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United States Patent 435/91.41:424/1991:536

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United States Patent 435/91.41:424/1991:536 USOO8802080B2 (12) United States Patent (10) Patent No.: US 8,802,080 B2 Warrington et al. (45) Date of Patent: Aug. 12, 2014 (54) RAAV EXPRESSION SYSTEMS FOR 2002/0132336 A1 9, 2002 Dwarki et al. GENETIC MODIFICATION OF SPECIFIC 2002/0194630 Al 12/2002 Manning, Jr. et al. 2003/0.148506 A1* 8, 2003 Kotin et al. ................ 435/320.1 CAPSD PROTENS 2003,0166284 A1 9, 2003 Srivastava et al. 2006/02921 17 A1* 12/2006 Loiler et al. ................. 424.93.2 (75) Inventors: Kenneth H. Warrington, Gainsville, FL Ole a (US); Shaun R. Opie, Phoenix, AZ FOREIGN PATENT DOCUMENTS (US); Nicholas Muzyczka, Gainesville, FL (US) AU 2003295312 4/2004 AU 2004226961 11, 2004 (73) Assignee: University of Florida Research A 3: 38. Foundation, Inc., Gainesville, FL (US) EP 1618 201 1, 2006 - WO WOOO. 54813 A2 9, 2000 (*) Notice: Subject to any disclaimer, the term of this WO WOO2,24234 A2 3, 2002 patent is extended or adjusted under 35 WO WO 2004/O27.019 A 4/2004 U.S.C. 154(b) by 1507 days. WO WO 2004/099423 A1 11, 2004 (21) Appl. No.: 10/513,059 OTHER PUBLICATIONS (22) PCT Filed: Mav 1, 2003 Chiorini et al. Cloning of Adeno-Associated Virus Type 4 (AAV4) y 1, and Generation of Recombinant AAV4 Particles. Journal of Virology. (86). PCT No.: PCT/USO3/13583 p. 6823-6833, Sep 1997. Hogue et al., “Chimeric Vius-like Particle Formation of Adeno-As S371 (c)(1), sociated Virus.” Biochem. Biophy's. Res. Comm., 266:371-76, 1999. (2), (4) Date: Oct. 2, 2005 Muralidhar et al., “Site-Directed Mutagenesis of Adeno-Associated Virus Type 2 Structural Protein Initiation Codons: Effects on Regu (87) PCT Pub. No.: WO2004/027019 lation of Synthesis and Biological Activity.” J. Virol. 68: 170-76, 1994. PCT Pub. Date: Apr. 1, 2004 Ruffing et al., “Assembly of Viruslike Particles by Recombinant O O Structural Proteins of Adeno-Associated Virus Type 2 in Insect (65) Prior Publication Data Cells,” J. Virol. 66:6922-30, 1992. Shi et al., “Insertional Mutagenesis of the Adeno-Associated Virus US 2006/OO88936A1 Apr. 27, 2006 Type 2 (AAV2) Capsid Gene and Generation of AAV2 Vectors Tar geted to Alternative Cell-Surface Receptors.” Human Gene Therapy, Related U.S. Application Data 12:1697-711, 2001. Warrington et al., “Adeno-Associated Virus Type 2 VP2 Capsid Pro (60) Provisional application No. 60/377,315, filed on May tein is Nonessential and Can Tolerate Large Peptide Insertions at Its 1, 2002. N Terminus. J. Virol. 78:6595-609, 2004. Wu et al., “Mutational Analysis of the Adeno-Associated Virus Type (51) Int. Cl. 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with CI2N 15/864 (2006.01) Altered Tropism.” J. Virol. 74:8635-47, 2000. CI2N 7/01 (2006.01) Yang et al., “Development of Novel Cell Surface CD34-Targeted CI2N 5/10 (2006.01) Recombinant Adenoassociated Virus Vectors for Gene Therapy.” CI2N 15/63 (2006.01) Human Gene Therapy, 9:1929-37, 1998. C07K I4/05 (2006.01) (Continued) (52) U.S. Cl. USPC ......435/91.41:424/1991:536/241,514.44 424/93.6; 435/456; 435/320.1; 435/69.1; R Primary Examiner Maria Marvich (58) Field of Classification Search (74) Attorney, Agent, or Firm — Haynes and Boone, LLP CPC ..................... C12N 2750/14145; C12N 15/86; C12N 2750/14143; C12N 2810/50; C07K (57) ABSTRACT 14/05; C07K 14/015; A61K 2139/5256 See application file for complete search history. Disclosed are improved recombinant adeno-associated viral (ra AV) vectors having mutations in one or more capsid pro (56) References Cited teins. Exemplary vectors are provided that have altered affin ity for heparin or heparin Sulfate, as well as vectors, expres U.S. PATENT DOCUMENTS sion systems, and raAV virions that lack functional VP2 5,741,683 A * 4, 1998 Zhou et al. .................... 435/457 protein expression, but are nevertheless, fully virulent. Also 5,910,488 A * 6/1999 Nabel et al. ...... ... 514/44 provided by the invention are rAAV vector-based composi 6,204,059 B1* 3/2001 Samulski et al. ............. 435/456 tions, virus particles, host cells, and pharmaceutical formu 6,329, 181 B1 12/2001 Xiao et al. ....... ... 435,9141 lations that comprise them useful in the expression of selected 6,692,815 B2* 2/2004 Edgman ........................ 428, 181 therapeutic proteins, polypeptides, peptides, antisense oligo 6,723,551 B2 * 4/2004 Kotin et al. ................ 435/235.1 6,943,153 B1 9/2005 Manning, Jr. et al. nucleotides and/or ribozymes in selected mammals, includ 6,962,815 B2 * 1 1/2005 Bartlett ......................... 435/455 ing organs, tissues, and human host cells. 7,220,577 B2* 5/2007 Zolotukhin ................ 435/320.1 2001/0034054 A1 10, 2001 Dwarki et al. 2001/005 1611 A1 12/2001 Srivastava et al. 28 Claims, 14 Drawing Sheets US 8,802,080 B2 Page 2 (56) References Cited Opie et al., “Identification of Amino Acid Residues in the Capsid Proteins of Adeno-Associated Virus Type 2 that Contribute to Heparan Sulfate Proteoglycan Binding.” J. Virology, The American OTHER PUBLICATIONS Society for Microbiology, 77(12): 6995-7006, Jun. 20, 2003. Rabinowitz et al., “Insertional Mutagenesis of AAV2 Capsid and the Girod et al., “Genetic Capsid Modifications Allow Efficient Re Production of Recombinant Virus.” Virology, 265(2): 274-285, Dec. Targeting of Adeno-Associated Virus Type 2.” Nature Medicine, 20, 1999. Steinbach et al., “Assembly of Adeno-Associated Virus Type 2 5(9): 1052-1056, Sep. 1999. Capsid in Vitro.” J. General Virology, 78(6): 1453-1462, 1997. Grifman et al., “Incorporation of Tumor-Targeting Peptides into Xiao et al., “Production of High-Titer Recombinant Adeno-Associ Recombinant Adeno-Associated Virus Capsids.” Molecular Therapy, ated Virus Vectors in the Absence of Helper Adenovirus,” J. Virology, 3(6): 946-975, Jun. 2001. The American Society for Microbiology, 72(3): 2224-2232, Mar. Kern et al., “Identification of a Heparin-Binding Motif on Adeno 1998. Associated Virus Type 2 Capsid.” J. Virology, 77(20): 11072-11081, Zhang et al., “Addition of Six-His-Tagged Peptide to the CTerminus Oct. 2003. of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Nicklin et al., “Efficient and Selective AAV2-Mediated Gene Trans Production.” J. Virology, 76(23): 12023-12031, Dec. 2002. fer Directed to Human Vascular Endothelial Cells.' Molecular Therapy, 4(2): 174-181, Aug. 2001. * cited by examiner U.S. Patent Aug. 12, 2014 Sheet 1 of 14 US 8,802,080 B2 p5 p19 ->p40 polyA M45 WP1 VP2 k P12 p5 p19 ->p40 polyA M45 VP1 k VP3 P13 p5 p19 ->p40 polyA M45 x VP2 VP3 P2,3 FIG. 1A-1 FIG A-2 p5 p19 ->p40 pOlyA plM45 VP1 VP2M203 M211 M235 FIG. 1B-1 FIG. 1B-2 U.S. Patent Aug. 12, 2014 Sheet 2 of 14 US 8,802,080 B2 p5 p19 ->p40 ATG polyA M45 VP1 VP2 VP3 P12 ATG FIG. 1 C-1 FIG. 1 C-2 p5 p19 ->p40 polyA plM45 VP1 k kkk VP1 p5 p19 p40 aCg polyA plusVP2 RET. V.,ATG ..." acg/ATG p5 p19 p40 polyA plM45 k k VP3 VP3 FIG. 2A FIG. 2B U.S. Patent Aug. 12, 2014 Sheet 3 of 14 US 8,802,080 B2 GFP M203 M21 Owl - - - - - - - - - - - - - M235 FIG. 3A-1 M203,211,235 FIG. 3B-1 FIG. 3B-2 VP1 VP2 FIG. 3C-1 FIG. 3C-2 U.S. Patent Aug. 12, 2014 Sheet 4 of 14 US 8,802,080 B2 p5 p19 p40 polyA plM45 VP VP2 VP3 VPO p5 p19 p40 polyA plM45 1k 138* 203,211,235* VPO p5 p19 p40 polyA psys VP1 k k kk VP1 p5 ->p19 ->p40 polyA M45 - REP H VP2 VP3 P2,3 p5 p19 p40 aCg polyA Py;M45-I In Er".REP "k VP2E ... V2, acg/ATG >p5 -p19 sp40 ATG polyA VP2A plm45 VP1 k VP3 VP1,3 p5 p19 p40 polyA plM45 k k VP3 VP3 VP3 p5 p19 ->p40 polyA M45 WP1 VP2 k.kk P12 -- pXX6 Ad helper functions TR -- TR pTRUF5Z GFP 2 FIG. 4 U.S. Patent Aug. 12, 2014 Sheet 5 of 14 US 8,802,080 B2 GFP O-O 9 p. VP3 to WP2A VP2 VP1 VPO F.G. 5A-2 GFP VP3 WP2A VP2A3 F.G. 5B-1 p5 p19 ->p40 138 polyA ATGlig-H REP HVP1 VE2ATG EM p5 p19 ->p40 polyA Pl: k * VP3 p5 p19 ->p40 138 polyA Pl: VP2SEMs ATG ATG p5 p19 ->p40 polyA M45 WP1 - VP3 P13 F.G. 6A U.S. Patent Aug. 12, 2014 Sheet 6 of 14 US 8,802,080 B2 1 2 3 4 5 VP1 VP2 sa VP3 L ... s anti-Capsid anti-Leptin FIG. 6B-1 FIG. 6B-2 1 2 3 4 5 VP1 VP2 anti-Capsid anti-GFP FIG. 60-1 F.G. 60-2 CC CC. Na R. 3 8 L LD l Na C on sg scs sg.sc sigsgs - sgs.g scs ss a Sa SSR N 92 S S 92 CON n SR 929 R. R. R 2 S2RNR St St 99St St S222 S2g N2 S 2.2S C2g SSN22 C2222 2S2 S E D C C C E O Sa Sa D. C. 2 CEE C E CE FIG. 7 U.S. Patent Aug. 12, 2014 Sheet 7 of 14 US 8,802,080 B2 L FT W E R447A as R459A - R484A H509A - R544A R566A R585A N587A R588A H526A, K527A R585A, R588A R585K R585K, R588K FIG. 9A U.S. Patent Aug. 12, 2014 Sheet 8 of 14 US 8,802,080 B2 Log Relative infectivity O 1 2 3 4.
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