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(12) United States Patent (10) Patent No.: US 7,279,318 B1 Seymour Et Al

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(12) United States Patent (10) Patent No.: US 7,279,318 B1 Seymour Et Al USOO7279318B1 (12) United States Patent (10) Patent No.: US 7,279,318 B1 Seymour et al. (45) Date of Patent: Oct. 9, 2007 (54) MODIFICATION OF BIOLOGICAL FOREIGN PATENT DOCUMENTS ELEMENTS WO WO96/21470 6, 1996 (75) Inventors: Leonard C. W. Seymour, Birmingham WO WO98,441.143 A1 * 10, 1998 (GB); Kerry David Fisher, Birmingham (GB) OTHER PUBLICATIONS Verma et al., Nature, vol. 389, 1997, pp. 239-242.* (73) Assignee: Hybrid Systems Limited, Birmingham Anderson, Nature, vol. 392-supplement, 1998, pp. 25-30.* Juengst, BMJ, vol. 326, 2003, pp. 1410–1411.* (GB) Robinson et al., “Effect of Polyethylene Glycol Conjugated to DNA-Transfecting Complexes Targeted at the Transferrin Receptor (*) Notice: Subject to any disclaimer, the term of this of HeLa Cells' Drug Delivery 4: 115-119 (1997). patent is extended or adjusted under 35 Wolfert et al., “Characterization of Vectors for Gene Therapy U.S.C. 154(b) by 604 days. Formed by Self-Assembly of DNA with Synthetic Block Co Polymers’ Human Gene Therapy 7:2123-2133 (Nov. 10, 1996). (21) Appl. No.: 10/009,347 Katayose et al., “Water-Soluble Polyion Complex Associates of DNA and Poly(ethyleneglycol)-Poly(L-lysine) Block Copolymer” (22) PCT Filed: Jun. 9, 1999 Bioconjugate Chem. 8:702-707 (1997). (86). PCT No.: PCT/GBOO/O2239 * cited by examiner Primary Examiner James Ketter S 371 (c)(1), (74) Attorney, Agent, or Firm—Pillsbury Winthrop Shaw (2), (4) Date: Jun. 26, 2002 Pittman, LLP (87) PCT Pub. No.: WO00/74722 (57) ABSTRACT PCT Pub. Date: Dec. 14, 2000 A method of modifying the biological and/or physicochemi (30) Foreign Application Priority Data cal properties of biological elements such as viruses and other micro-organisms is disclosed in which the biological Jun. 9, 1999 (GB) ................................. 99.13359.7 element is modified by providing it with a coating of a multivalent polymer having multiple reactive groups. This (51) Int. Cl. modification can enable Some biological elements to be CI2N 7/00 (2006.01) targeted or re-targeted to particular sites in a host biological (52) U.S. Cl. .................................... 435/235.1; 530/350 system and can be useful in connection with viral vectors for (58) Field of Classification Search ............. 435/320.1, gene therapy or antitumor therapy. In other cases the modi 435/235.1, 4, 6; 514/44; 536/23.1; 424/93.1 fication can be useful for enhancing or improving the See application file for complete search history. efficiency of viruses or bacterial micro-organisms used for (56) References Cited example in pest control. degradation and dispersal of oil deposits and various other industrial, environment or medi U.S. PATENT DOCUMENTS cal applications. 5,521,291 A * 5/1996 Curiel et al. ............. 530.391.7 5,656,611 A 8, 1997 Kabanov et al. 35 Claims, 13 Drawing Sheets U.S. Patent Oct. 9, 2007 Sheet 1 of 13 US 7,279,318 B1 o o t N r g in s CD trus o C o N o C C c ad c o o o o o d es c o c o d cd d o d C O ra co wr Y N r w Sdn of ougue fugueue 9, U.S. Patent US 7.279,318 B1 (2O) uuOLt OO U.S. Patent Oct. 9, 2007 Sheet 4 of 13 US 7,279,318 B1 do?KojouogonpòYI‘*’0.1tiñ!!! U.S. Patent US 7.279,318 B1 U.S. Patent Oct 9, 2007 Sheet 6 of 13 US 7,279,318 B1 snu?AouapepºleooVINGIHdJoso?s?1910e.Je?O[20]SÁ??:99in3) U.S. Patent US 7,279,318 B1 (n) do U.S. Patent (TA) do U.S. Patent Oct. 9, 2007 Sheet 9 of 13 US 7,279,318 B1 O Cp1 O CN o g) CC t SO e 1. s 2p C 2 o C siasts CD - l CD D e 8 s uo. s (n-ay) did U.S. Patent Oct. 9, 2007 Sheet 10 of 13 US 7,279,318 B1 Figure 10 AS) A9-21 A549 450 900 90 400 BOO 8O 35O 700 70 3OO SOO SO 250 500 50 2OO 400 40 150 3OO 3O OO 2OO 20 50 100 10 O O O W PC FGF VEGF V PC FGF VEGF v Yst. 7O HUWE 90 60 80 5O 70 SO 40 50 30 40 20 30 2O O O O O v. PC FGF VEGF FGF VEGF W V V --OFGF --OVEGF--of GF -FGF --oVEGF U.S. Patent Oct. 9, 2007 Sheet 11 of 13 US 7.279,318 B1 I0.in?ji? d39/gp?/-O- da??jsp7--5-IG-5- "—————————1–1—1–––––)-L) ----~---------1------ 000?.00600900100900900?y00800?00||0 000009 00009€. 00000€ 00009? 00000Z 00009! 00009 f &—00000! U.S. Patent Oct. 9, 2007 Sheet 12 of 13 US 7,279,318 B1 00||0000£ C Cd Se LI190Idau Jed ?h U.S. Patent Oct. 9, 2007 Sheet 13 of 13 US 7,279,318 B1 ver O US 7,279,318 B1 1. 2 MODIFICATION OF BIOLOGICAL but primarily reflect the inadequate levels, duration and ELEMENTS distribution of expression of therapeutic genes achieved. In short, the Successful application of Sophisticated treatment CROSS REFERENCE TO RELATED strategies is limited by inadequate vectors for gene delivery APPLICATIONS 5 and expression. Two main types of vectors for use in gene therapy This application is the National Phase of International applications have been explored so far non-viral (usually Application PCT/GB00/02239, filed Jun. 9, 2000 which based on cationic liposomes) and viral (usually retroviruses, designated the U.S., and claims priority from British Patent adenoviruses, latterly adeno-associated viruses (aav) and Application No. 9913359.7, filed Jun. 9, 1999, and which 10 lentiviruses). are incorporated herein by reference. Viruses are the obvious choice as vectors for gene deliv The present invention relates to methods of modifying the ery since this is essentially their sole function in nature. biological and/or physico-chemical properties of micro Consequently viruses have seen considerable use in gene organisms referred to hereinafter by the collective term therapy to date, forming the majority of vectors employed in “biological elements’ of which viruses constitute one impor- 15 clinical studies. The main feature of adenoviruses which tant category. The invention also relates to Such biological limits their successful application is their immunogenicity. elements which have been modified to bring about a modi Although they are professional pathogens, evolved over fication or change in their biological and/or physico-chemi millions of years as highly efficient gene delivery vectors, cal properties in accordance with the invention, processes their hosts have similarly developed very effective protec for their preparation and their use in various biotechnology 20 tion mechanisms. Serum and ascites fluid from cancer strategies in various fields, including agriculture, the petro patients contain antibodies that can completely prevent viral chemical industry, environmental science and medicine. infection in vitro even at high dilution. Typical human protocols involving adenovirus lead to significant inflam BACKGROUND matory responses, as well as inefficient infection of target 25 cells. Micro-organisms, including viruses, find many applica Although the non-viral systems have a much better safety tions throughout the broad fields of biotechnology. They are record, and are easier to produce in large quantities, they involved in medicine, agriculture, industrial production pro have low specific transfection activity and efficiency of gene cesses (including notably the oil and brewing industries) and expression in target tissues has been a major problem. bioremediation. Many useful applications and functions 30 Another major limitation to successful application of have been identified and developed for such biological presently available vectors for treatment of disease is a agents. However, often the development or enhancement of requirement for their administration directly to the site of their activities is limited by their precise properties, restrict disease, either by direct application or by intra-arterial ing their ability to fulfil tasks that are theoretically possible administration. No vectors are capable of targeting to spe but practically beyond their scope. In this situation, which is 35 cific cells following intravenous injection. Cationic lipid quite commonly encountered, it would often be desirable to systems occlude the first capillary bed they encounter, the re-engineer the properties of the virus or micro-organism to pulmonary bed, while adenoviruses/retroviruses are rapidly endow it with properties more appropriate for its required taken up by the liver and (in animal studies) mediate local purpose. toxicities. Although local administration can be feasible for Thus, biological insecticides for example such as bacu- 40 treatment of certain diseases (e.g. bronchial epithelial cystic loviruses may be restricted in their usefulness through fibrosis), other diseases have a more widespread distribution inappropriate target specificity and adverse Survival charac (notable clinical cancer and atherosclerosis) and intravenous teristics in the environment, Sulphur metabolising bacteria targeted gene delivery is crucial to embrace the possibility of may be limited in their useful application in the petrochemi Successful gene therapy. cal industry through inadequate patterns of dispersion and 45 One approach disclosed in WO 98/44143 for facilitating distribution; and in the context of human or veterinary gene clinical use of viruses has been to modify the surface of the therapy, viruses intended to mediate delivery of therapeutic viruses with a mono-functional polymer Such as poly(eth genes may be limited in their usefulness through inefficiency ylene-glycol) (PEG). This can lead to significantly of transgene expression in target tissues. decreased neutralisation of infection by serum antibodies. The field of Somatic cell gene therapy has attracted major 50 This approach retains normal receptor-binding and infection interest in recent years because it promises to improve in respect of target cells (via the CAR receptor for adenovi treatment for many different types of disease, including both rus), but presents a problem in that it does not facilitate genetic diseases (e.g.
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