may be slightly advanced into fat layer 1718, thus dermal tissue, for example, a relatively high rotational separating the harvested DMO from tissue surrounding speed, for example, a speed higher than 1,000 RPM, the DMO. The harvested DMO may remain within coring e.g., between 1,000 RPM and 10,000 RPM. For example, tube 1716 as it is withdrawn from the body. The config- 30 tool 1808 may be rotated at a rotational speed higher uration of Apparatus 1700 may eliminate the need for than 2,000 RPM, e.g., approximately 7,000 RPM, Tool forming an "exit" incision in the skin, e.g., as described 1808 may then be advanced e.g., manually or automat- above, thus enabling the harvesting of a DMO with only ically, for example, until it passes beyond the end of win- a single incision. dow cutout 1802, to cut DMO 1830 within notch 1802. [0121] According to some exemplary embodiments of 35 When complete, the forward and rotational movements the invention, apparatus 1700 may also include a drill of tool 1808 may be stopped, and cutting tool 1808 may stopper 1708 to enable manually advancing coring tube be retracted with harvested DMO 1830 within it. SHT 1716 for a predetermined distance along chamber 1701, 1806 may then be removed from the harvest site. DMO e.g., to a position in which coring tube 1716 has slightly 1830 may be removed from cutting tool 1808, e, g., using advanced into fat tissue 1718. 40 a syringe to flush sterile fluid, for example saline, through [0122] Reference is now made to Fig. 18, which sche- tool 1808, or a vacuum source to draw out DMO 1830 matically illustrates aharvesting apparatus 1800, accord- from a back end (not shown) of cutting tool 1808. ing to yet another exemplary embodiment of the inven- [0125] It will be appreciated by those skilled in the art tion, and to Fig.19, which schematically illustrates a cross that apparatus 1800 may enable harvesting of the DMO sectional view of apparatus 1800 being implemented for 45 by forming only one incision. Furthermore, apparatus harvesting a DMO 1830. 1800 may be efficiently applied for harvesting a DMO [0123] According to some exemplary embodiments, from areas having relatively thick skin, e.g., from a region core biopsy devices with similarities to the devices used, of the donor’s back. for example, in breast cancer biopsy applications, as de- [0126] It will be appreciated by those skilled in the art scribed below, may be utilized for harvesting a DMO, 50 that the harvesting methods and/or apparatuses accord- Apparatus 1800 may include a cutting tool 1808, e.g., as ing to embodiments of the invention, e.g., as described described above, and a Subcutaneous Harvest Trocar above, may include introducing thin tissue cutting devic- (HST) 1806, e, g., a hypodermic needle with a sharpened es within the dermis. Thus, the harvesting methods tip 1804 and a suitable inner diameter, e.g., being slightly and/or apparatuses according to embodiments of the in- larger than the outer diameter of cutting tool 1808, such 55 vention may enable harvesting the DMO with relatively that cutting tool 1808 may be inserted into and substan- minimal damage to the outer skin surface, end therefore tially coaxially within HST 1806.HST 1806 may include may provide a minimally invasive method of harvesting a notch cutout ("window") 1802 of a suitable depth, e, g., the desired tissues.

12 23 EP 2 377 401 A1 24

[0127] Although some embodiments of the invention may be optionally administered at an intended implanta- described herein may refer to methods and/or appara- tion site. tuses for harvesting a DMO, it will be appreciated by [0132] As indicated at block 2004, according to some those skilled in the art that according to other embodi- exemplary embodiments of the invention, the DTMO, op- ments of the invention at least some of the methods5 tionally together with surrounding sterile saline fluid may and/or apparatuses may be implemented for any other be aspirated into a carrier, for example, an implantation procedures, e.g., plastic surgical procedures, dermato- needle, e.g., attached to a syringe. The needle may have logical procedures, or any other procedures including any suitable diameter, for example, between 17-gauge harvesting of tissues, For example, the methods and/or and 12-gauge. Optionally, a tip of the needle may have apparatuses according to embodiments of the invention 10 a short extension of silicon tubing, or the like, affixed to may be implemented for harvesting dermal tissue to be it, to facilitate the aspiration of the DTMO into the needle used,e.g., in asubsequent implantation, as filler material. cannula while retracting the plunger of the syringe. [0128] According to some embodiments of the present [0133] As indicated at block 2006, with the loaded DT- invention, a system and method are provided for ex- vivo MO, the implantation needle, may be pushed into the ("in vitro") handling or processing of dermal micro organs. 15 skin, e.g., without the silicon tubing extension, into the Dermal tissue that has been harvested as a direct MO subcutaneous destination, along a distance approxi- may be left on their inner guide as a mount for the MO. mately equivalent to the length of the DTMO. In these embodiments, the inner guide may be used to [0134] As indicated at block 2008, according to some maintain position and orientation of the MOs during sub- embodiments, the implantation needle may exit through sequent processing. In other embodiments, the dermal 20 the skin surface at a distal end of the implantation site. MOs may be removed from the inner guide and directly [0135] According to some exemplary embodiments of placed into tissue culture wells or transduction chambers the invention, the method may include applying pressure of a bioreactor, as described in detail below, e.g., with on the aspirated dermal therapeutic micro organ such reference to Fig. 22. In some embodiment, e.g., if the that the dermal therapeutic micro organ exits from the DMO remains in the coring tube as it is with drawn from 25 carrier into the implantation site. the skin, the DMO may be flushed out of the coring tube [0136] As indicated at block 2010, the tip of the DTMO by the use of biologically compatible fluid, e.g., saline or may be grasped at the exit point with a gripping tool, for growth medium, applied to the back end of the coring example tweezers. tube. The flushing of the DMO may be such that it is [0137] As indicated at block 2012, the implantation flushed directly into a chamber of the bioreactor, e.g., as 30 needle may be retracted through the subcutaneous described below. Alternatively, vacuum may be applied space, releasing the DTMO from the implantation needle to a back end of the coring tube to "draw out" the DMO, and laying the DTMO linearly along the needle tract. As- e.g., directly into a chamber of the bioreactor. sistance may be given to help release the DTMO, if need- [0129] According to some embodiments of the present ed, for example by gently pushing down on the syringe invention, a system and method are provided for implan- 35 plunger during retraction. tation of DTMOs. After producing and/or processing of a [0138] As indicated at block 2014, once the DTMO has DMO, for example, by genetically modifying the DMO, been left in place, the tip of the DTMO may be released the modified DMO or DTMO may be implanted back into by the gripping tool. the patient, for example, for protein or RNA based ther- [0139] According to some embodiments of the present apy. The number of full or partial DTMOs that are im- 40 invention, a system and method are provided for in-vivo planted may be determined by the desired therapeutic demarcation and localization of the implanted dermal mi- dosing of the secreted protein. DTMOs may be implanted cro organs. Identification of the location of a subcutane- subcutaneously or at any other locations within the body. ous implantation or implantation at any other location in Subcutaneous implantation by use of a needle trocar, for the body, of processed tissue, such as a DTMO, may be example, may enable the DTMO to remain in a linear 45 important, for example, in the case where it is necessary form in the subcutaneous space. The linear form of im- to stop the protein treatment, or to decrease the dosage plantation may help facilitate localization in case later of the secreted protein. For example, termination or titra- ablation of the DTMO is required, for example, in order tion of dosage may be performed by removing one or to stop treatment or reduce the dose of therapeutic pro- more DTMOs entirely and/or by ablating one, a portion tein. Other known geometrical implantation patterns50 of one, or more than one of the implanted DTMOs. In could be used. The linear implantation may also assist order to identify a subcutaneously implanted DTMO, ac- in the integration of the dermal tissue to the surrounding cording to one embodiment, the DMO may be colored tissue. prior to implantation by an inert, biocompatible ink or stain [0130] Reference is now made to Fig. 20, which sche- containing, for example, a chromophore, which may be matically illustrates a flowchart of a method of implanting 55 visible to the naked eye or may require special illumina- a DTMO according to some exemplary embodiments of tion conditions to visualize it. In this way a DTMO may the invention. be distinguished from its surrounding tissue by visual in- [0131] As indicated at block 2002 a local anesthetic spection and/or by use of enhanced imaging means.

13 25 EP 2 377 401 A1 26

[0140] According to one embodiment, the peripheral removal. At block 2104 an inner guide may be inserted surface of a DTMO may be coated with, for example, subcutaneously along the length of the DTMO, to harvest biocompatible carbon particles, biocompatible tattoo ink, a core of tissue, which includes the DTMO. At block 2106 or other suitable materials. Once implanted subcutane- a coring needle, of the same or larger diameter than that ously, the DTMO may be visible with the naked eye or 5 of the implantation needle (for example, 11- gauge or sim- with a suitable enhanced imaging device. Other ways to ilar), may be inserted concentrically over the inner guide. enhance the visibility of an implanted DTMO may include At block 2108 a core of tissue that includes the DTMO using a strong light source above the skin surface, or may be harvested. At block 2110 the inner guide with the pinching the skin and directing the light source at the skin cored of tissue and the coring needle may be extracted from one side, such that the skin may appear translucent 10 from the skin, with the DTMO. In one embodiment, such and the dyed DTMO may be more visible. Alternatively, a coring approach may be combined with vacuum suction the stain may be fluorescent, visible only when illuminat- to help remove the cut material from the body. ed using UV light, such as using fluorescent plastic [0145] According to an embodiment of the present in- beads. vention, minimally invasive or non-invasive methods of [0141] According to another embodiment, the location 15 ablating the DTMO in- situ may be used to make the pro- of a subcutaneously implanted DTMO may be identified cedure less traumatic and less invasive for the patient. by co-implanting a biocompatible structure along with the In one embodiment, in the case of the dyed DTMO, a DTMO. An example of such a biocompatible structure is laser, for example, a non- invasive Yag laser may be used a non-absorbable single stranded nylon suture common- The energy of the Yag laser, for example, may be selec- ly used in many surgical procedures. Such a suture may 20 tively absorbed by the chromophore,such that the energy be implanted in the same implantation tract with the DT- is primarily directed to the DTMO, with minimum damage MO, or may be implanted directly above the DTMO in caused to the surrounding tissue. Other light energy the upper dennis, such that the spatial location of the sources may also be used. DTMO may be determined by the suture location. Fur- [0146] According to another embodiment, the DTMO ther, the depth of the DTMO may be known to be at the 25 may be ablated by delivering destructive energy from a depth of the subcutaneous space. The suture may be minimally invasive probe inserted into the subcutaneous visible to the naked eye, observed with the assistance of space along the length of the DTMO. Such a probe may illumination means, and/or observed with The aid of other enable delivery of a variety of energy types, including suitable imaging means, such as ultrasound. Alternative- radio frequency, cryogenic, microwave, resistive heat, ly, the suture can be fluorescent, and visible through the 30 etc. A co-implanted structure, such as a suture, may be skin under appropriate UV illumination. The suture may used to determine the location of the DTMO, thereby en- alternatively be of an absorbable material, so that it may abling the probe to be inserted subcutaneously, for ex- enable determination of localization for a desired period ample, along or directly below the suture. In such a case, of time, such as a few months. for example, the destructive energy may be delivered [0142] According to another embodiment, the DTMO 35 while the suture is still in place. Alternatively, the suture may be genetically modified or engineered to include a may be removed after placement of the probe and before gene to express a fluorescent marker or other marker delivery of the destructive energy. The amount of energy capable of being visualized. For example, the DTMO can applied may be either that required to denature the pro- be modified with the GFP (Green Fluorescent Protein) teins in the tissue such as during coagulation by diather- gene or Luciferase reported gene, which, for example, 40 my. Additionally or alternatively, the amount of energy may be expressed along with the gene for the therapeutic applied may be as much as is used in electro-surgical protein. In this manner, the DTMO may be visualized cutting devices, which char tissue. Of course, other non-invasively using appropriate UV or other suitable il- means of localization and other means of delivering de- lumination and imaging conditions. structive energy may be used. [0143] According to some embodiments of the present 45 [0147] After a DMO is harvested, e.g., according to invention, a system and method are provided for removal embodiments of the present invention, the DMO is op- or ablation of implanted DTMOs. In a case, for example, tionally genetically altered. Any methodology know in the where DTMO-based therapy to a patient must be termi- art can be used for genetically altering the tissue. One nated, or if the protein secretion must be decreased, each exemplary method is to insert a gene into the cells of the implanted DTMO may be partially or entirely removed, 50 tissue with a recombinant viral vector. Any one of a or partially or entirely ablated. One embodiment for re- number of different vectors can be used, such as viral moval of a DTMO is by means of a coring tube similar vectors, plasmid vectors, linear DNA, etc., as known in to, or slightly larger in diameter than, that used for direct the art, to introduce an exogenous nucleic acid fragment harvesting of the DMO. encoding for a therapeutic agent into target cells and/or [0144] As can be seen with reference to Fig. 21, at55 tissue. These vectors can be inserted, for example, using block 2102 the location of the implanted subcutaneous any of infection, transduction, transfection, calcium- DTMO may be determined, At block 2103, a local anes- phosphate mediated transfection, DEAE-dextran medi- thetic may be optionally administered at the site of DTMO ated transfection, electroporation, liposome-mediated

14 27 EP 2 377 401 A1 28 transfection, biolistic gene delivery, liposomal gene de- refers to either the D or L stereoisomer form of the amino livery using fusogenic and anionic liposomes (which are acid, unless otherwise specifically designated. Also en- an alternative to the use of cationic liposomes), direct compassed within the scope of this invention are equiv- injection, receptor-mediated uptake, magnetoporation, alent proteins or equivalent peptides, e.g., having the bi- ultrasound and others as known in the art. This gene 5 ological activity of purified wild type tumor suppressor insertion is accomplished by introducing the vector into protein. "Equivalent proteins" and "equivalent polypep- the vicinity of the DMO so that the vector can react with tides" refer to compounds that depart from the linear se- the cells of the DMO. Once the exogenous nucleic acid quence of the naturally occurring proteins or polypep- fragment has been incorporated into the cells, the pro- tides, but which have amino acid substitutions that do duction and/or the secretion rate of the therapeutic agent 10 not change it’s biologically activity. These equivalents encoded by the nucleic acid fragment can be quantified, can differ from the native sequences by the replacement [0148] According to some exemplary embodiments of of one or more amino acids with related amino acids, for the invention, the genetic modification of the DMO may example, similarly charged amino acids, or the substitu- modify the expression profile of an endogenous gene. tion or modification of side chains or functional groups. This may be achieved, for example, by introducing an 15 [0153] The protein, peptide, polypeptide glycoprotein enhancer, or a repressible or inducible regulatory ele- or lipoprotein can be, without being limited, any of the ment for controlling the expression of the endogenous following proteins or various combinations thereof: pro- gene. tease, a lipase, a ribonuclease, a deoxyribonuclease, a [0149] In another embodiment, the invention provides blood clotting factor, a cytochrome p450 enzyme, a tran- a method of delivering a gene product of interest into a 20 scription factor, a MHC component, a cytokine, an inter- subject by implanting the genetically modified DMO of leukin, a BMP, a chemokine, a growth factor, a hormone, the invention into a subject. an enzyme, a monoclonal antibody, a single chain anti- [0150] As indicated above, the DMO may be in contact body, an oxidoreductas, a p450, a peroxydase, a hydro- with a nutrient solution during the process. Thus, a ther- genase, a dehydrogenas, a catalase, a transferase, a apeutic agent generated by the DTMO may be secreted 25 hydrolase, an isomerase, a ligase, an aminoacyl-trna into the solution where its concentration can be meas- synthetase, a kinase, a phosphoprotein, a mutator trans- ured. The gene of interest may be any gene which en- poson, an oxidoreductas, a cholinesterase, a glucoamy- codes to any RNA molecule (sense or antisense), pep- lase, a glycosyl hydrolase, a transcarbamylase, a nucle- tide, polypeptide, glycoprotein, lipoprotein or combina- ase, a meganuclease, a ribonuclease, an atpase, a pepti- tion thereof or to any other post modified polypeptide. In 30 dase, a cyclic nucleotide synthetase, a phosphodieste- one embodiment of the invention, the gene of interest rase, a phosphoprotein, a DNA or RNA associated pro- may be naturally expressed in the tissue sample. In an- tein, a high mobility group protein, a paired box protein, other embodiment of this invention, the tissue sample a histone, a polymerase, a DNA repair protein, a ribos- may be genetically engineered so that at least one cell omal protein, an electron transport protein, a globin, a will express the gene of interest, which is either not nat- 35 metallothionein, a membrane transport protein, a struc- urally expressed by the cell or has an altered expression tural protein, a receptor, a cell surface receptor, a nuclear profile within the cell. receptor, a G-protein, an olfactory receptor, an ion chan- [0151] As used herein, the term "nucleic acid" refers nel receptor, a channel, a tyrosine kinase receptor, a cell to polynucleotide or to oligonucleotides such as deoxpri- adhesion molecule or receptor, a photoreceptor, an ac- bonucleic acid (DNA), and, where appropriate, ribonu- 40 tive peptide, a protease inhibitor, a chaperone, a chap- cleic acid (RNA) or mimetic there of The term should also eronin, a stress associated protein, a transcription factor be understood to include, as equivalents, analogs of ei- and a chimeric protein, ther RNA or DNA made from nucleotide analogs, and, [0154] In one embodiment the amount of protein se- as applicable to the embodiment being described, single creted by the DMO of the invention is at least 1.6P g/ (sense or antisense) and double-stranded polynucle- 45 DTMO/day at the pre-implantation day. otide. This term includes oligonucleotides composed of [0155] In one embodiment of this invention, the gene naturally occurring nucleobases, sugars and covalent in- of interest may encode to erythropoietin or to equivalent ternucleoside (backbone) linkages as well as oligonucle- protein thereof. otides having non-naturally-occurring portions which [0156] In another embodiment of the invention, the function similarly. Such modified or substituted oligonu- 50 gene of interest may encode, without limitation, to any of cleotides are often preferred over native forms because the following proteins, any combination of the following of desirable properties such as, for example, enhanced proteins and any equivalents thereof: insulin, trypsino- cellular uptake, enhanced affinity for nucleic acid target gen, chymotrypsinogen, elastase, amylase, serum thym- and increased stability in the presence of nucleases. ic factor, thymic humoral factor, thymopoietin, gastrin, [0152] As is known to those of skill in the art, the term 55 secretin, somatostatin, substance P, growth hormone, a "protein", "peptide" or "polypeptide" means a linear pol- somatomedin, a colony stimulating factor, erythropoietin, ymer of amino acids joined in a specific sequence by epidermal growth factor, hepatic erythropoietic factor peptide bonds. As used herein, the term "amino acid" (hepatopoietin), a liver-cell growth factor, an interleukin,

15 29 EP 2 377 401 A1 30 a negative growth factor, fibroblast growth factor and growth factor 16, Fibroblast growth factor 17, Fibroblast transforming growth factor of the β family, Interferon α, growth factor 18, Fibroblast growth factor 19, Fibroblast Interferon .β Interferon .γ, human growth hormone, G- growth factor 2, Fibroblast growth factor 20, Fibroblast CSF, GM-CSF, TNF-receptor, PDGF, AAT, VEGF, Su- growth factor 3, Fibroblast growth factor 4, Fibroblast per oxide dismutase, Interleukin, TGF-β, NGF, CTNF, 5 growth factor 5, Fibroblast growth factor 6, Fibroblast PEDF, NMDA, AAT, Actin, Activin beta-A, Activin beta- growth factor 7, Fibroblast growth factor 8, Fibroblast B, Activin beta-C Activin beta-E Adenosine Deaminase growth factor 9, Fibronectin, focal-adhesion kinase adenosine deaminase Agarase-Beta, Albumin HAS Al- (FAK), Follitropin alfa, Galactose Oxidase, Galactosi- bumin, Alcohol Dehydrogenase Aldolase, Alfimeprase dase, Beta, gamaIP-10., gastrin, GCP, G-CSF, Glial de- Alpha 1-Antitrypsin Alpha Galactosidase Alpha-1-acid 10 rived Neurotrophic Factor (GDNF), Glial fibrillary acidic Glycoprotein (AGP), Alpha-1-Antichymotrypsin, Alpha- Protein, Glial filament protein (GFP), glial-derived neu- 1Antitrypsin AT, Alpha-1-microglobulin A1M, Alpha-2- rotrophic factor family receptor (GFR), globulin, Glucose Macroglobulin A2M, Alpha- Fetoprotein, Alpha-Galactos- Oxidase, Glucose-6-Phosphate Dehydrogenase, Glu- idase, Amino Acid Oxidase, D-, Amino Acid Oxidase, L-, cosidase, Alpha, Glucosidase, Beta, Glucuronidase, Be- Amylase, Alpha, Amylase, Beta, Angiostatin, Angi-15 ta, Glutamate Decarboxylase, Glyceraldehyde-3-Phos- otensin, Converting Enzyme, Ankyrin, Apolipoprotein, phate Dehydrogenase, Glycerol Dehydrogenase, Glyc- APO-SAA, Arginase, Asparaginase, Aspartyl Ami- erol Kinase. Glycogen Phosphorylase ISO BB, Granulo- notransferase, Atrial Natriuretic factor (Anf), Atrial Natri- cyte Macrophage Colony Stimulating Factor (GM- CSF), uretic Peptide, Atrial natriuretic peptide (Anp), Avidin, Be- growth stimulatory protein (GRO), growth hormone, ta-2-Glycoprotein 1, Beta-2-microglobulin, Beta-N- 20 Growth hormone releasing hormone, Hemopexin, hepat- Acetylglucosaminidase B-NAG, beta amyloid, Brain ic erythropoietic factor (hepatopoietin), Heregulin alpha, natriuretic protein (Bnp), Brain- derived neurotrophic fac- Heregulin beta 1, Heregulin beta 2, Heregulin beta 3, tor (BDNF), Cadherin E, Calc a, Calc b, Calcitonin, Cal- Hexokinase, Histone. Human bone morphogenetic pro- cyclin, Caldesmon, Calgizzarin, Calgranulin A, Calgran- tein, Human relaxin H2, Hyaluronidase, Hydroxysteroid ulin C, Calmodulin, Calreticulin, Calvasculin, Carbonic 25 Dehydrogenase, Hypoxia-Inducible Factor- 1 alpha (HIF- Anhydrase, Carboxypeptidase, Carboxypeptidase A, 1 Alpha), I-309/TCA-3, IFN alpha, IFN beta, IFN gama, Carboxypeptidase B, Carboxypeptidase Y, CARDIAC IgA, IgE, IgG, IgM, Insulin, Insulin Like Growth Factor I TROPONIN I, CARDIAC TROPONIN T, Casein, Alpha, (IGF-I), Insulin Like Growth Factor II (IGF- II), Interferon, Catalase, Catenins, Cathepsin D, CD95L, CEA, Cellula- Interferon-inducible T cell alpha chemoattractant (I- se, Centromere Protein B, Ceruloplasmin, Ceruplasmin, 30 TAC), Interleukin, Interleukin 12 beta, Interleukin 18 bind- cholecystokinin, Cholesterol Esterase, Cholinesterase, ing protein, Intestinal trefoil factor, IP 10, Jagged 1, Jag- Acetyl, Cholinesterase Butyryl, Chorionic Gonadotrophin ged 2, Kappa light chain, Keratinocyte Growth Factor (HCG), Chorionic Gonadatrophin Beta CORE (BchCG), (KGF), Kissl, La/SS-B, Lactate Dehydrogenase, Lactate Chymotrypsin, Chymotrypsinogen, Chymotrypsin, Chy- Dehydrogenase, L-, Lactoferrin, Lactoperoxidase, lamb- motrypsin, Creatin kinase, K-BB, CK-MB (Creatine Ki- 35 da light chain, Laminin alpha 1, Laminin alpha 2, Laminin nase-MB), CK-MM, Clara cell phospholipid binding pro- beta 1 Laminin beta 2, Laminin beta 3, Laminin gamma tein, Clostripain, Clusterin, CNTF, Collagen, Colla- 1, Laminin gamma 2, LD78beta, Leptin, leucine Ami- genase, Collagens (type 1- VI), colony stimulating factor, napeptidase, Leutenizing Hormone (LH), LIF, Lipase, liv- Complement C1q Complement C3, Complement C3a, er-cell growth factor, liver-expressed chemokine (LEC), Complement C3b-alpha, Complement C3b-beta , Com- 40 LKM Antigen,TNF, TNF beta, Luciferase, Lutenizing hor- plement C4, Complement C5, Complement Factor B, mone releaseing hormone, Lymphocyte activation gene- Concanavalin A, Corticoliberin, Corticotrophin releasing 1 protein (LAG-1), Lymphotactin, Lysozyme, Macro- hormone, C-Reactive Protein (CRP), C-type natriuretic phage Inflammatory Protein 1 alpha (MIP-1 Alpha), Mac- peptide (Cnp), Cystatin C, D-Dimer, Delta 1, Delta-like rophage-Derived Chemokine (MDC), Malate Dehydro- kinase 1 (Dlk1), Deoxyribonuclease, Deoxyribonuclease 45 genase, Maltase, MCP(macrophage/monocyte chemo- I, Deoxyribonuclease II, Deoxyribonucleic Acids, Der- tactic protein)-1, 2 and 3, 4, M- CSF, MEC (CCL28), Mem- salazine, Dextranase, Diaphorase, DNA Ligase, T4, brane-type frizzled-related protein (Mfrp), Midkine, MIF, DNA Polymerase I, DNA Polymerase, T4, EGF,MIG (monokine induced by interferon gamma), MIP 2 to Elastase, Elastase, Elastin, Endocrine-gland-derived 5, MIP-1beta, Map40; P40 T-cell and mast cell growth Vascular endothelial growth factor (EG-VEGF), Elastin 50 factor, Myelin Basic Protein Myeloperoxidase, Myoglob- Endothelin Elastin Endothelin 1 Elastin Eotaxin Elastin, in, Myostatin Growth Differentiation Factor-8 (GDF-8), Epidermal growth factor (EGF), Epithelial Neutrophil Ac- Mysoin, Mysoin LC, Mysoin HC, ATPase, NADase, NAP- tivating Peptide-78 (ENA-78) ,Erythropoietin (Epo), Es- 2, negative growth factor, nerve growth factor (NGF), triol, Exodus, Factor IX, Factor VIII, Fatty acid-binding Neuraminidase, Neuregulin 1, Neuregulin 2, Neuregulin protein, Ferritin, fibroblast growth factor, Fibroblast55 3, Neuron Specific Enolase, Neuron-Specific Enolase, growth factor 10, Fibroblast growth factor 11, Fibroblast neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), Neuturin, growth factor 12, Fibroblast growth factor 13, Fibroblast NGF, NGF-Beta, Nicastrin, Nitrate Reductase, Nitric Ox- growth factor 14, Fibroblast growth factor 15, Fibroblast ide Synthesases, Nortestosterone, Notch 1, Notch 2,

16 31 EP 2 377 401 A1 32

Notch 3, Notch 4, NP-1, NT-1 to 4, NT-3 Tpo, NT- 4, Nu- terin, CNTF, Collagen, Collagenase, Collagens (type 1- clease, Oncostatin M, Ornithine transcarbamoylase, Os- VI), colony stimulating factor, Complement C1q Comple- teoprotegerin, Ovalbumin, Oxalate Decarboxylase, P16, ment C3, Complement C3a, Complement C3b-alpha, Papain, PBP, PBSF, PDGF, PDGF-AA, PDGF-AB, Complement C3b-beta , Complement C4, Complement PDGF-BB, PEDF, Pepsin, Peptide YY (PYY), Peroxi- 5 C5, Complement Factor B, Concanavalin A, Corticoliber- dase, Persephin, PF-4, P-Glycoprotein, Phosphatase, in, Corticotrophin releasing hormone, C- Reactive Protein Acid, Phosphatase, Alkaline, Phosphodiesterase I, (CRP), C-type natriuretic peptide (Cnp), Cystatin C, D- Phosphodiesterase II, Phosphoenolpyruvate Carboxyla- Dimer, Delta 1, Delta-like kinase 1 (Dlkl), Deoxyribonu- se, Phosphoglucomutase, Phospholipase, Phospholi- clease, Deoxyribonuclease I, Deoxyribonuclease II, De- pase A2, Phospholipase A2, Phospholipase C, Phospho- 10 oxyribonucleic Acids, Dersalazine, Dextranase, Diapho- tyrosine Kinase, Pituitary adenylate cyclase activating rase, DNA Ligase, T4, DNA Polymerase I, DNA Polymer- polypeptide, Placental Lactogen, Plakoglobin, Plakophi- ase, T4, EGF, Elastase, Elastase, Elastin, Endocrine- lin, Plasma Amine Oxidase, Plasma retinol binding pro- gland-derived vascular endothelial growth factor (EG- tein, Plasminogen, Pleiotrophin (PTN), PLGF-1, PLGF- VEGF), Elastin Endothelin Elastin Endothelin 1 Elastin 2, Pokeweed Antiviral Toxin, Prealbumin, Pregnancy as- 15 Botaxin Elastin, Epidermal growth factor (EGF), Epithe- soc Plasma Protein A, Pregnancy specific beta 1 glyco- lial Neutrophil Activating Peptide-78 (ENA-78) ,Erythro- protein (SP1), Prodynorphin, Proenkephalin, Progester- poietin (Epo), Estriol, Exodus, Factor IX, Factor VIII, Fatty one Proinsulin, Prolactin, Pro-melanin-concentrating acid-binding proteinFerritin Ferritin, fibroblast growth fac- hormone (Pmch), Pro-opiomelanocortin, proorphanin, tor,Fibroblast growth factor 10, Fibroblast growth factor Prostate Specific Antigen PSA, Prostatic Acid Phos-20 11, Fibroblast growth factor 12, Fibroblast growth factor phatase, PAP, Prothrombin, PSA-A1, Pulmonary sur- 13, Fibroblast growth factor 14, Fibroblast growth factor factant protein A, Pyruvate Kinase, Ranpirnase,15, Fibroblast growth factor 16, Fibroblast growth factor RANTES, Reelin, Renin, Resistin, Retinol Binding Glob- 17, Fibroblast growth factor 18, Fibroblast growth factor ulin RBP, RO SS-A 60kda, RO/SS-A 52kda, S100 (hu- 19, Fibroblast growth factor 2, Fibroblast growth factor man, brain) (BB /AB), S100 (human) BB homodimer, Sa- 25 20, Fibroblast growth factor 3, Fibroblast growth factor posin, SCF, SCGF-alpha, SCOF-Beta, SDF-1 alpha, 4, Fibroblast growth factor 5, Fibroblast growth factor 6, SDF-1 Beta, Secreted frizzled related protein 1 (Sfrp1), Fibroblast growth factor 7, Fibroblast growth factor 8, Fi- Secreted frizzled related protein 2 (Sfrp2), Secreted friz- broblast growth factor 9, Fibronectin, focal-adhesion ki- zled related protein 3 (Sfrp3), Secreted frizzled related nase (FAK), Follitropin alfa, Galactose Oxidase, Galac- protein 4-(Sfrp4), Secreted frizzled related protein 530 tosidase, Beta, gamaIP-10, gastrin, GCP, G-CSF, Glial (Sfrp5), secretin, serum thymic factor, Binding Globulin derived Neurotrophic Factor (GDNF), Glial fibrillary acid- (SHBG), somatomedin, somatostatin, Somatotropin, s- ic Protein, Glial filament protein (GFP), glial-derivad neu- RankL, substance P, Superoxide Dismutase, TGF alpha, rotrophic factor family receptor (GFR), globulin, Glucose TGF beta, Thioredoxin, Thrombopoietin (TPO), Throm- Oxidase, Glucose-6-Phosphate Dehydrogenase, Glu- bospondin 1, Thrombospondin 2, Thrombospondin 3, 35 cosidase, Alpha, Glucosidase, Beta, Glucuronidase, Be- Thrombospondin 4, Thrombospondin 5, Thrombospon- ta, Glutamate Decarboxylase, Glyceraldehyde-3-Phos- din 6, Thrombospondin 7, thymic humoral factor, thy- phate Dehydrogenase, Glycerol Dehydrogenase, Glyc- mopoietin, thymosin al, Thymosin alpha-1, Thymus and erol Kinase, Glycogen Phosphorylase ISO BB, Granulo- activation regulated chemokine (TARC), Thymus-ex- cyte Macrophage Colony Stimulating Factor (GM- CSF), pressed chemokine (TECK), Thyroglobulin Tg, Thyroid 40 growth stimulatory protein (GRO), growth hormone, Microsomal Antigen, Thyroid Peroxidase, Thyroid Per- Growth hormone releasing hormone, Hemopexin, hepat- oxidase TPO, Thyroxine (T4) , Thyroxine Binding Globu- ic erythropoietic factor (hepatopoietin), Heregulin alpha, lin TBG, TNFalpha, TNF receptor, Transferin, Transferrin Heregulin beta 1, Heregulin beta 2, Heregulin beta 3, receptor, transforming growth factor of the b family, Tran- Hexokinase, Histone, Human bone morphogenetic pro- sthyretin, Triacylglycerol lipase, Triiodothyrorzine (T3), 45 tein, Human relaxin H2, Hyaluronidase, Hydroxysteroid Tropomyosin alpha, tropomyosin-related kinase (trk), Dehydrogenase, Hypoxia-Inducible Factor-1 alpha HIF- Troponin C, Troponin I, Troponin T, Trypsin, Trypsin In- 1 Alpha), I-309/TCA-3. IFN alpha, IFN-beta, IFN gama, hibitors, Trypsinogen, TSH, Tweak, Tyrosine Decarbox- IgA, IgE, IgG, IgM, Insulin, Insulin Like Growth Factor I ylase, Ubiquitin, UDP glucuronyl transferase, Urease, (lGF-I), Insulin Like Growth Factor II (IGF-II), Interferon, Uricase, Urine Protein 1, Urocortin 1, Urocortin 2, Uro- 50 Tntarferon-induazble T cell alpha chemoattractant (I- cortin 3, Urotensin II, Vang-like 1 (Vangl1), Vang-like 2 TAC), Interleukin, Interleukin. 12 beta, Interleukin 18 (Vangl2), Vascular Endothelial Growth Factor (VEGF), binding protein, Intestinal trefoil factor, IP 10, Jagged 1, Vasoactive intestinal peptide precursor, Vimentin, Vita- Jagged 2, Kappa light chain, Keratinocyte Growth Factor mine D binding protein, Von Willebrand factor, Wnt1, (KGF), Kiss1. La/SS- B, Lactate Dehydrogenase, Lactate Wnt10a Wnt10b, Wnt11, Wnt12, Wnt13 , Wnt14, Wnt15, 55 Dehydrogenase, L-, Lactoferrin, Lactoperoxidase, lamb- Wnt16, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, da light chain, Laminin alpha 1, Laminin alpha 2, Laminin Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9, Xanthine Oxidase, beta 1 Laminin beta 2, Laminin beta 3, Laminin gamma Clara cell phospholipid binding protein, Clostripain, Clus- 1, Laminin gamma 2, LD78beta, Leptin, leucine Ami-

17 33 EP 2 377 401 A1 34 nopeptidase, Leutenizing Hormone (LH), LIF, Lipase, liv- mopoietin, thymosin a1 Thymosin alpha-1, Thymus and er-cell growth factor, liver-expressed chemokine (LEC), activation regulated chemokine (TARC), Thymus-ex- LKM Antigen, TNFbeta, Luciferase, Lutenizing hormone pressed chemokine (TECK), Thyroglobulin Tg, Thyroid releaseing hormone, Lymphocyte activation gene- 1 pro- Microsomal Antigen, Thyroid Peroxidase, Thyroid Per- tein (LAG-1), Lymphotactin, Lysozyme, Macrophage In- 5 oxidase TPO, Thyroxine (T4) , Thyroxine Binding Globu- flammatory Protein 1 alpha (MIP-1 Alpha), Macrophage- lin TBG, TNFalpha, TNF receptor, Transferin, Transferrin Derived Chemokine (MDC), Malate Dehydrogenase. receptor, transforming growth factor of the b family, Tran- Maltase, MCP(macrophage/monocyte chemotactic pro- sthyretin, Triacylglycerol lipase, Triiodothyronine (T3), tein)-1, 2 and 3, 4, M-CSF, MEC (CCL28), Membrane- Tropomyosin alpha, tropomyosin-related kinase (trk), type fiizzled-related protein (Mfrp), Midkine, MIF, MIG 10 Troponin C, Troponin I, Troponin T, Trypsin, Trypsin In- (monokine induced by interferon, gamma), MIP 2 to 5, hibitors, Trypsinogen, TSH. Tweak, Tyrosine Decarbox- MIP-1beta, Mp40; P40 T- cell and mast cell growth factor, ylase, Ubiquitin, UDP glucuronyl transferase, Urease, Myelin Basic Protein Myeloperoxidase, Myoglobin, My- Uricase, Urine Protein 1, Urocortin 1, Urocortin 2, Uro- ostatin Growth Differentiation Factor-8 (GDF- 8), Mysoin, cortin 3, Urotensin II, Vang-like 1 (Vangl1), Vang-like 2 Mysoin LC, Mysoin HC, ATPase, NADase, NAP- 2, neg- 15 (Vangl2), Vascular Endothelial Growth Factor (VEGF), ative growth factor, nerve growth factor (NGF), Neurami- Vasoactive intestinal peptide precursor, Vimentin, Vita- nidase, Neuregulin 1, Neuregulin 2, Neuregulin 3, Neu- mine D binding protein, Von Willebrand factor, Wnt1, ron Specific Enolase, Neuron-Specific Enolase, neuro- Wnt10a, Wnt10b, Wnt11, Wnt12, Wnt13, Wnt14, Wnt15, trophin-3 (NT-3), neurotrophin-4 (NT-4), Neuturin, NGF, Wnt16, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, NGF-Beta, Nicastrin, Nitrate Reductase, Nitric Oxide20 Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9 and Xanthine Oxi- Synthesases, Nortestosterone, Notch 1, Notch 2, Notch dase. 3, Notch 4, NP-1, NT-1 to 4, NT-3 Tpo, NT-4, Nuclease, [0157] Following the genetic modification process, the Oncostatin M, Ornithine transcarbamoylase, Osteopro- tissue sample may be then analyzed in order to verify tegerin, Ovalbumin, Oxalate Decarboxylase, P16, Pa- the expression of the gene of interest by the tissue sam- pain, PBP, PBSF, PDGF, PDGF- AA, PDGF-AB, PDGF- 25 ple. This could be done by any method known in the art, BB, PEDF, Pepsin, Peptide YY (PYY), Peroxidase, for example by ELISA detection of proteins or Northern Persephin, PF-4, P-Glycoprotein, Phosphatase, Acid, blot for RNA. The efficacy of a particular expression vec- Phosphatase, Alkaline, Phosphodiesterase I, Phos- tor system and method of introducing nucleic acid into a phodiesterase II, Phosphoenolpyruvate Carboxylase, cell can be assessed by standard approaches routinely Phosphoglucomutase, Phospholipase, Phospholipase 30 used in the art. For example, DNA introduced into a cell A2, Phospholipase A2, Phospholipase C, Phosphotyro- can be detected by a filter hybridization technique (e.g., sine Kinase, Pituitary adenylate cyclase activating Southern blotting) and RNA produced by transcription of polypeptide, Placental Lactogen, Plakoglobin, Plakophi- introduced DNA can be detected, for example, by North- lin, Plasma Amine Oxidase, Plasma retinol binding pro- ern blotting, RNase protection or reverse transcriptase- tein, Plasminogen, Pleiotrophin (PTN), PLGF-1, PLGF- 35 polymerase chain reaction (RT-PCR). The gene product 2, Pokeweed Antiviral Toxin, Prealbumin, Pregnancy as- can be detected by an appropriate assay, for example soc Plasma Protein A, Pregnancy specific beta 1 glyco- by immunological detection of a produced protein, such protein (SP1), Prodynorphin. Proenkephalin, Progester- as with a specific antibody, or by a functional assay to one Proinsulin, Prolactin, Pro-melanin-concentrating detect a functional activity of the gene product, such as hormone (Pmch), Pro-opiomelanocortin, proorphanin, 40 an enzymatic assay. If the gene product of interest to be Prostate Specific Antigen PSA, Prostatic Acid Phos- expressed by a cell is not readily assayable, an expres- phatase PAP, Prothrombin, PSA-A1, Pulmonary sur- sion system can first be optimized using a reporter gene factant protein A, Pyruvate Kinase, Ranpirnase,linked to the regulatory elements and vector to be used. RANTES, Reelin, Renin, Resistin, Retinol Binding Glob- The reporter gene encodes a gene product which is easily ulin RBP, RO SS-A 60kda, RO/SS-A 52kda, S100 (hu- 45 detectable and, thus, can be used to evaluate efficacy of man brain) (BB /AB), S100 (human) BB homodimer, Sa- the system. Standard reporter genes used in the art in- posin, SCF, SCGF-alpha, SCGF-Beta, SDF-1 alpha, clude genes encoding β-galactosidase, chloramphenicol SDF-1 Beta, Secreted frizzled related protein 1 (Sfrp1), acetyl transferase, luciferase, GFP/EGFP and human Secreted frizzled related protein 2 (Sfrp2), Secreted friz- growth hormone. 50 zled related protein 3 (Sfrp3), Secreted frizzled related [0158] The invention contemplates, in one aspect, the protein 4 (Sfrp4), Secreted frizzled related protein 5 use of the genetically modified DTMO for transplantation (Sfrp5), secretin, serum thymic factor, Binding Globulin in an organism. As used herein the terms "administering", (SHBG), somatomedin, somatostatin, Somatotropin, s- "introducing", "implanting" and "transplanting" may be RankL, substance P, Superoxide Dismutase, TGF alpha, used interchangeably and refer to the placement of the TGF beta, Thioredoxin, Thrombopoietin (TPO), Throm- 55 DTMO of the invention into a subject, e.g., an autologous, bospondin 1, Tbrombospondin 2, Thrombospondin 3, allogeneic or xenogeneic subject, by a method or route Thrombospondin 4, Thrombospondin 5, Tluombospon- which results in localization of the DTMO at a desired din 6, Thrombospondin 7, thymic humoral factor, thy- site. The DTMO is implanted at a desired location in the

18 35 EP 2 377 401 A1 36 subject in such a way that at least a portion of the cells cryogenically preserved by methods known in the art, for of the DTMO remain viable. In one embodiment of this example, without limitation, gradual freezing (0°C, -20°C, invention, at least about 5%, in another embodiment of -80°C, -196°C) in DMEM containing 10% DMSO, imme- this invention, at least about 10%, in another embodiment diately after being formed from the tissue sample or after of this invention, at least about 20%, in another embod- 5 genetic alteration. iment of this invention, at least about 30%, in another [0162] In accordance with an aspect of some embod- embodiment of this invention, at least about 40%, and in iments of the invention, the amounts af tissue sample another embodiment of this invention, at least about 50% including a genetically modified cell(s) to be implanted or more of the cells remain viable after administration to are determined from one or more of: Corresponding a subject. The period af viability of the cells after admin- 10 amounts of the therapeutic agent of interest routinely ad- istration to a subject can be as short as a few hours, e.g., ministered to such subjects based on regulatory guide- twenty-four hours, to a few days, to as long as a few lines, specific clinical protocols or population statistics weeks to months or years. To facilitate transplantation for similar subjects. Corresponding amounts of the ther- of the cell populations within a tissue which may be sub- apeutic agent such as protein of interest specifically to ject to immunological attack by the host, e.g., where xe- 15 that same subject in the case that he/she has received nogenic grafting is used, such as swine-human trans- it via injections or other routes previously, Subject data plantations, the DTMO may be inserted into or encapsu- such as weight, age, physical condition, clinical status. lated by biocompatible immuno-protected material such Pharmacokinetic data from previous tissue sample which as rechargeable, non-biodegradable or biodegradable includes a genetically modified cell administration to oth- devices and then transplanted into the recipient subject. 20 er similar subjects. Response to previous tissue sample Gene products produced by such cells/tissue can then which includes a genetically modified cell administration be delivered via, for example, polymeric devices de- to that subject. signed for controlled delivery of compounds, e.g., drugs, [0163] In accordance with an aspect of some embod- including proteinaceous biopharmaceuticals. A variety of iments of the invention, only some of the DTMOs are biocompatible polymers (including hydrogels, for exam- 25 used in a given treatment session. The remaining DT- ple), including both biodegradable and non-degradable MOs may be returned to maintenance (or stored cryo- polymers, can be used to form an implant for the sus- genically or otherwise), for later use. tained release of a gene product of the cell populations [0164] There is thus provided in accordance with an of the invention at a particular target site. The generation embodiment of the invention, a method of determining of such implants is generally known in the art. See, for 30 the amount of a therapeutic dermal micro organ to be example, Concise Encyclopedia of Medical & Dental Ma- implanted in a patient, the method including determining terials, ed. By David Williams (MIT Press: Cambridge, a secretion level of a therapeutic agent by a quantity of MA, 1990); the Sabel et al. US Patent No. 4,883,666; the DTMO in vitro; estimating a relationship between in Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et vitro production and secretions levels and in vivo serum al. U.S. Patent No. 5,106,627; Lim U.S. Patent No.35 levels of the therapeutic agent; and determining an 4,391,909; and Sefton U.S. Patent No. 4,353,888. Cell amount of DTMO to be implanted, based on the deter- populations within the DTMO of the present invention mined secretion level and the estimated relationship. Op- can be administered in a pharmaceutically acceptable tionally, the relationship is estimated based one or more carrier or diluent, such as sterile saline and aqueous buff- factors chosen from the following group of factors: er solutions. The use of such carriers and diluents is well 40 known in the art. a) Subject data such as weight, age, physical con- [0159] The secreted protein such as, for example with- dition, clinical status; out limitation, may be any protein according to the em- b) Pharmacokinetic data from previous DTMO ad- bodiments of the invention described above. The protein ministration to other similar subjects; and of interest may be, in one embodiment of this invention, 45 c) Pharmacokinetic data from previous DTMO ad- erythropoietin. In another embodiment of this invention, ministration to that subject. the method of the invention may be used for the expres- sion and secretion of each and any protein known in the [0165] Optionally, the relationship is estimated based art and combinations thereof. In addition, the method of on at least two of said factors. Optionally, the relationship the invention may be used for the expression of RNA50 is based on three of said factors. molecules (sense or antisense) [0166] In an embodiment of the invention, determining [0160] Alternatively, the DMO, which includes geneti- an amount of a DTMO to be implanted in a patient is also cally modified cells can be kept in vitro and the therapeu- based on one or both of: tic agent, left in the supernatant medium surrounding the tissue sample, can be isolated and injected or applied to 55 corresponding amounts of the same therapeutic pro- the same or a different subject. tein routinely administered to such subjects based [0161] Alternatively or additionally, a dermal micro or- on regulatory guidelines, specific clinical protocols gan which includes a genetically modified cell can be or population statistics for similar subjects; and

19 37 EP 2 377 401 A1 38

corresponding amounts of the same therapeutic [0171] Reference in now made to Figure 22, which agent specific to that same subject in the case the schematically illustrates a system 2207 for processing a subject has received it previously via injections or harvested DMO 2204, according to some exemplary em- other administration routes. bodiments of the invention. 5 [0172] According to some exemplary embodiments of [0167] In an embodiment of the invention, the method the invention, system 2207 may include a bioreactor includes preparing an amount of DTMO for implantation, 2200 having one or more processing chambers 2202, in accordance with the determined amount. each adapted to accommodate a DMO 2204. Bioreactor [0168] There is also provided in accordance with an 2200, which in one exemplary embodiment has a number embodiment of the invention, method of adjusting the 10 of chambers equal to the number of DMOs harvested dosage of a therapeutic agent produced by a DTMO im- from a particular subject, may be adapted to provide one planted in a subject and excreting a therapeutic agent, or more of processing chambers 2202 with a suitable including (a) monitoring level of therapeutic agent in the fluid or fluids, e.g., a growth medium, from a local fluid subject; (b) comparing the level of agent to a desired reservoir 2208 and/or discharge the fluid of one or more level; (c) if the level is lower than a minimum level, then 15 of processing chambers 2202, e.g., to a waste container implanting additional DTMO; (d) and if the level is higher 2210, as described below. The fluid may be supplied to than a maximum level, then inactivating or removing a reservoir 2208 via an inlet line 2242, e.g., connected by portion of the implanted DTMO . Optionally, the method a sterile connector 2258 to reservoir 2208, as described includes periodically repeating (a) - (d). Alternatively or below. additionally, inactivating or removing consists of remov- 20 [0173] DMO 2204 may be transferred to chamber 2202 ing a portion of the implanted DTMO. Optionally, remov- using a cutting tool used for harvesting DMO 2204, e.g., ing includes surgical removal. Alternatively or addition- as described above. The DMO transfer into chamber ally, inactivating or removing includes inactivating. Op- 2202 may be preferably performed directly after harvest- tionally, inactivating includes killing a portion of the im- ing DMO 2204 and while maintaining sterile conditions. planted DTMO. Optionally, inactivating includes ablating 25 Processing chamber 2202 may include a DMO insertion a portion of the implanted DTMO. port 2201 adapted for receiving DMO 2204. For example, [0169] As described above with reference to Fig. 1, at port 2201 may include a sterile septum interface capable least part of the process of sustaining the DMO during of receiving a blunt cannula, e.g., a SafeLine 4 Injection the genetic alteration, as well as the genetic alteration Site marketed by B. Braun Medical Inc. Once the tip of itself, may be performed in a bioreactor, as described 30 the cutting tool is inserted through the septum, DMO 2204 below. may be gently flushed into chamber 2202 in a generally [0170] According to some embodiments of the inven- sterile manner, e.g., using a syringe connected to the tion, the bio- reactor may have some or all of the following back end of the cutting tool. According to one exemplary properties: embodiment, DMO 2204 may be flushed into a medium 35 bath 2206 within chamber 2202. Alternatively, if, for ex- Allow for the provision of nutrients and gasses to the ample, DMO 2204 was harvested with an inner guide, surfaces of the DMO so that they may diffuse into e.g., described above, a lid 2232 fitted over chamber the DMO and the DMO may remain viable. Thus, 2202, e.g., as described below, may be removed, DMO significant areas and volumes of the DMO may not 2204 may be gently removed from the inner guide and be blocked from coming into contact with a surround- 40 placed within chamber 2202, and lid 2232 may be re- ing fluid. turned and sealed over chamber 2202 to maintain sterility Allow for the maintenance of the DMO at a desired of chamber 2202. temperature. [0174] Bioreactor 2200 may be adapted to apply, e.g., Allow for the maintenance of a desired pH and gas in a generally identical manner, one or more processes composition in the vicinity of the DMO . 45 to DMOs being accommodated within at least some of Allow for the removal of waste products from the the processing chambers. According to exemplary em- DMO and/or from the bioreactor. bodiments of the invention, bioreactor 2200 may be Allow for a simple method of inserting the genetically adapted to fluidically separate the contents of one or modifying vector without substantial danger that the more of the processing chambers from the contents of inserting vector will contaminate the surroundings. 50 one or more other processing chambers, as described Allow for the removal of excess unused vector. below. Allow for measurement of the amount of therapeutic [0175] According to exemplary embodiments of the in- agent generated. vention, bioreactor 2200 may also include a mechanism Allow for removal of substantially sterile therapeutic for controlling the flow of a fluid into and/or out of process- agent. 55 ing chamber 2202, as described below. Allow for easy insertion of the DMO and removal of [0176] According to an exemplary embodiment, biore- all or measured amounts of DTMO. actor 2200 may include a sterile buffer 2222 fluidically connected to a non-sterile syringe pump 2214, which

20 39 EP 2 377 401 A1 40 may be adapted to inject air into buffer 2222 and/or dis- [0181] According to some exemplary embodiments of charge air from buffer 2222 in a sterile manner, e.g., via the invention, air may be used to purge fluid located in a sterile filter 2220, e.g. a 0.45 Pm pore air filter. Biore- one or more "passage lines", e.g., fluidically connecting actor 2200 may also include a control valve 2212 able to between inlet reservoir 2208, waste container 2210 be moved between at least four positions, e.g., an inlet- 5 and/or chamber 2202, for example, in order to "flush" the buffer position wherein inlet reservoir 2208 is fluidically passage lines after transferring fluid to/from chamber connectedto buffer 2222, an outlet- buffer positionwhere- 2202, inlet reservoir 2208, and/or buffer 2222. This as- in waste container 2210 is fluidically connected to buffer pect may be useful, for example, in order to reduce a 2222, a chamber-buffer position wherein chamber 2202 "dead volume" of fluid, which may be "trapped" in one or is fluidically connected to buffer 2222, and/or a no-con- 10 more of the passage lines. For example, controller 2286 nectionposition wherein buffer 2222, chamber 2202, inlet may controllably activate motor 2216 to move syringe reservoir 2208, and waste container 2210 are fluidically plunger 2218 such that a predetermined volume of air is disconnected from each other. A piston 2226 may con- drawn into buffer 2222, before drawing the fluid from res- nect between valve 2212 and a motor 2224 adapted to ervoir 2208 into buffer 2222. Buffer 2222 may have a move valve 2212 between the different positions. Option- 15 geometry such that the air will rise above the fluid within ally, a bellows diaphragm 2228 may be fitted over piston buffier 2222, such that upon actuation of syringe pump 2226 such that there is substantially no transfer of non- 2214 the fluid in buffer 2222 may be discharged first, sterile air from into the sterile buffer 2222, e.g., during followed by the air, which will act to flush the passage motion of piston 2226. lines of some or all of the fluid remaining therein. [0177] System 2201 may also include a motor 2216 to 20 [0182] According to some exemplary embodiments of actuate a plunger 2218 of syringe pump 2214. If biore- the invention; a bottom surface 2230 of chamber 2202 actor 2200 includes more than one chamber, then either may include a plurality of holes, or a mesh-like pattern, one motor may be implemented for simultaneously ac- e.g., configured to enable the fluid to be transferred into tuating each one of the plungers associated with the and/or out of chamber 2202 in a substantially uniform chambers, or a plurality of motors may be implemented, 25 manner, and/or to allow discharging substantially most each able to actuate one or more of the plungers. of the fluid from chamber 2202 This configuration may [0178] According to exemplary embodiments of the in- also enable reducing the occurrence of "dead- spots", i.e., vention, system 2201 may include a controller 2286 able areas of chamber 2202 in which the fluid remains stag- to control the operation of motor 2216 and/or motor 2224, nant and/or is not refreshed. e.g., as described below. 30 [0183] According to some exemplary embodiments, lid [0179] According to exemplary embodiments of the in- 2232 may be a removable sterile lid, such as a membrane vention, fluid from reservoir 2208 may be controllably affixed by a detachable adhesive, silicon plug material, transferred into chamber 2202, e.g., in order to fill cham- or the like. Lid 2232 may be adapted to maintain a sterile ber 2202. For example, controller 2286 may activate mo- "barrier" between chamber 2202 and the environment. tor 2224 to position valve 2212 at the inlet- buffer position, 35 Optionally, a sterile air filter 2234, e.g., a 0,45Pm pore and controllably activate motor 2216 such that syringe air filter, may be implemented to fluidically connect cham- pump 2214 evacuates a predetermined quantity of air ber 2202 and the environment, thus enabling equilibra- from buffer 2222. As a result a predetermined volume of tion of pressures while maintaining a sterile barrier be- fluid corresponding to the predetermined volume of air tween chamber 2202 and the environment. Alternatively, may be "drawn" from inlet reservoir 2208 into buffer 2222. 40 lid 2232 may include a "breathable" material, such that Controller 2286 may then controllably activate motor pressure equilibration may be enabled through lid 2232. 2224 to move valve 2212 to the chamber- buffer position, [0184] Reservoir 2208 and/or waste container 2210 and controllably activate motor 2216 such that syringe may be commonly connected, e.g., via one or more man- pump 2214 discharges the fluid of buffer 2222 into cham- ifolds (not shown), to one or more of processing cham- ber 2202. In a similar manner, the syringe pump and con- 45 bers 2202 for a specific subject. Alternatively, inlet res- trol valve may be controlled to discharge the contents of ervoir 2208 and/or waste container 2210 may be individ- chamber 2202, or a partial amount thereof, into waste ually connected to each one of the processing chambers. container 2210. Inlet reservoir 2208 and/or waste container 2210 may [0180] According to some exemplary embodiments of includea mechanism for equilibrating pressure in asterile the invention, the fluid in chamber 2202 may be control- 50 manner. For example, inlet reservoir 2208 and/or waste lably stirred and/or mixed, e.g., in order to assist viral container 2210 may be fluidically connected to the envi- transduction and/or any other ex-vivo maintenance pro- ronment via a sterile air filter 2236 and/or a sterile air filter cedure applied to DMO 2204. For example, controller 2238, respectively. Filter 2236 and/or filter 2238 may in- 2286 may controllably activate motor 2216 and/or motor clude, for example, a 0.45 Pm pore air filter. Alternatively, 2224, e.g., as described above, to periodically discharge 55 waste container 2210 may include an expandable waste the fluid, or a part thereof, from chamber 2202 into buffer, container, such that no pressure equilibration is required and thereafter to inject the fluid in buffer 2222 back into and, therefore, no sterile air filter need be used for it. chamber 2202. [0185] According to an exemplary embodiment of the

21 41 EP 2 377 401 A1 42 invention, bioreactor 2200 may be adapted to enable di- have a storage shelf life of 9 days at refrigerated 4°C rect injection of fluid or discharging of fluid to/ from cham- conditions. Thus, a refrigeration system (not shown) may ber 2202. A sampling septum port 2240 may be used, be employed to maintain the fluid of tanks 2244 and/or for example, for direct injection of viral vector fluid, or for 2246 at a temperature, which may be lower than the in- sampling of growth medium to test for various bioreactor 5 cubation temperature of reservoir 2208. Accordingly, in- parameters, such as ELISA, glucose uptake, lactate pro- let line 2242 may pass through an interface between re- duction or any other indicative parameter. Septum port frigerator conditions to incubator conditions. After the 2240 may include a standard silicon port adapted for nee- shelf life has expired, tank 2244 and/or tank 2246 may dle insertion or a cannula port, e.g., as described above be replaced by new tanks. with reference to DMO insertion port 2201. A syringe (not 10 [0190] According to an exemplary embodiment, at shown) may be detachably inserted through septum port least some of the elements of bioreactor 2200 may be 2240. The syringe may be driven by a motor, e.g., similar formed of disposable sterile plastic components. Accord- to motor 2216, which may be activated manually or au- ing to these embodiments, bioreactor apparatus 2200 tomatically, e.g., by controller 2286. may include a single-use sterilely packaged bioreactor [0186] According to exemplary embodiments of the in- 15 apparatus, which may be conveyed to a DMO harvesting vention, at least some, and in some exemplary embodi- site and may be opened in a sterile environment and ments all, components of bioreactor 2200 may be main- prepared on site such that growth medium is injected into tained at predetermined conditions, e.g., incubator con- each bioreactor chamber 2202. The tool used for har- ditions, including a temperature of approximately 37°C, vesting the DMOs may be inserted through the DMO in- a gaseous atmosphere of approximately 90-95% air and 20 sertion ports 2201 to flush the DMOs into chambers 2202 approximately 5-10% CO2, and/or a relatively high de- in a sterile fashion, as described above. Bioreactor ap- gree of humidity, e.g., 85-100%. According to one exem- paratus 2200 may be transported, e.g., under incubator plary embodiment, only chamber 2202 may be main- conditions, to a processing site where it may be connect- tained in the incubator conditions. As described above, ed to other components of system 2207, e.g., connector these incubator conditions may be required, e.g., for25 2258, motors 2216 and/or 2224, pinch valves 2252, maintaining the vitality of the DMO tissue culture. and/or peristaltic pump 2256. Controller 2286 may then [0187] According to exemplary embodiments of the in- control the maintenance and transduction of the DMOs vention, a fluid supply arrangement may be implemented during the entire ex-vivo processing in which the DTMO for supplying fluid to inlet line 2242 from at least one fluid is produced from the harvested DMO. The dosage need- tank, e.g., fluid tanks 2244 and 2246. In one exemplary 30 ed for the specific subject may be determined by use of embodiment, tanks 2244 and 2246 may contain the same the pharmacokinetic model, e.g., as described herein. fluid, e.g., a growth medium, in which case one tank may Bioreactor apparatus 2200 may then be detached from be used as a backup reservoir for the other tank. In an- system 2207 and transported, e.g., under incubator con- other exemplary embodiment, tanks 2244 and 2246 may ditions, to the site of implantation. In order to implant a contain two different types of fluids, such as two types of 35 specific DTMO, e.g., according to the implantation meth- growth medium to be used at different stages of DMO ods described above, bioreactor chamber 2202 for the processing. Tank 2244 and/or tank 2246 may include a specific DTMO may be opened by removing lid 2232 and sterile air filter to equilibrate pressure in a sterile manner, the DTMO may be removed from the chamber. e.g., as described above with reference to reservoir 2208. Alternatively, tank 2244 and/or tank 2246 may include a 40 Examples collapsible tank, e.g., a sterile plastic bag as is known in the art. Example 1 [0188] According to exemplary embodiments of the in- vention, each of tanks 2244 and 2246 may be fluidically In vitro secretion levels of human erythropoietin by connected to a combining connector 2254 via a valve 45 DTMO -hEPO 2252, e.g., a pinch valves, a septum port connector 2248 and a penetration spike 2250. Connector 2254 may in- [0191] Experiments were conducted to assay the var- clude, for example a Y-shaped or a T- shaped connector iability of in vitro hEPO secretion level between DTMOs- as is known in the art. Valve 2252 may be adapted to hEPO obtained from different human skin samples. control the flow of fluid from tank 2244 and/or tank 2246 50 to connector 2254. A pump, e.g., a peristaltic pump, 2256 Experimental Procedure may be located between connector 2254 and connector 2258, along inlet line 2242. Controller 2286 may be used [0192] DTMO-hEPO was prepared (in triplicates) from to control the amount and/or flow-rate of the fluid provided skin samples obtained from six different human subjects to reservoir 2208 by controllably actuating motor 2257 55 and hEPO secretion levels were measured at various and/or valves 2252. point in time, as indicated in Figure 4, after the viral vector [0189] According to one exemplary embodiment, the was washed. fluid contained within tanks 2244 and/or tank 2246 may

22 43 EP 2 377 401 A1 44

Experimental Results and physiological response were identified in the two ex- perimental groups while, as expected, the control group [0193] The DTMO-hEPO secretion levels were similar mice had no hEPO in their blood. among the different human skin samples. In addition, the [0199] In all experimental groups, an elevation of he- DTMO-hEPO secretion levels were similar to the secre- 5 matocrit can be seen as early as 15 days post-implanta- tion levels of hEPO previously obtained from split thick- tion and is maintained for more than 5 months, while the ness TMO-hEPO (data not shown). MO/lavZ control mice do not show such an elevation in hematocrit level. DTMO-hEPO seems to result in similar Example 2 secretion levels for similar time periods when compared 10 to split thickness derived TMO-hEPO. Histology Example 5 [0194] In order to verify that the DTMO contains mainly dermal components, a histological analysis was per- DTMO-hEPO do not form keratin cysts when im- formed. MOs were prepared from either split thickness 15 planted sub-cutaneously skin or dermal skin samples and histological analysis was performed by a dermato-pathologist. As can be seen on Experimental Procedure the left side of Figure 5, the DTMO contains dermal layers and dermal components without residual basal and/or [0200] DTMO-hEPO and split thickness derived TMO- epidermal layers, In comparison, the split thickness TMO, 20 HEPO were implanted S.C. in SCID mice and keratin shown on rightside of Figure 5, containsall the skin layers cyst formation was monitored by clinical and histological including the basal and epidermal layers. analysis.

Example 3 Experimental Results 25 Immunocytochemistry studies [0201] As can be clearly seen in Figure 8, keratin cyst formation was observed while implanting the split thick- [0195] To study which cells are transduced in the DT- ness derived TMO-hEPO 76 and 141 days post implan- MO-hEPO tissue, a histological immunohistochemistry tation. In contrast, no cyst formation was observed in analysis of DTMO-hEPO was performed on day 9 post- 30 SCID mice with the DTMO- hEPO 113 days post implan- harvesting, using an anti-hEPO monoclonal antibody (1: tation. 20 dilution). Analysis revealed strong staining of dermal fibroblasts, as shown in Figure 6. The staining was Example 6 spread throughout the entire DTMO. 35 Split thickness derived and DMO integration in Example 4 healthy human subjects

Comparison of long term hEPO hematopoietic ac- Experimental Procedure tivity in SCID mice derived from DTMO-HEPO and Entire TMO 40 [0202] Human Dermal MO and human split thickness derived Split thickness derived TMO were obtained using [0196] An experiment was performed to examine and a commercially available dermatome (Aesculap GA 630). comparethe longterm effects of subcutaneously implant- Prior to harvesting, topical and local anesthesia for both ed DTMO- HEPO and Split thickness derived TMO- hEPO the donor and recipient site were performed using Emla in SCID mice. 45 lotion (topical anesthesia) and subcutaneous injections of Marcain+Adrenalin (local anesthesia). Experimental Procedure [0203] Two types of skin samples were harvested in order to produce human Dermal MO and human split [0197] Human DTMO-hEPO and human Split thick- thickness derived MO. For human split thickness derived ness derived TMO-hEPO were prepared and implanted 50 MO, a strip of healthy skin was excised from the lower subcutaneously in two groups of SCID mice (five mice part of the abdomen. From this skin section, six linear per group). A control group was implanted with human MOs were prepared as previously described. Simultane- DTMO and Split thickness derived TMO transduced with ously, slits of specific dimensions were made in the im- an Ad/lacZ viral vector. plantation site using an adjustable slit maker, and MOs 55 were grafted shortly after into the skin slits. For preparing Experimental Results Human Dermal MO, skin was harvested in two steps. First, a skin flap of 200 Pm in depth was harvested and [0198] As is shown in Figure 7, similar secretion levels kept on moist gauze. From this harvest site, a 1 mm deep

23 45 EP 2 377 401 A1 46 dermis skin strip was harvested. Following skin harvest- changed every 2-4 days and analyzed for the presence ing, the 200 Pm skin flap was placed back on the donor of secreted hEPO using a specific ELISA kit (Cat. # site serving as a biological dressing. From the dermis DEP00, Quantikine IVD, R&D Systems). strip harvested above, four dermal MOs were prepared [0207] The above described miniature swine skin utilizing an identical procedure as for the split thickness 5 hEPO linear TMOs were implanted both sub- cutaneously derived Split thickness derived TMO MO. The human and grafted as skin grafts in several immune competent Dermal MO were implanted subcutaneously shortly after, miniature swines (in two of the miniature swine, the using a trocar. The donor and implantation sites were TMOs-hEPO were implanted subcutaneously, and in two dressed using Bioclusive4 transparent membrane different miniature swine, TMOs-hEPO were grafted in (Johnson& Johnson, USA). After one week the dressing 10 1mm deep slits). A sufficient number of TMOs-hEPO was changed and the implants were examined to check were implanted in each miniature swine so that their com- graft integration. Two to three weeks following the MO bined pre-implantation secretion level in each pig was implantation, the scheduled abdominoplasty procedure approximately 7 micrograms per day. Elevated serum was performed and a section of skin, including the graft hEPO levels (Figure 3A) determined by an ELISA assay and implantation area was excised. A clinical evaluation 15 and reticulocyte count elevation were obtained for seven was performed on the graft area including photographs days after implantation. and histological examination to determine MO integra- [0208] It will thus be clear, the present invention has tion. been described using non-limiting detailed descriptions of embodiments thereof that are provided by way of ex- Experimental Results 20 ample and that are not intended to limit the scope of the invention. For example, only a limited number of genetic [0204] A clinical inspection, which was performed one changes have been shown. However, based on the week after implantation, and histological analysis, which methodology described herein in which live tissue is re- was performed soon after abdominaplasty (2-3 weeks planted in the body of the patient, and the viability of that after grafting), revealed excellent integration of the graft- 25 tissue in the body after implantation, it is clear that virtu- ed MOs into the skin slits and at the dermal MOs subcu- ally any genetic change in the tissue, induced by virtually taneous implantation sites (Figure 9). No indication of any known method will result in secretions of target pro- inflammation or swelling was found on either split thick- teins or other therapeutic agents in the patient. ness derived MOs that were implanted into the slits or [0209] Variations of embodiments of the invention, in- Dermal Mos that were implanted subcutaneously. 30 cluding combinations of features from the various em- bodiments will occur to persons of the art. The scope of Example 7 the invention is thus limited ony by the scope of the claims. Furthermore, to avoid any question regarding the Autologous implantation of miniature swine skin lin- scope of the claims, where the terms "comprise" "in- ear split thickness TMOs, expressing human eryth- 35 clude," or "have" and their conjugates, are used in the ropoietin (hEPO into immuno competent animals) claims, they mean "including but not necessarily limited to". [0205] Linear (30.6 mm long and 0.6 micrometer wide) [0210] The present application further encompasses miniature swine (Sinclar swine) skin micro organs were the following embodiments: prepared from fresh skin tissue samples obtained from 40 live animals under general anesthesia procedures. Tis- 1. A dermal micro organ comprising a plurality of sue samples of 0.9-1.1mm split skin thickness (depth) dermal components, which substantially retain the were removed using a commercial dermatome (Aescu- micro-architecture and three dimensional structure lap GA630) and cleaned using DMEM containing of the dermal tissue from which they are derived, glutamine and Pen-Strep in Petri dishes (90 mm). 45 having dimensions selected so as to allow passive [0206] In order to generate the linear micro organs, the diffusion of adequate nutrients and gases to cells of above tissue samples were cut by a press device using said dermal micro organ and diffusion of cellular a blade structure as described above, into the desired waste out of said cells so as to minimize cellular tox- dimensions: 30.6 mm x 600 micrometers. The resulting icity and concomitant death due to insufficient nutri- linear micro-ograns were placed, one per well, in a 24- 50 tion and accumulation of waste in said dermal micro well micro-plate containing 500 Pl per well of DMEM (Bi- organ. ological Industries - Beit Haemek) in the absence of se- 2. The dermal micro organ of 1, wherein said dermal rum under 5 % CO2 at 37°C for 24 hours. Each well un- micro organ produces not more than a negligible derwent a transduction procedure in order to generate a amount of keratin. miniature swine skin therapeutic micro organ (pig skin- 55 3. The isolated dermal organ according to 1 or 2 hav- TMO) using an adeno viral vector (1x10 10IP/ml) carrying ing a length of approximately 5-100 mm. the gene for human erythropoietin (Adeno- hEPO) for 24 4. The dermal micro organ of any one of 1-3, wherein hours while the plate was agitated. The medium was said dermal micro organ includes part of the cross-

24 47 EP 2 377 401 A1 48 section of the dermis. any one of 14-18, wherein said at least one recom- 5. The dermal micro organ of 4, wherein said dermal binant gene product is not naturally produced by the micro organ includes most of the cross-section of organ from which the dermal micro- organ is derived. the dermis. 21. The genetically modified dermal micro organ of 6. The dermal micro organ of 5, wherein said dermal 5 any one of 14-20, wherein said genetically modified micro organ includes substantially the entire cross- dermal micro organ comprises an in-vivo demarca- section of the dermis. tion. 7. The dermal micro organ of any one of 1-6, wherein 22. The genetically modified dermal micro organ of said dermal micro organ further comprises fat tissue. any one of 14-21, wherein said genetically modified 8. The dermal micro organ of any one of 1-7, wherein 10 dermal micro organ is encapsulated in an immuuno- said dermal micro organ further comprises tissue protective housing. from at least one epidermal layer. 23. A method of inducing a local or systemic physi- 9. The dermal micro organ of 8, wherein said at least ological effect in a subject comprising implanting in one epidermal layer comprises a basal epidermal the body a dermal micro organ according to any one layer. 15 of 1-22. 10. The dermal micro organ of any one of 1-9, where- 24. The method of 23, wherein implanting said der- in said dermal micro organ is maintainable in-vitro mal micro organ comprises implanting said dermal for at least several days. micro organ into or underneath the skin. 11. The dermal micro organ of 10, wherein said der- 25. A method of delivering a gene product of interest mal micro organ is maintainable in- vitro in for at least 20 to a subject comprising implanting in the body the several weeks. genetically modified dermal micro organ of any one 12. The dermal micro organ of any one of 1-11, of 14-24. wherein said dermal micro organ comprises an in- 26. The method of 25, wherein implanting said ge- vivo demarcation. netically modified dermal micro organ comprises im- 13. The dermal micro organ of any one of 1-12,25 planting said genetically modified dermal micro or- wherein said dermal micro organ is encapsulated in gan into or underneath the skin. an immunoprotective housing. 27. The method of any one of 23-26, wherein said 14. A genetically modified dermal micro organ ex- dermal micro organ is derived from said subject. pressing at leastone recombinantgene product, said 28. The method of any one of 23-26, wherein said dermal micro organ comprising a plurality of dermal 30 dermal micro organ is derived from a donor. components,which retain themicro- architectureand 29. The method of 28, wherein said donor is a human three dimensional structure of the dermal tissue from being. which they are derived, having dimensions selected 30. The method of 28, wherein said donor is a non- so as to enable passive diffusion of adequate nutri- human animal. ents and gases to cells of said dermal micro organ 35 31. A method of determining the amount of a thera- and diffusion of cellular waste out of said cells so as peutic dermal micro organ to be implanted in a pa- to minimize cellular toxicity and concomitant death tient, the method comprising: due to insufficient nutrition and accumulation of waste in said dermal micro organ, wherein at least determining a production and/or secretion level some of said cells express at least a portion of at 40 of a therapeutic agent by a quantity of the dermal least one recombinant gene product. micro organ in vitro; 15. The genetically modified dermal micro organ of estimating a relationship between in vitro pro- 14, wherein said dermal micro organ includes part ductionand/or secretion and in vivo serum levels of the cross-section of the dermis. of the therapeutic agent; and 16. The genetically modified decimal micro organ of 45 determining an amount of therapeutic dermal 15, wherein said dermal micro organ includes most micro organ to be implanted, based on the de- of the cross-section of the dermis. termined production and/or secretion level and 17. The genetically modified dermal micro organ of the estimated relationship. 16, wherein said dermal micro organ includes sub- stantially the entire cross-section of the dermis. 50 32. A method of implanting a dermal therapeutic mi- 18. The genetically modified dermal micro organ of cro organ at an implantation site comprising: any one of 14- 17, wherein said dermal micro organ further comprises fat tissue. inserting into said implantation site a carrier hav- 19. The genetically modified dermal micro-organ of ing said dermal therapeutic micro organ aspirat- any one of 14-18, wherein said at least one recom- 55 ed therein; binant gene product is naturally produced by an or- withdrawing said carrier while holding said der- gan from which the dermal micro-organ is derived. mal therapeutic micro organ in said implantation 20. The genetically modified dermal micro-organ of site.

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33. The method of 32, wherein said implantation site gan, the apparatus comprising: is within or under the skin. 34. A method of implanting a dermal therapeutic mi- a support configuration to support a skin-related cro organ at an implantation site comprising: tissue structure from which said dermal micro 5 organ is to be harvested; and inserting into said implantation site a carrier hav- a cutting tool able to separate said dermal micro ing said dermal therapeutic micro organ aspirat- organ from said skin-related tissue structure. ed therein; applying pressure on the aspirated dermal ther- 44. The apparatus of 43, wherein said support con- apeutic micro organ such that said dermal ther- 10 figuration comprises a first tubular element, and apeutic micro organ exits from said carrier into wherein said cutting tool comprises a second tubular said implantation site. element adapted to be inserted along and substan- tially coaxially with said first element. 35. The method of 34, wherein said intended implan- 45. The apparatus of 44, wherein said first tubular tation site is intra the body. 15 element comprises an inner guide needle. 36. A method of adjusting the dosage of a therapeutic 46. The apparatus of 44 or 45, wherein said first tu- agent produced by a therapeutic dermal micro organ bular element is adapted to extract said dermal micro implanted in a subject and excreting a therapeutic organ from said second tubular element. agent, comprising: 47. The apparatus of any one of 44-46, wherein said 20 second tubular element comprises a coring tube able monitoring a level of therapeutic agent in the to cut through said skin-related tissue structure when subject; and advanced along a cutting axis. controlling the amount of therapeutic dermal mi- 48. The apparatus of 47, wherein said coring tube cro organ in the subject based on a comparison comprises a rotatable coring tube. between said level of therapeutic agent and at 25 49. The apparatus of 47 or 48, wherein at least one least one threshold level. of an inner surface and an outer surface of said cor- ing tube is at least partially coated with a low friction 37. A method according to 36, wherein said at least material. one threshold level comprises a minimum level and 50. The apparatus of 49, wherein said low friction a maximum level, and wherein controlling the30 material comprises Teflon or Parylene. amount of therapeutic dermal micro organ compris- 51. The apparatus of 44, wherein said first tubular es: element comprises a notch cutout. 52. The apparatus of any one of 43- 51, wherein said implanting additional therapeutic dermal micro support structure comprises a clamping mechanism organ if said level of therapeutic agent is lower 35 to support said skin-related tissue structure. than said minimum level; and 53. The apparatus of 43, wherein said support con- inactivating or removing a portion of the implant- figuration comprises a vacuum chamber having an ed therapeutic dermal micro organ if said level inner support surface able to maintain said skin-re- of therapeutic agent is higher than said maxi- lated tissue structure at a desired shape and position mum level. 40 to enable said cutting tool to separate said dermal micro organ from said skin-related tissue structure. 38. A method according to 37 further comprising pe- 54. The apparatus of 53, wherein said skin related riodically repeating said monitoring and said control- tissue structure is located inside said chamber when ling until the level of said agent is between said min- a vacuum condition is applied to said chamber. imum and said maximum levels. 45 55. The apparatus of 53 or 54 comprising a guiding 39. A method according to 37 or 38, wherein inacti- channel connected to said vacuum chamber and vating or removing said portion comprises removing configured to maintain said cutting tool, when insert- said portion. ed into said guiding channel, at a predetermined dis- 40. A method according to 37 or 38, wherein inacti- tance from said support surface. vating or removing said portion comprises surgically 50 56. The apparatus of 55, wherein said distance is removing said portion. predetermined based on a desired location within 41. A method according to 37 or 38, wherein inacti- said skin-related tissue structure for harvesting said vating or removing said portion comprises ablating dermal micro organ. said portion. 57. The apparatus of any one of 53-56 comprising 42. A method according to 37 or 38, wherein inacti- 55 one or more vacuum channels to fluidically connect vating or removing said portion comprises killing said at least part of said surface with at least one vacuum portion. source. 43. An apparatus for harvesting a dermal micro or- 58. The apparatus of any one of 53-57 comprising a

26 51 EP 2 377 401 A1 52 clamping configuration for clamping said skin-relat- supporting a skin-related tissue structure from ed tissue structure when said skin-related tissue which said dermal micro organ is harvested; and structure is supported by said support surface. separating said dermal micro organ from said 59. The apparatus of any one of 53-58, wherein at skin-related tissue structure. least one dimension of said chamber is predeter- 5 mined based on at least one intended dimension of 74. A method according to 73, wherein said support- said dermal micro organ. ing comprises inserting an inner guide into said skin- 60. The apparatus of any one of 53-59, wherein said related tissue structure, and wherein said separating cutting tool is adapted to extract said dermal micro comprises inserting a cutting tool along and coaxially organ from within said chamber. 10 with said inner guide. 61. The apparatus of any one of 53-60, wherein said 75. A method according to 74 wherein said support- vacuum chamber comprises an elevated protrusion ing comprises clamping said skin-related tissue able to maintain a plateau of skin generally above structure. the trajectory of the cutting tool, such that the har- 76. The method of 74 or 75 further comprising with- vested dermal micro organ is separated from the 15 drawing the inner guide so as to extract said dermal body with only one lance cut. micro organ. 62. The apparatus of any one of 43-61 comprising a 77. The method of any one of 74-76, wherein insert- rotation mechanism able to rotate said cutting tool. ing said inner guide comprises 63. The apparatus of 62, wherein said rotation mech- inserting said inner guide generally in parallel with a anism rotates said cutting tool at a speed of at least 20 skin surface of said skin-related tissue structure. 1,000 RPM. 78. The method of any one of 74-77, wherein said 64.The apparatus of 63, wherein said rotation mech- separating further comprises rotating said cutting anism rotates said cutting tool at a speed of at least tool. 2000 RPM. 79. The method of 78, wherein said rotating com- 65. The apparatus of 64, wherein said rotation mech- 25 prises rotating said cutting tool at a speed higher anism rotates said tool at a speed of approximately than 1,000 RPM. 7,000 RPM. 80. The method of 79, wherein said rotating com- 66. The apparatus of claim any one of 43-65, wherein prises rotating said cutting tool at a speed higher said skin-related tissue structure has a generally cy- than 2,000 RPM. lindrical shape. 30 81. The method of 80, wherein said rotating com- 67. The apparatus of any one of 43-66, wherein said prises rotating said cutting tool at a speed of approx- skin-related tissue structure comprises components imately 7,000 RPM. of the epidermal tissue and components of the der- 82. The method of 73, wherein supporting comprises mal tissue. applying to said skin-related tissue structure a vac- 68. The apparatus of 67, wherein said skin-related 35 uum condition. tissue structure further comprises at least some fat 83. The method of any one of 73-82, wherein said tissue and muscle tissue. skin-related tissue structure comprises components 69. The apparatus of any one of 43-68, wherein said of the epidermal tissue, and components of the der- harvested dermal micro organ comprises at least mal tissue. part of a cross-section of the dermis. 40 84. The method of 83, wherein said skin- related tis- 70. The apparatus of 69, wherein said dermal micro sue structure further comprises at least some fat tis- organ further comprises fat tissue. sue and muscle tissue. 71. The apparatus of 69 or 70, wherein said dermal 85. The method of any one of 73-84, wherein said micro organ further comprises epidermal tissue. dermal micro organ comprises at least part of the 72. The apparatus of any one of 43-71, wherein said 45 cross-section of the dermis. dermal micro organ comprises a plurality of dermal 86. The method of 85, wherein said dermal micro components, which substantially retain the micro-ar- organ further comprises fat tissue. chitecture and three dimensional structure of the der- 87. The method of any one of 73-86 comprising form- mal tissue from which they are derived, having di- ing at least one lance cut. mensions selected so as to allow passive diffusion 50 88. The method of any one of 73-87, wherein said of adequate nutrients and gases to cells of said der- dermal micro organ comprises a plurality of dermal mal micro organ and diffusion of cellular waste out components, which substantially retain the micro-ar- of said cells so as to minimize cellular toxicity and chitecture and three dimensional structure of the der- concomitant death due to insufficient nutrition and mal tissue from which they are derived, having di- accumulation of waste in said dermal micro organ. 55 mensions selected so as to allow passive diffusion 73. A method of harvesting a dermal micro organ of adequate nutrients and gases to cells of said der- from a subject, comprising: mal micro organ and diffusion of cellular waste out of said cells so as to minimize cellular toxicity and

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concomitant death due to insufficient nutrition and 10. The genetically modified dermal micro-organ of accumulation of waste in said dermal micro organ. claim 9, wherein said genetically modified dermal organ is 20-40 mm in length.

Claims 5 11. The genetically modified dermal micro-organ of any one of claims 1-10, wherein at least one dimension 1. A genetically modified dermal micro- organ express- of the cross- section of said genetically modified der- ing at least one recombinant gene product, wherein mal organ is 0.5-3.5 mm. said dermal micro-organ consisting essentially of a plurality of dermal components and not including ep- 10 12. A genetically modified dermal micro-organ accord- idermal layers, which maintain the micro-architec- ing to any one of claims 1-11 for use in a method of ture and three dimensional structure of the dermal inducing a local or systemic physiological effect in a tissue from which they are derived, having dimen- subject. sions selected so as to enable passive diffusion of adequate nutrients and gases to cells of said dermal 15 13. The genetically modified dermal micro-organ of micro-organ and diffusion of cellular waste outof said claim 12, wherein said dermal micro- organ is derived cells so as to minimize cellular toxicity and concom- from said subject. itant death due to insufficient nutrition and accumu- lation of waste in said dermal micro organ, wherein 14. A genetically modified dermal micro-organ accord- at least some of said cells of said genetically modified 20 ing to any one of claims 1-11 for use in a method of dermal micro-organ express at least a portion of at delivering erythropoietin to a subject. least one recombinant gene product, wherein said recombinant gene product is erythropoietin. 15. The genetically modified dermal micro-organ of claim 14, wherein said dermal micro- organ is derived 2. The genetically modified dermal micro-organ of 25 from said subject. claim 1, wherein said dermal micro-organ includes part of the cross-section of the dermis. 16. The genetically modified dermal micro-organ of any one of claims 12  15, wherein said use comprises 3. The genetically modified dermal micro-organ of implanting said genetically modified dermal micro- claim 2, wherein said dermal micro-organ includes 30 organ into or under the skin. most of the cross-section of the dermis.

4. The genetically modified dermal micro-organ of claim 3, wherein said dermal micro-organ includes substantially the entire cross-section of the dermis. 35

5. The genetically modified dermal micro-organ of any one of claims 1-3, wherein said dermal micro- organ further includes fat tissue. 40 6. The genetically modified dermal micro-organ of any one of claims 1-5, wherein said genetically modified dermal micro-organ comprises an in-vivo demarca- tion. 45 7. The genetically modified dermal micro-organ of claim 6, wherein said in-vivo demarcation comprises an ink or stain on the peripheral surface of said micro- organ. 50 8. The genetically modified dermal micro-organ of claim 6, wherein said in-vivo demarcation comprises a green fluorescent protein (GFP) gene or a luci- ferase reporter gene expressed by said micro- organ. 55 9. The genetically modified dermal micro-organ of any one of claims 1-8, wherein said genetically modified dermal organ is 10-60 mm in length.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• IL 020880 W [0004] • US 5106627 A, Aebischer [0158] • US 4883666 A, Sabel [0158] • US 4391909 A, Lim [0158] • US 4892538 A, Aebischer [0158] • US 4353888 A, Sefton [0158]

Non-patent literature cited in the description

• Concise Encyclopedia of Medical & Dental Materials. MIT Press, 1990 [0158]

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