US008835617B2

(12) United States Patent (10) Patent No.: US 8,835,617 B2 Luban et al. (45) Date of Patent: Sep. 16, 2014

(54) POLYNUCLEOTIDES ENCODINGA HUMAN 2007/01051 22 A1 5/2007 Ota et al. TRIM-CYPFUSION POLYPEPTIDE, 2007/O141679 A1 6/2007 Sodroski et al. 2007/O185O17 A1 8/2007 Aggarwal et al. COMPOSITIONS THEREOF, AND METHODS 2008/004.5454 A1 2/2008 Luban et al. OF USING SAME (75) Inventors: Jeremy Luban, New York, NY (US); FOREIGN PATENT DOCUMENTS Martha Neagu, Gaithersburg, MD (US) EP 336523 A1 10, 1989 (73) Assignee: The Trustees of Columbia University WO WO-9011092 A1 10, 1990 in the City of New York, New York, NY WO WO-2006/O14422 2, 2006 (US) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Brennan et al. TRIMCyp expression in Old World Macaca patent is extended or adjusted under 35 nemestrina and Macaca fascicularis. PNAS, Mar. 4, 2008, vol. 105, U.S.C. 154(b) by 429 days. No. 9, pp. 3569-3574.* (21) Appl. No.: 13/128,143 Liao et al. A novel fusion , TRIM5-Cyclophilin A in the pig tailed macaque determines its susceptibility to HIV-1 infection. (22) PCT Filed: Nov. 6, 2009 AIDS 2007, vol. 21, Suppl. 8, S19-S26.* (86). PCT No.: PCT/US2O09/063481 Extended European Search Report mailed Apr. 10, 2012 for Euro pean Application No. 09825443.6, filed Nov. 6, 2009 (9 pages). S371 (c)(1), Neagu et al., “Potent inhibition of HIV-1 by TRIM5-cyclophilin (2), (4) Date: Dec. 1, 2011 fusion engineered from Human components.” The Journal of (87) PCT Pub. No.: WO2010/054141 Clinical Investigation, vol. 119, pp. 3035-3047 (Oct. 2009). Nisole et al., “A Trim5-cycliphilin A fusion found in owl PCT Pub. Date: May 14, 2010 monkey kidney cells can restrict HIV-1.” PNAS, vol. 101, pp. 13324 (65) Prior Publication Data 13328 (Sep. 7, 2004). Sayah et al., “Cyclophilin A retrotransposition into TRIM5 explains US 2012/0095444 A1 Apr. 19, 2012 owl monkey resistance to HIV-1.” Nature, vol. 430, pp. 569-573 (Jul. 2004). International Search Report mailed on May 27, 2010, for Interna Related U.S. Application Data tional Application No. PCT/US09/63481 filed Nov. 6, 2009. Liao et al., Full = Trim57 cyclophilin A V1 Fusion protein. Jan. 9. (60) Provisional application No. 61/112,013, filed on Nov. UniProt Accession No. AOSE27 MACNE. 6, 2008, provisional application No. 61/240,505, filed Mortellaro et al., “Ex. vivo gene therapy with lentiviral vectors res on Sep. 8, 2009. cues adenosine deanimase (ADA)-deficient mice and corrects their immune and metabolic defects.” Blood, vol. 108, pp. 2979-2988 (51) Int. Cl. (Nov. 1, 2006). C7H 2L/04 (2006.01) Written Opinion mailed on May 27, 2010, for International Applica C07K 9/00 (2006.01) tion No. PCT/US09/63481 filed Nov. 6, 2009. Ambrose, Z., KewalRamani, V.N., BieniaSZ, P. D., and Hatziioannou, CI2N 5/62 (2006.01) T. (2007). HIV/AIDS: in search of an animal model. Trends in CI2N 15/63 (2006.01) biotechnology 25, 333-337. A6 IK 4.8/00 (2006.01) An, D. S., et al. Stable reduction of CCR5 by RNAi through C07K I4/47 (2006.01) hematopoietic stem transplant in non-human primates. Proc Natl CI2N 9/90 (2006.01) Acad Sci 104, 13110-13115 (2007). A61 K38/00 (2006.01) (Continued) (52) U.S. Cl. CPC ...... C07K 14/47 (2013.01); C12N 2830/85 Primary Examiner — Louise Humphrey (2013.01); A61K 48/005 (2013.01); A0IK (74) Attorney, Agent, or Firm — Wilmer Cutler Pickering 221 7/15 (2013.01); A6 IK 38/00 (2013.01); Hale and Dorr LLP CI2N 284.0/206 (2013.01); A0IK 2227/105 (57) ABSTRACT (2013.01); C12N 9/90 (2013.01); C07K A nucleic acid is provided which encodes a human TRIM 2319/00 (2013.01); A0IK 2207/12 (2013.01); cyclophilin A fusion sequence encoding a human TRIM CI2N 2740/16043 (2013.01); C12N 2830/60 CypA fusion protein which is active as an anti-viral agent, and (2013.01); A0I K2267/0337 (2013.01); A0IK in particular, as an anti-HIV-1 agent. Also provided is a nucleic acid encoding a polypeptide having both TRIM activ 2217/052 (2013.01); C12N 2740/1601 1 ity and cyclophilin activity. Also provided is an isolated poly (2013.01) nucleotide encoding a human TRIM-CypA fusion protein, or USPC ... 536/23.4:530/350; 530/387.9; 435/320.1; variants thereof retaining the TRIM and cyclophilin A activi 435/325 ties. Also provided are compositions thereof, antibodies that (58) Field of Classification Search specifically bind thereto, and vectors and host cells compris CPC. A61 K48/005; C07K 14/47; C07K 2319/00; ing the nucleic acid or polypeptide. In addition, methods are C12N 15700; C12N 15/63 provided for treating or preventing viral infection, or reducing See application file for complete search history. viral load in a subject comprising administering the nucleic acid, polypeptide, vector, or composition to the Subject in an (56) References Cited amount effective to treat or prevent the viral infection. In some embodiments, the viral infection is HIV-I infection, U.S. PATENT DOCUMENTS hepatitis C infection, pox virus infection, vaccinia virus infec 5,399,346 A 3, 1995 Anderson et al. tion, or HTLV infection. 2006/0134663 A1 6, 2006 Harkin et al. 12 Claims, 75 Drawing Sheets US 8,835,617 B2 Page 2

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U.S. Patent Sep. 16, 2014 Sheet 8 Of 75 US 8,835,617 B2

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0.1 0.1 OOOOL OOO. O.O. O. 1. OOOO. O.O. O.O. 0.1 1. -8-A ic --sarasp --sa -- WSS sciet - - 298 SGGEriker Cyphis - &--S34 - *-W2ss garmarker OO O

O O

O.

O.O. O.O. O. OOOOL OOOL O.O. 0. 1.

FIG. 8

U.S. Patent Sep. 16, 2014 Sheet 11 Of 75 US 8,835,617 B2

1. O

-8- empty vector k& AOC 1. SaR332P -S S32 - S - S322Cyphi.126C

O. 1.

Days post-infection

FIG 11 U.S. Patent Sep. 16, 2014 Sheet 12 of 75 US 8,835,617 B2

COrd blood 105 (transduced) cells Challenge A. in neonatal liver With HIV-1

Gate: CD34"/CD38 Negative

intransduced

FIG. 12 U.S. Patent Sep. 16, 2014 Sheet 13 Of 75 US 8,835,617 B2

CCCC titCCtC taggaaaatt cCtttgtc.ca CatCaCCCCC gtCC attCCt CactCaat CC talaccacgtc titcc.ctggcc tgtc.ttcact cittctic ccca gaat CaCCaC ttctgcactg 12 gtgtctgaag gtg tattgag tgattttgtg CaCCCC agaa gtaggaagtC tittggga Caa 18 aactd tattt aCCttggCat CtCtcaaCaa CaggaaCCtC aCCaCCCaCC a CaCCCaCCa 24 gCagtggaat agctactatg gCttctggaa toctogttaa tgtaaaggag gaggtgaCCt 3 O gcc.ccatctg cctggaactic Ctgacacaac ccctgagcct ggaCtgCCCC cacagottct 36 CCCaag CatC CCtCaCtgca aaCCaCaaga agtCcatgct agaCaaagga CaCagtag Ct 42 gCCCtgtgtg CCC gat Cagt taCCagcCtg agaaCataCC gCCtaatcgg Catgtag CCa A 81 act tagtgga gaag ClCagg gaggtoaagt tgagcc.ca.ga gggg Cagaaa gttgat catt 54 gtCCaCGC Ca tggagagaaa CttCtaCtCit tCtgtCagga CCaCCCCaaC gtcatttgct 6 O ggCtttgttga gCGgtCtCag gaCCaCCgtg gtCaCCaCaC gttcc.ccaca gaggaggttg 66 CCCaggagta CCaagttgaag CtCCagg Cag CtCtggagat gCtgagg Cag aaCCaCCaCC 72 aag Citgaaga gttggaag Ct CaCatCagag aag agaaag C ttCCtggaag aCt CaaataC 78 agtatga Caa aaCCaaCgtC ttgg Cagatt ttgag CaaCt gagaga CatC CtggaCtggg 84 aCCaCaCCaa tgag CtgCaa aaCCUggaga aCCaCCaCCa aga cattctg aaaag CCtta 9 O CqaactCtga aaCtgagatg gtCCaCCaCa cccagtc.cct gag agaCCtC at Ctcagat C 96 tggag Cat CC gCtgCagggg tCagtgatgg agctgct tca gggtgttggat ggCClCatala 1 O2 aaag CaCCC a gaaCglgaCC ttgaagaag C Cagaaactitt to Caaaaaat CalaaCCaCaC 1 O 8 tgttt CQagC tCCtgatCtg alaaggaatgC tagaagttgtt taga gag Ctg a CaCal CuCC 114 gaCg CtaCtg ggttgatgtg a CagtggCtC Calaa Cala Ca tt catgtgct gtcatttctg 12 Ol aagataagag a CaaCl CaCC tCtCCC aaaC CaCagataat atatgggg Ca CCaCCCaCaa 126 gata CC agaC d tgaat t Cadt tatt gtaCt99 Cat CCtgggCtCt Caddgu du Ca 132 CatCagggaa a. C t 9 C C Claga CC tgtcCaagaa aaCtgCttgg at CCtggggg 138 tatgtgCtgg tgCaatgt gtaatattga aaaaaal Caa aattat CaaC 144 Citadata CQQ dCCCuldig dCCddCCdCl tadatgtagt gCtttCC agg 15 O atagttcCtt a. t C C t tgttccitt toattgtgcc cct citctgtg attat 15 6 Ctgat CQtgt tagaCtatg aggCttgCaC tgtctcattic tt Caa 162 CadaCCatgg d taagttitt Ctcactgttc tttittctoag cctgtatttic 168 Catatttaaa CClagaaaa C C C a. C CC ccatgactict gtgCtCaCCa agCtCttgaa If 4. CCtt Citta Ca aCl CagcCC tCtgta Ca gCaCCtCttg CCaC ClCCa totCataCaC 18O tgaact cat tgcatcatt aac Cat Ct titt.ccttgct gtcticcctitc tittctatttg 186 cgt.cct tc tcatcagt a. a. a. t C taat aattgccttg tgccatattg tcCCCaatat 192 tattgaCa CC tgatagca ttittitt Ca toattittcc.g taCtCCtaag CaaaaCl CaC 1981 at a CCtca taaaatgaga tta gg tattactt CtgCC agata tittatcacco 2O4 a. ttgcct ct gaCaCtgaCt aagaagatga agaaaag Ctt tt Caa CagCC tittctatat c 21 Ol tCgtgtgat aattgttcac Caatgaatga gtCCttagcC CtgtgtCagt ttacCCtCga 21 6 gcc cittatt tgtgagttaa agagaaaata tdataaatgg a tact citta a glala Cagg 222 tttgtatict agaggatCtC agttcaactic citgtct citcc atataCCag C agtgtaaCtg 228 gaataaCat aCttaaatgg Ctgtgctitat titCCttitt Ct tottt titt Ctttitt ttitt 234 ttittgaga tgaagttittg citcttgttcc CCagg Ctgga C tgCaatggC aCCatClCgg 24 Ol cactgcaa. cotccacct c tcagattcaa gcaattcticc gccticagoc tCCCaagtag 24 6 CCC attac CaCCaCCCCt. CCCtaaattit C tattittcag taCaCaCCCC 252 titcCC Cat gtt citcgtctaga acctctgacc Caggtgat C CaCCCCCCtC 258 CCtCCCaa agtgCtggga ttacaggcgt. gag CCaCQ gC C cc.ca.gcctg tgcttattitt 264 aaaataa titt aaaaaCtt Ca Cat-taaataa C tCCtaatgt tittattocat 27 O tagggtga Caataa CCta ttgcatatat tittgaaatag Ctagaa.gaga 27 61 attittgaa agttct caac a Caaagaaac gacaca tatt tgaggtgatg gatatgctaa 282 taCCCtcCt toggttatta CCCaatgitat aCatctatoa aaa CatCaCa C 288 a. a. a. t al t C t a. tatt tatt at ttgtcaatta aaag Caaaat adala Caaaad al 2941 a. a. tactittgg at cattgttga aaaaataa at tcctgaag ta taaag Catct S

FIG. 13

U.S. Patent Sep. 16, 2014 Sheet 15 Of 75 US 8,835,617 B2

L gaaCgtggta taaaagggg C CCCaCCCCaC gCtCGtgCCC ttittgcagac gCCaCCCCCC aCCaaaa CCC tgtactatta gCCatggtCa aCCCCaCCCt gttctitcgac attgCCgtCG 121 a CCCCC agcC CttgggCCgC gtctoctittg agctgtttgc aga Caaggto CCaaaga Cag 181 cagaaaattit tcgtgctctg agCaCtggag agaaaggatt tggittataag ggttcctgct 241 ttCaCagaat tatt CCaCCC tittatctgtc a CCC, tCC, tCa CttCaCaCCC CataatCCCa 3O1 Ctggtgg Caa gtcCatctat gCC gagaaat ttgaagatga gaactitcatc Ctaaag Cata 361 Coggtoctog CatcttgtcC atggcaaatg Ctgg accCaa Cacaaatggt. toccagttitt 421 tCatctgcac tgCCaag act gag togttgg atCCCaag Ca tgtggtgttt CCCaaagttga A 81 aagaagg Cat gaatattgttg gaggCCatgg agCCCtttgg gtCCaggaat CCC aaga CCa 541 gCaagaagat caccattgct gaCtgtggaC aactCCaata agtttgacitt gtgttittatc 6O1 ttaaCCaCCa gat cattcct tctgtagctic aggaga gCaC CCCCCCCCC catttgctcg 661 cagtaticcita gaatctttgt gctictogctg cagttccCtt gttitt.ccttg 21 titCCCtcCCa tgCCtagCtg gattgCagag taagttitat taaaaaCtaa 781 ataacaattg to citcgtttg agittaag agt ttgatgtag gctittattitt aag Cagtaat 841 gggittact tc tgaaa catca cittgtttgct aatt CtaCa cagtact tag atttitt titta 9 O1 Ctttccagtic CCaCCaaCtC tdaatgtttg CaCtCCaa tattoaaaat CtagCCaCCa 961 aCtggg Catg gtggCtCaCt gtCtgtaatg attacctga CCCaCaa CaC CaC Clga CCC 1021 taggagt caa gatcagcCtg ggcaa.ca tag gaga CCCtg totCta Caaa. aaataattag 1 O 81 CCtCCCCtCC tggtgCatCC CtagtCCtag tgat Ctgga CCCtgaCC, tC CCaCCattCC 1141 ttgag CCtag agtgagctat tat catgcca tgta CagcC tgggtgtt Ca cagatcttgt 12O1 gtCtCaaagg a CCCaCaCC CaCCaaaaCC aCCaCCCaC aallaa CaCC ttgggtCagt 1261 CtgCagtgag tt catgcat tagaggtgtt tt Caagatg actaatgtca d d c ttgaga 1321 catctgttgc ggitttittitt titttittittitt C C C t C C a. a. gCagtgg CQt a. t C tCagCt 38 CaCtgCagCC tCCCCCtCC gggttcaagt tCtagtg CCtCagCCtC CaCtaCCt 1441 gCCataatgg CCC totgCCa CCatCCCCaC taatttittg tatttittagt tagatgggg 15O1 titt cat catt ttgaCCagg C tggtctcaaa tottgacct cctgccttg 15 61 gCCtCCCaaa CtCCtgaga tacagatgtg gCCaCCCCa tttctgtaa 1621 Caaag Ctaag CttgaaCaC gttgatgttc CaCCCaaC ttagg Ctgt 1681 aggtoaagtt tata CatCt. aattatggtg gaatticcitat alaa CCCaCC 1741 taCttggtCC taCagt Cag CtCCCtgCag agggittalagg CQC agaCtaC tgCagtgag 18O1 gagg tactCC ttgtag Cata tagag cct ct cc ctagottt ggittatgag C t t t C a. C C t 1861 tittgCaaaCC tgaCCaatitt aag CCatalag at CtggtCaa a CCCala CCC CCCaCtaa 1921 ggaCttggitt tCt Caggada ttatatgtac agtgCttgCt gg Cagttaga gtCagga Ca 1981 at Ctaag Ctg agaaaa CCCC ttctotg.ccc aCCttaaCag aCCtCtaggg Cittaa CCC 2O41 aCCaat CaaC tttgccitatc CtaCaCC, tCC Cggatttgat Catttggtgt CttCCC Caat 21 O1 ttttgttitta ctgtctggitt ccttctg.cgt. gaattaccac CaCCaCCaCt tgtgcatcto 21 61 agtc.ttgttgt gttgtctggit tacgitatt co Ctgggtgata ccattcaatg tottaatgta 2221 CttgttggCtC agaCCtgagt gCaaggtgga aataaa CatC aaaCat Cttt to atta (SE ID NO : 3)

FIG. 15

1 mVnptviffdi avogeplgrV s felfadkVp kitaenfrals togekgfgykg scfhriipgf 61 mcdc.gclftrh notogksiyo ekfedenfill khtopgilism anagpntings of fictakte 121 Wildgkhv Vfg kVkegmnive amerfgs ring kitskkitiad cqcle (SEQ ID NO : 4)

F.G. 16

U.S. Patent Sep. 16, 2014 Sheet 19 Of 75 US 8,835,617 B2

SCALPS-GFP vector GTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGAT GCCGCATAGTTAAGCCAGTATCTGCTCCCTG CTTGTGTGTTGGAGGTCGCTGAG TAGTGCGC GAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTG CTTA. GGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGCTGTGGAATGTGT GTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCAT CT CAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCA. AAGCATGCATCT CAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCC TAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCA GAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGG CCTAGGCTTTTG CAAAAAGCTTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTA. CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAA CGACCCCCGCCCATTCACGTCAATAATGACGTATGTTCCCAT AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGT AAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTA CATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGC GTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGAC GCAAATCGGCGCTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGCGCGTTTTCCCTCTACT GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCT CAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCT CAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGG CTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTT GACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAA TTAGATCGCGATGGGAAAAAATTCGGTTAAG GCCAGGGGGAAAGAAAAAATATAAATTAAAA CATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAAC ATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAAC CATCCCTTCAGACAGGATCAGAAG AACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATA AAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGC ACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAG AAGTGAATTATATAAATATAAAG TAGTAAAAATTGAACCATTAGGAG TAGCACCCACCAAGG CAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGG TTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGT CAATGACGCTGACGGTACAGGCCAG ACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTCCTGAGGGCTATTGAGCCCCAAC AGCATCTGTTGCAACT CACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTG GAAAGATAC CTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACT CATTTG CACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATC ACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTA ATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATG GGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAA TGATAG TAGGAGCCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGA GTTAGGCAGGGATATTCACCATTATCGTTTCACACCCACCTCCCAACCCCGAGGGG ACCCGA CAGGCCCGAAGGAAT AGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAG TGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAAATGGCAGTATTCATCCACAATTT TAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAG TAGACATAATAG CAA Fig. 20A U.S. Patent Sep. 16, 2014 Sheet 20 Of 75 US 8,835,617 B2

CAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTAT TACAGGGACAGCAGAGATCCAGTTTGGTTAAT tala CTGCAGCCCCGATAAAATAAAAGATTT TATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCT GCAGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAAT AGAGAAGTTCAGATC AAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTT CGGCCCCGGCCCGGGGCCAAGAACAGATGGT CACCGCAGTTTCGGCCCCGGCCCGAGGCCAA GAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTT CCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGC TTCTCCCTTCTGTTCGCGCGCTTCTCCTTCCCGAGCTCTATAAAAGAGCT CACAACCCCTCA CTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGGGG tAGAAGCGCTGGATCCGTT TAAACGCGGCCGCCCAGCACAGTGGCTCGAGCCGCGGGTTAACTGGCCAGaatt citcq acct to a C to CCCCCCCCaCCtt CaCOCC to Cott CCCCtcadtt CCCCCCtctgtcCaat CCCC a gaCCCCCCtcatCCCttgaCaacgo CCC aaga CCCCCCCCCCttCCC, tCtcCCCCC to CCCC CocCatgctgcCCaCCCCC gttcCg CactgaCCCtcCCCCotgCCCC gCdt CCC gtaCtgCC gCCCCCCCCC gagt CCC at CCCCC a gCC a CCCCC a C9 gag CCCCC agg C9ggala CCt CCCt c CCCCCCttacCCCCCaC gCCCCCCt catgtgtCct CCCC at Cag CCCCCCCtt CCC, tC tata CCCCaCat CCaC totCaCtCtcCCCaadt CCCaCaCCCC atto CCCCCCaCCCaCCCCCCaC accqggittgcggg.cggggCOGAACGTGGTATAAAAGGGGCGGGAGGCCAGGCTCGTGCCGTT TTGCAGACGCCACCGCCGAGGAAAACCGTGTACTATTAGCCaC gCdtgCCaCCATGGCCCAG TCCAAGCACGGCCTGACCAAGGAGATGACCATGAAGTACCGCATGGAGGGCTGCGTGGACGG CCACAAGTTCGTGAT CACCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCA ACCTGTGCGTGGTGGAGGGCGGCCCCTTGCCCTTCGCCGAGGACAT. CTTGTCCGCCGCCTTC ATGTACGGCAACCGCGTGTTCACCGAGTACCCCCAGGACATCGTCGACTACTTCAAGAACTC CTGCCCCGCCGGCTACACCTGGGACCGCTCCTTCCTGTTCGAGGACGGCGCCGTGTGCATCT GCAACGCCGACAT CACCGTGAGCGTGGAGGAGAACTGCATGTACCACGAGTCCAAGTTCTAC GGCGTGAACTTCCCCGCCGACGGCCCCGTGATGAAGAAGATGACCGACAACTGGGAGCCCTC CTGCGAGAAGATCATCCCCGTGCCCAAGCAGGGCATCTTGAAGGGCGACGTGAGCATGTACC TGCTGCTGAAGGACGGTGGCCGCTTGCGCTGCCAGTTCGACACCGTGTACAAGGCCAAGTCC GTGCCCCGCAAGATGCCCGACTGGCACTTCATCCAGCACAAGCTGACCCGCGAGGACCGCAG CGACGCCAAGAACCAGAAGTGGCAC CTGACCGAGCACGCCATCGCCTCCGGCTCCGCCTTGC CCTGAgcc atcgcTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTT AACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTT GTATCATGCTAT TCCT. TCCCCTATGGCT, TTCATTTTCTCCTCCTTGTATAAATCCTGGTTCCTCTCTCT, TTATG AGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACC CCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACT CATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGC TGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAgcto a CGTCCTTTCCaTGGCTGCTC GCCTGTGTTCCCACCTCGATTCTCCGCCGCACGTCCTTCTCCTACGTCCCTTCCCCCCT CAA TCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC TTCGCCCTCAGACGAGTCGGATCTCCCtttggg.ccgcct coccgcTTAATCGCGTCGAGACC TAGAAAAACATGGAGCAATCACAAG TAG CAATACAGCAGCTACCAATGCTGATTGTGCCTGG CTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACAC CTCAGGTACCTTTAAGACC AATGACTTACAAGGCAGCTG TAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAG GGCTAATTCACTCCCAA CGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGC TACTTCCCTGATTGGCAGAACTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGG ATGGTGCTACAAGCTAGTACCAGTTGAGCAAGAGAAGGTAGAAGAAGCCAATGAAGGAGAGA ACACCCGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTATTA Fig. 20B U.S. Patent Sep. 16, 2014 Sheet 21 Of 75 US 8,835,617 B2

GAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACATGGCCCGAGAGCTGCATCCGGACTG TACTGGGTCTCTCTCCTTAGACCAGATCTGAGCCTGGGAGCTCTCTCGCTAACTAGGGAACC CACTGCTTAAGCCT CAATAAAGCTTGCCTTGAGTGCTTCAAG TAGTGTGTGCCCGTCTGTTG TGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG GGCCCGTTTCATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGC TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGT GCGCTCTCC TGTTCCCACCCTCCCCCTTACCGGATACCTGTCCCCCTTTCTCCCTTCGGCAA GCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC GGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC CTTCGGAAA AAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT CAAGAAGATCCTTTGATCTTTTCTACG GGGTCTGACGCTCAGTGGAA CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA AAGGATCTTCACC TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATAT ATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCT CACCGGCTCCAG ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTA TCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAG TAGTTCGCCAGTTAA TAGTTTGCGCAA CGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGC AAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTT ATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCT TTTCTGTGACTGGTGAGTACT CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGT TGCTCTTGCCCGGCGT CAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTCCT CATCATTGGAAAACGTTCTTCGGGGCCAAAACTCTCAAGGATCTTACCCCTGTTGAGATCCA GTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTT TCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA ATGTTGAATACTCATACTCTTCCTTTTT CAATATTATTGAAGCATTTATCAGGGTTATTGTC TCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTGAC (SEO ID NO:8)

Fig. 20O U.S. Patent Sep. 16, 2014 Sheet 22 Of 75 US 8,835,617 B2

SCALPS-hT5Cyp - GFP GTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGAT GCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGC GAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTA GGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGCTGTGGAATGTGT GTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCAT CT CAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCA AAGCATGCATCT CAATTAGTCAGCA ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCC TAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCA GAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAG TAGTGAGGAGGCTTTTTTGGAGG CCTAGGCTTTTGCAAAAAGCTTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGT AAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTA CATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGC GTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGAC G CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAG CAGCGCGTTTTGCCTGTACT GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCT CAATAAAGCTTGCCTTGAGTGCTTCAAG TAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGC GCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGG CTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTT GACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAA TTAGATCGCGATGGGAAAAAATTCGGTTAAG GCCAGGGGGAAAGAAAAAATATAAATTAAAA CATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAAC ATCAGAAGGCTG TAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAG AACTTAGATCATTATATAATACA GTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATA AAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGC ACAGCAAGCC gccGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAG AAGTGAATTATATAAATATAAAG TAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGG CAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGG TTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAG ACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAAC AGCATCTGTTGCAACT CACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTG GAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACT CATTTG CACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATC ACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTA ATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATG GGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAA TGATAG TAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAAT AGA GTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGA Fig. 21A U.S. Patent Sep. 16, 2014 Sheet 23 Of 75 US 8,835,617 B2

CAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAG TGAACGGATCGGCACTGCGTGCGCCAATTCTGCAGACAAATGGCAGTATTCATCCACAATTT TAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAG TAGACATAATAGCAA CAGACATACA AACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTAT TACAGGGACAGCAGAGATCCAGTTTGGTTAAT tala CTGCAGCCCCGATAAAATAAAAGATTT TATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCT GCAGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAAT AGAGAAGTTCAGATC AAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTT CGGCCCCGGCCCGGGGCCAAGAACAGATGGT CACCGCAGTTTCGGCCCCGGCCCGAGGCCAA GAACAGATGGTCCCCAGATATGGCCCAACCCT CAGCAGTTTCTTAAGACCCATCAGATGTTT CCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGC TTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCT CACAACCCCTCA CTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGGGGtctagagccaccATGGCTTCT GGAATCCTGGTTAATGTAAAGGAGGAGGTGACCTGCCCCATCTGCCTGGAACTCCTGACACA ACCCCTGAGCCTGGACTGCGGCCACAGCTTCTGCCAAGCATGCCT CACTG CAAACCACAAGA AGTCCATGCTAGACAAAGGAGAGAG TAGCTGCCCTGTGTGCCGGATCAGTTACCAGCCTGAG AACATACGGCCTAATCGGCATGTAGCCAACATAGTGGAGAAGCTCAGGGAGGTCAAGTTGAG CCCAGAGGGGCAGAAAGTTGATCATTGTGCACGCCATGGAGAGAAACTTCTACTCTTCTGTC AGGAGGACGGGAAGGTCATTTGCTGGCTTTGTGAGCGGTCTCAGGAGCACCGTGGT CACCAC ACGTTCCT CACAGAGGAGGTTGCCCGGGAGTACCAAGTGAAGCTCCAGGCAGCTCTGGAGAT GCTGAGGCAGAAGCAGCAGGAAGCTGAAGAGTTGGAAGCTGACATCAGAGAAGAGAAAGCTT CCTGGAAGACT CAAATACAGTATGACAAAACCAACGTCTTGGCAGATTTTGAGCAACTGAGA GACATCCTGGACTGGGAGGAGAGCAATGAGCTG CAAAACCTGGAGAAGGAGGAGGAAGACAT TCTGAAAAGCCTTACGAACTCTGAAACTGAGATGGTGCAGCAGACCCAGTCCCTGAGAGAGC TCATCTCAGATCTGGAGCATCGGCTGCAGGGGTCAGTGATGGAGCTGCTTCAGGGTGTGGAT GGCGTCATAAAAAGGACGGAGAACGTGACCTTGAAGAAGCCAGAAACTTTTCCAAAAAATCA AAGGAGAGTGTTTCGAGCTCCTGATCTGAAAGGAATGCTAGAAGTGTTTAGAGAGCTGACAG ATGTCCGACGCTACTGGGTTGATGTGACAGTGGCTCCAAACAACATTTCATGTGCTGTCATT TCTGAAGATAAGAGACAAGTGAGCTCTGTCAACCCCACCGTGTTCTTCGACATTGCCGTCGA CGGCGAGCCCTTGGGCCGCGTCTCCTTTGAGCTGTTTGCAGACAAGGTCCCAAAGACAGCAG AAAATTTTCGTGCTCTGAGCACTGGAGAGAAAGGATTTGGTTATAAGGGTTCCTGCTTTCAC AGAATTATTCCAGGGTTTATGTGTCAGGGTGGTGACTTCACACGCCATAATGGCACTGGTGG CAAGTCCATCTATGGGGAGAAATTTGAAGATGAGAACTTCATCCTAAAGCATACGGGTCCTG GCATCTTATCGATGGCAAATGCTGGACCCAACACAAATGGTTCCCAGTTTTTCATCTGCACT GCCAAGACTGAGTGGTTGGATGGCAAGCATGTGGTGTTTGGCAAAGTGAAAGAAGGCATGAA TATTGTGGAGGCGATGGAGCGCTTTGGGTCCAGGAATGGCAAGACCAGCAAGAAGAT CACCA TTGCTGACTGTGGACAACTCGAATAAg GATCCGTTTAAACGCGGCCGCCCAGCACAGTGGCT CGAGCCGCGGGTTAACTGGCCAGalatt CtCgaCCttgaCtggCdgCCCgaCCttgaggCCtg C gtt CGC Ctcagtt GCCCCCC to to Calatggggaga CGCGCC to at CGC tuga CaldCGGCC Caaga CCCCCCCCCCtt CCC to tCCCCCC, tCCCCCCCCC at CCt CCCCaCCCCCCtt CCCC a Cto a CCCtcCCCCC to CCCCCCC, tCCCG tact gCCCCCCCCCCCCC agtCCCatgCCC Cag C CaCCCCCaCC gag CCCCCaCCCCC gala CCtgC CtcCCCCCCtta CCCCCCaCCCCCCCCtca togtotCdt CCCC at Cag CJCCC gCtt CCC, tCtatagg CCagatgCaCtgtCaCtCtC gCdaa CtCCCaC a CCCC attcCCCCCCaCCCaCCCCCCaCaCCCCCtt CCCCCCCCCCCCGAACGTG GTATAAAAGGGGCGGGAGGCCAGGCTCGTGCCGTTTTGCAGACGCCACCGCCGAGGAAAACC GTGTACTATTAGCC accCdt CCCaCCATGGCCCAGTCCAAGCACGGCCTGACCAAGGAGATG Fig.21B U.S. Patent Sep. 16, 2014 Sheet 24 Of 75 US 8,835,617 B2

ACCATGAAGTACCGCATGGAGGGCTGCGTGGACGGCCACAAGTTCGTGAT CACCGGCGAGGG CATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAAC CTGTGCGTGGTGGAGGGCGGCCCCT TGCCCTTCGCCGAGGACATCTTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAG TACCCCCAGGACATCGTCGACTACTTCAAGAACTCCTGCCCCGCCGGCTACAC CTGGGACCG CTCCTTCCTGTTCGAGGACGGCGCCGTGTGCATCTGCAACGCCGACAT CACCGTGAGCGTGG AGGAGAACTGCATGTACCACGAGTCCAAGTTCTACGGCGTGAACTTCCCCGCCGACGGCCCC GTGATGAAGAAGATGACCGACAACTGGGAGCC CTCCTGCGAGAAGATCATCCCCGTGCCCAA GCAGGGCATCTTGAAGGGCGACGTGAGCATGTACCTGCTGCTGAAGGACGGTGGCCGCTTGC GCTGCCAGTTCGACACCGTGTACAAGGCCAAGTCCGTGCCCCGCAAGATGCCCGACTGGCAC TTCATCCAGCACAAGCTGACCCGCGAGGACCGCAGCGACGCCAAGAACCAGAAGTGGCACCT GACCGAGCACGCCATCGCCTCCGGCTCCGCCTTGCCCTGAgCdatcgCTAATCAACCTCTGG ATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGT GGATACGCTGCTTTAATGCCTTT GTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTC CTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAAC GTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACC TGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACT CATCGC CGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGT TGTCGGGGAAgctgaCGTCCTTTCCaTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGC GGGACGTCCTTCTGCTACGTCCCTTCGGCCCT CAATCCAGCGGACCTTCCTTCCCGCGGCCT GCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCT CAGACGAGTCGGATCTCCC tittggg.ccgcctCCCCCCTTAATCGCGTCGAGACCTAGAAAAACATGGAGCAATCACAAGTA GCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTG GGTTTTCCAGTCACACCT CAGGTACCTTTAAG ACCAATGACTTACAAGGCAGCTGTAGATCT TAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAG ATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTGGCAGAACTACACA CCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGA GCAAGAGAAGGTAGAAGAAGCCAATGAAGGAGAGAA CACCCGCTTGTTACACCCTGTGAGCC TGCATGGGATGGATGACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCA TTTCAT CACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTAGACCAGAT CTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCT CAATAAAGCTTGC CTTGAGTGCTTCAAG TAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGGGCCCGTTTCATGTGAGCAAAAGGCCA GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCC CTGACGAGCAT CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCT TACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCT GTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCCCC GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACA CGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGT ATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAA AAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAAC TCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC TAGATCCTTTTAAA Fig. 21C U.S. Patent Sep. 16, 2014 Sheet 25 Of 75 US 8,835,617 B2

TTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC AATGCTTAATCAGTGAGGCAC CTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC TGACTCCCCGTCGTG TAGATAACTACGATACGGGAGGGCTTAC CATCTGGCCCCAGTGCTGC AATGATACCGCGAGACCCACGCT CACCGGCTCCAGATTTATCAG CAATAAACCAGCCAGCCG GAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGT TGCCGGGAAGCTAGAGTAAG TAGTTCGCCAGTTAATAGTTTGCGCAAC GTTGTTGCCATTGC TACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAAC GATCAAGGCGAGTTACATGATCCCCCATGTTGTG CAAAAAAGCGGTTAGCTCCTTCGGTCCT CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCA TAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT CAACCA AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGT CAATACGGGAT AATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCG AAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTCCACCCA ACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAA AATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACT CATACTCTTCCTTTT TCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTA TTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC (SEO ID NO: 9)

Fig. 21D U.S. Patent Sep. 16, 2014 Sheet 26 Of 75 US 8,835,617 B2

A. linear scale t H. Wector g 75 -X Ses. - V - T5c R332P 3. 5. SCR332-332 -- Rhisis 9 25. -- Ao 5Cyp S. -- hi5Cyp -O-128

Re R its

B Logarithmic scale

f -Hyfector Xh So i V. ScaR332P - SR332-332 -O-Rih Scy - -- Ac5Cyp - T50yg 13-2 -O-H28{R 8-2 -: i8 3. 2 Re R its

FIG. 22 U.S. Patent Sep. 16, 2014 Sheet 27 Of 75 US 8,835,617 B2

Linear Scale H. Wector - X:T3c. -V htSar332 -- T5:R323-332 -O-RhSc. -- his Cyp -- AoT5Cyp -O-hTSCyp 126Q

Days post-infection

Entpy Wector Logarithmic scale -O- T5CypH 26Q 3. -X ht5e VT5a R332P TSct R323-332 -O-RhT5cs - ht5Cyp -- AoSCyp

sy. y s. 2) Days post-infection

FIG. 23 U.S. Patent Sep. 16, 2014 Sheet 28 Of 75 US 8,835,617 B2

A ------is ge------ASVNVKEEC.C.E.R.S.CGSECQACAKEKKSYK. GESSCRVCRISYQRENERENREVANEVEKE, REVXSPEGRVHCARE&E ~ 8 838 2. ~~ S a C --- & - --S&S K.L.F.C.E. (KVICWICERSEERGETFREEWAQEYQWKQAAEMR C defial Cide fg. & Cié fg. KQEAEEEARESKASWKQIYKNWA: E.8. ESSNE. gas de fg. & CC & fg. & Co. ei Sao C. de fga. ŠS$$. N.EKEEE ISKS, INSEEVSREISEERGSW.E.E.CW SSS RSsy. SarSS DGVIKRTENVELKKPETFPKNQRRVFRAPOLKGMiEVFRELTD RyS&S &S vivAPNNISCAVISEDKROVssPKPQITYGARGTRYQTFVNFNYCTGII.G S ------

------

B 2 102 3.

$10 Ol s O ick 8109 {0 A3Cyp 00 Syg S-S322-ys 10- 0: 0-2 - 08 O2 0-2 O-1 00 02 R is R is

FG, 24

U.S. Patent Sep. 16, 2014 Sheet 30 Of 75 US 8,835,617 B2

A. 102 isy. G83. o, if

101 *: Ot s 3. 10 O tack 100 O lock is 10- 0 AoS{y Discypy h5Cy: Visscypt; 26Q

0-2 O-3 O-2 O-1 o 11 102 10-2 0-1 Oo O) 102 t is is 102 RV-y 102 W- 3Sf 88ff.8

s Ot O

3. Wector SO s OWector CSA is 10 SCy: 3BS $5Cyg sA Ori to-m-m-m-m 0-2 O-1 00 0 2 10-2 10-1 00 Ot 102 - ifs is 102 if Yi

3 O 33 s e 10° OWectar 4 AoSCy O'SCyp 10 O 5 Asts,

FIG. 26 U.S. Patent Sep. 16, 2014 Sheet 31 Of 75 US 8,835,617 B2

A. 3. O2 2 ity-2

O; l 3 to s 00 Ort

-2 10-1 0-2 10-1 O) O1 02 0-2 Ol 00 101 O2 its its O2 Sivia 10 Five O C1

10 D s 10-1 O-1 SS to-2 0-2

O-3 C-3 10- 100 O2 0-2 10-i 0 101 02 R its is 10 siv,251

s t! () Vector A h50 & oo () AoT5Cyp hT5Cyp

10-2 10-1 00 102 its

FIG. 27

U.S. Patent Sep. 16, 2014 Sheet 33 Of 75 US 8,835,617 B2

Á A:

A& m SAS

8.33' 3 ces {& ces:

7.0. Wi. 7.0 i. 2

6.0 60

50 5.0 4.0 4.) hr5Cyp 3.0 2,0 O hT5CypH250 1.0 0.

0 0. 20 5 15 25 3S day 33st-irfection day post-infection a spitages: acrophages: 5.5 say 2.0 saw 5.0 4.5 4.0 1.5 3.5 3. O ht5Cyp

15 O.S 1.0 0.5 O O 20 30 40 5 5 25 35 day post-infectio day post-infection 80 (3t ces % {{sts 4 hi5Cyp 3. O ht5CypH126Q R its 2 hT5Cyp O h5Cyphi 26Q

sustairs 10 20 cay post-infectic

FIG. 29 U.S. Patent Sep. 16, 2014 Sheet 34 of 75 US 8,835,617 B2

A pist CR4 ceis 8

8y : Citiciate i.). 40 Rsg3*g. g a g a $38.8: 4 Gy; 3.5 x 3S o & 100 transitiec CE3's ceis i.i. TSCygii; l 1. 99.3 so. SCypiff SCygii.263

10 4.

Aaiysis

lossypies 11.1,

o 10% o 30 to o2 10 g to 45 GF

FIG. 30 U.S. Patent Sep. 16, 2014 Sheet 35 of 75 US 8,835,617 B2

A. & 102 107 g () s 34. S 101 mo g 106 as 10 O esO 10 O 10' O SS S s 10 s 10:2 103

G 102 103 * 105 0 12 13 104 to

83 oxg34 (24.

FIG. 31

U.S. Patent Sep. 16, 2014 Sheet 37 Of 75 US 8,835,617 B2

A 8 ''Jurkat vector 100 CRik O hi5CypH126G 30 W hT5ct -O 75 A his sagasa 2 20 O RhT5c. 50 s (). Aot 5Cyp 10 hitscyp - 25

O O 2O 30 40 SO O 10 20 30 40 50 E. is is 60 O2 50 O1 s 40 Vector 30 O RhT5c. 100 s 20 hT5Cyp {0 AoT5Cypt O

0-2 20 30 40 0-2 is is O2 10. s 100 Vector Luc KD O Vector CypA KD 10-1 hT5Cyp Luc KD O ht5Cyp CypA KD 0-2 0-3 O O-1 100 101 02 is

FIG. 33 U.S. Patent Sep. 16, 2014 Sheet 38 of 75 US 8,835,617 B2

A 10°E CD4+ T cells scALPS-GFP s ce s 101 Mock hT5Cyp hT5CypH126Q

s d

100 hTRIMCyp GFP B SCALPS: (sr) ove) grp) SCAPS dsRED ser) areol ove) grp)

i

FIG. 34 U.S. Patent Sep. 16, 2014 Sheet 39 Of 75 US 8,835,617 B2

A 84* ces

5ty 288

PRAftonomycin-treated soo 60

16 o i o S&AS ck SAS is: SCyp iSCypi- 283

------be

o o

FIG. 35 U.S. Patent Sep. 16, 2014 Sheet 40 of 75 US 8,835,617 B2

hT5Cyp O hT5CypH126Q 5 O

O O 20 3D day post-infection iRay 2 :ay 34

FIG. 36 U.S. Patent Sep. 16, 2014 Sheet 41. Of 75 US 8,835,617 B2

precirassia 6 weeks post-tasia fi

rasciscai Ces 3e arcs. ---

Sys Nede

10° (s porter-Terror Tre 12 8 1. 5 ( * 1. y 15 G

FIG. 37

lyrigh ce

38 C3 (iii.34 crp3&

FIG 38 U.S. Patent Sep. 16, 2014 Sheet 42 of 75 US 8,835,617 B2

fe post- re- Si

- ific

FIG. 39 U.S. Patent Sep. 16, 2014 Sheet 43 of 75 US 8,835,617 B2

is RSSS

8

SS ". -

Šs Assettiy & itsis *e S X& at . . . s R&S & : Transcription & ŠsŠssssss

S

XX S. W

s s ss s N.M. integration Sixpressiae

SS

FIG. 40 U.S. Patent Sep. 16, 2014 Sheet 44 of 75 US 8,835,617 B2

A Owl Monkey: TRIMCyp

Niffe six Sis E3 Multirnerization CA Birding

83 : x is exi

Resis Rits isfire if 8-8

s

Stë Sssssssssatistae: basey Š...... SSSSSSSSS &:ESSy: STSX & Sis S.S. 853 iss

ississsssss SS SSSSSSSSSSSSSS*:S is Yi's Swiss...' assasSSSSSSSSSSSSS -xx as SYSY's: & S.S.R&s&SSSS SSS: ss. SSSSSSSSSSSSS

FIG. 41

U.S. Patent Sep. 16, 2014 Sheet 46 of 75 US 8,835,617 B2

A. fusicis Ecteis. Seiker residiesey A. Westfitti AcTSCy ...RYin AAAWEASAny pa s Sty ...RY-wavivar NNIsraw is cyps it's Cyp. ...RYS-GKSKSYK PCS.S.-Cyå. Scy. ... Yi-GSESAMARS RETASTS-typs Siirectly ...RYS-Cyps h's (sees cyp RYSir SessSGSSSSS3Kirya

FR

who is -H his directoylp Kohlfs (SGSSGyp -- T5oCyp e - SSCyp. "A his Cyp the AeSCyp , "Is " " ' " ' "

FG. 43 U.S. Patent Sep. 16, 2014 Sheet 47 of 75 US 8,835,617 B2

MASGELVNVKEEVECPICLELLTFLSLoCGRSFCRACLEANRKKSM.DYK GEsscPvcRISYQPENIRPNRHvANLVEKLREvKLSPEggkvpHCARHGE

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US 8,835,617 B2 1. 2 POLYNUCLEOTDESENCODNGA HUMAN 70%, 75%, 80%, 85%, 90%. 95% or 99% identity to SEQID TRIM-CYPFUSION POLYPEPTIDE, NO:1, SEQID NO:3, SEQID NO:5, SEQID NO:6, or SEQ COMPOSITIONS THEREOF, AND METHODS ID NO:7. Another embodiment provides a nucleic acid which OF USING SAME comprises a sequence complementary to a nucleic acid pro vided by an embodiment of the invention. This application is a U.S. National Phase application under Provided herein is a nucleic acid encoding a polypeptide 35 U.S.C.X371 of International Patent Application No. PCT/ comprising SEQID NO:4 and SEQID NO:2, or a polypep US2009/063481 filed Nov. 6, 2009, which claims priority to tide comprising SEQID NO:10, 11, or 12. Application Ser. No. 61/112,013 filed Nov. 6, 2008 and Provided herein is a nucleic acid encoding a human Application Ser. No. 61/240,505 filed Sep. 8, 2009, the con- 10 TRIM5-cyclophilin Afusion protein. In one embodiment, the tents of which applications are hereby incorporated in their polynucleotide encodes a polypeptide comprising SEQ ID entirety. NO:2 and SEQID NO:4, wherein the last amino acid of SEQ The invention disclosed herein was made with Govern ID NO:2 is serine at position 309, serine at position 322, or ment support under NIH Grant Nos. RO1AI36199 and alanine at position 331. In another embodiment, the nucleic RO1AI59159 from the Department of Health and Human 15 acid encodes a polypeptide consisting essentially of SEQID Services. Accordingly, the U.S. Government has certain NO:4 and SEQID NO:2, wherein the last amino acid of SEQ rights in this invention. ID NO:2 is serine at position 309, serine at position 322, or This patent disclosure contains material that is subject to alanine at position 331. In one embodiment, the last amino copyright protection. The copyright owner has no objection to acid of SEQ ID NO:2 is serine at position 309, serine at the facsimile reproduction by anyone of the patent document 20 position 322, or alanine at position 331. In one embodiment, or the patent disclosure, as it appears in the U.S. Patent and the last amino acid of SEQID NO:2 is any one amino acid Trademark Office patent file or records, but otherwise residue from about residue 280 to about residue 400, or from reserves any and all copyright rights. about residue 280 to about residue 290, or from about residue Throughout this application, patent applications, pub 308 to about residue 311; or from about residue 321 to about lished patent applications, issued and granted patents, texts, 25 residue 346; or from about residue 366 to about residue 371; and literature references are cited. The disclosures of these or from about residue 381 to about residue 393. In one publications in their entireties are hereby incorporated by embodiment, the last residue of SEQID NO:2 is residue 493. reference into this application. The last residue of SEQID NO:2 can be fused to Cyp A (for example, at residue 2 of SEQID NO:4). In another embodi BACKGROUND 30 ment, the nucleic acid encodes a polypeptide comprising a sequence that is at least 60%. 65%, 70%, 75%, 80%, 85%, Several million people die each year as a consequence of 90%. 95%, or 99% identical to SEQID NO.4 and a sequence HIV-1 infection. Currently used antiviral therapies suppress that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, HIV-1 replication and resultant disease but these antiviral or 99% identical to SEQID NO:2, or a sequence that is at least therapies are plagued with complications and cannot elimi- 35 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% iden nate the virus. Gene therapy is an alternative to life-long tical to SEQ ID NO:10, or a sequence that is at least 60%, pharmacotherapy. Ideally, gene therapy should potently Sup 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to press HIV-1 replication without eliciting viral resistance. SEQID NO:11, or a sequence that is at least 60%. 65%, 70%, While all steps of the viral life cycle are potential genetherapy 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID targets, blocking the virus before reverse transcription (RT) 40 NO:12, and the polypeptide has the function of TRIM5 and would preclude the genetic diversity that permits emergence specifically binds a capsid protein of HIV. of viral resistance. Additionally, targeting the virus before In another embodiment, the fusion protein comprises a HIV-1 clNA is ligated into host chromosomal DNA would polypeptide linker sequence, wherein (i) TRIM5 is located prevent the virus from becoming a heritable genetic element upstream of the linker sequence, so that the N-terminal amino in that cellular lineage. The discovery that certain TRIM5 45 acid of the linker sequence is attached to the C-terminal (T5) orthologues inhibit HIV-1 infection immediately after amino acid of TRIM5, and (ii) cyclophilin A is located down the virus enters otherwise susceptible cells raised the prospect stream of the linker sequence, so that the C-terminal amino that these host factors might be exploited in HIV-1 gene acid residue of the linker sequence is attached to the N-ter therapy. minal amino acid residue of cyclophilin A. In one embodi HIV-1 infection is a serious problem throughout the world 50 ment, the linker sequence comprises from about 10 to about and there is a great need for a composition that will prevent 20 amino acids. In one embodiment, wherein the linker infection of a subject by HIV-1 and for a composition that sequence comprises the consecutive amino acids treats orameliorates the effects of HIV-1 infection in humans. SGGSGGSGGSGG. There is a need for life-long anti-HIV-1 pharmacotherapy, Provided herein is a polypeptide encoded by a nucleic acid and therapies that treat or prevent HIV-1 associated morbidity 55 of the invention. Also provided is a polypeptide comprising and mortality. SEQ ID NO.4 and SEQID NO:2, or a polypeptide compris ing SEQID NO:10, 11, or 12. In one embodiment, the last SUMMARY (C-terminal)amino acid of SEQID NO:2, which can be fused to cyclophilin, is serine at position 309, serine at position 322, Provided herein is a nucleic acid which comprises a 60 oralanine at position 331. In one embodiment, the last amino sequence selected from the group consisting of SEQ ID acid of SEQ ID NO:2 is any one amino acid residue from NO:1, SEQID NO:3, SEQID NO:5, SEQID NO:6, SEQID about residue 280 to about residue 400, or from about residue NO:7 and a variant having at least about 50% identity to SEQ 280 to about residue 290, or from about residue 308 to about ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or residue 311; or from about residue 321 to about residue 346; SEQID NO:7, wherein the nucleic acid encodes a polypep- 65 or from about residue 366 to about residue 371; or from about tide having both TRIM activity and cyclophilin activity. In residue 381 to about residue 393. In one embodiment, the last one embodiment, the variant has at least about 55%, 60%, residue of SEQID NO:2 is residue 493. In one embodiment, US 8,835,617 B2 3 4 the last residue of SEQID NO:2 is 280, 281,282,283, 284, ated introduction, or liposome-mediated introduction of a 285, 286, 287, 288, 289, 290, 308, 309, 310,311, 321, 322, polynucleotide encoding a human TRIM5-Cyp A fusion 323,324, 325, 326, 327, 328,329, 330, 331, 332, 333,334, polypeptide. In one embodiment, the vial infection is an HIV 335, 336,337, 338,339, 340,341, 342, 343, 344, 345, 346, infection, an HTLV infection, a pox virus infection, a retro 366, 367, 368, 369, 370, 371,381,382,383,384, 385, 386, 5 virus infection, a malaria infection, a hepatitis C virus infec 387,388, 389, 390, 391,392,393, or 493. The last residue of tion, ahepatitis B virus infection, or a vaccinia virus infection. SEQID NO:2 can be fused to Cyp A (for example, at residue Provided herein is a method for treating HIV infection or 2 of SEQID NO:4). Also provided is a polypeptide compris preventing HIV infection of a subject which comprises intro ing a sequence that is at least 60%. 65%, 70%, 75%, 80%, ducing into cells of the subject a human TRIM5-Cyp A fusion 85%, 90%. 95%, or 99% identical to SEQ ID NO.4 and a 10 sequence that is at least 60%. 65%, 70%, 75%, 80%, 85%, polypeptide. In one embodiment, the human TRIM5-Cyp A 90%. 95%, or 99% identical to SEQID NO:2, or a sequence fusion polypeptide binds to a capsid protein of the HIV and that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, subsequently degrades the HIV, thereby treating or prevent or 99% identical to SEQID NO:10, or a sequence that is at ing HIV infection in the subject. least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% 15 Provided herein is a peptidomimetic comprising an amino identical to SEQID NO:11, or a sequence that is at least 60%, acid sequence Substantially identical to the amino acid 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to sequence of a polypeptide of the invention and wherein the SEQID NO:12. peptidomimetic comprises TRIM5 function and cyclophilin Provided herein is an antibody that specifically binds to a A function. polypeptide of the invention. Also provided is an antibody Provided herein is a cell comprising a nucleic acid of the that specifically binds to a polypeptide encoded by a nucleic invention. In one embodiment, the cell is a stem cell. In acid provided by the invention. In one embodiment, the anti another embodiment, the cell is a T cell. body is a polyclonal antibody, a monoclonal antibody, or a Provided herein is a method for imparting resistance to chimeric antibody. HIV to a subject or to cells of a subject, the method compris Provided herein is a method of producing a polypeptide of 25 ing administering to the cells of the Subject a nucleic acid of the invention, which comprises: (a) introducing a nucleic acid the invention, a vector of the invention, or a polypeptide of the encoding the polypeptide into a host cell under conditions invention. Provided herein is a method for reducing or inhib that are compatible with expression of the polypeptide by the iting lentiviral replication, including but not limited to HIV host cell, and (b) recovering the polypeptide. replication, to a Subject or to cells of a Subject, the method Provided herein is a nucleic acid vector, which comprises a 30 comprising administering to the cells of the Subject a nucleic nucleic acid of the invention. In one embodiment, the vector acid of the invention, a vector of the invention, or a polypep comprises a viral vector. In one embodiment, the vector is a tide of the invention. Provided herein is a method for reducing lentiviral vector, an adenovirus vector, a retroviral vector, or viral load, including but not limited to HIV, to a subject or to an adeno-associated viral (AAV) vector. Also provided is a cells of a subject, the method comprising administering to the lentiviral vector comprising a nucleic acid of the invention. 35 cells of the subject a nucleic acid of the invention, a vector of Also provided is a lentiviral vector encoding a human the invention, or a polypeptide of the invention. Also provided TRIM5-cyclophilin A fusion protein, wherein the vector is a therapeutic composition comprising a nucleic acid of the comprises a promoter from a spleen focus-forming virus invention, a polypeptide of the invention, or a peptidomimetic (SFFV) long terminal repeat (LTR), a promoter from a human of the invention, and a therapeutically acceptable carrier. In cyclophilin A gene, or both. Provided herein is a nucleic acid 40 one embodiment, the carrier comprises a vector, a liposome, vector having SEQID NO:8 or SEQID NO:9. or a viral vector. Provided herein is a host organism comprising a vector of Provided herein is a method for ex vivo gene therapy, the the invention. In one embodiment, the host is a prokaryote, a method comprising: (a) removing bone marrow cells from a eukaryote or a fungus. subject; (b) transfecting the removed bone marrow cells with Provided herein is a method for preparing a pharmaceutical 45 a nucleic acid of the invention in vitro; and (c) transplanting composition which comprises admixing a polypeptide of the the transfected bone marrow back into the subject. Also pro invention or a fragment thereof, thereby preparing the phar vided is a method for reducing viral burden or load in a subject maceutical composition. Also provided is a pharmaceutical infected by a virus, the method comprising administering to composition comprising a nucleic acid of the invention, a the Subject a nucleic acid of the invention, a polypeptide of the polypeptide of the invention, or a vector of the invention, and 50 invention, or a vector of the invention. a carrier. Provided herein is a topical composition for prevention or Provided herein is a method for treating a subject suffering treatment of a viral infection which comprises a nucleic acid from a disease or condition, the method comprising admin of the invention or a polypeptide of the invention, and a istering to the Subject a nucleic acid of the invention, a topical carrier. In one embodiment, the composition is for use polypeptide of the invention, or a vector of the invention. Also 55 on the skin, in the vagina, in the nose, in the mouth, or on any provided is a method for treating a subject Suffering from a skin or muscosal Surface. disease or a condition, the method comprising administering Provided herein is a method for deliveringatherapeutically to the subject a polypeptide or a fragment thereof of the effective amount of a human TRIM5-cyclophilin A fusion invention, so as to treat the Subject. protein systemically to a subject, comprising administering to Provided herein is a method for preventing retroviral infec 60 the subject a vector of the invention under conditions that are tion of a subject, or for treating a subject with retroviral compatible with expression of the nucleic acid in the subject. infection, the method comprising administering to the Subject In one embodiment, the vector comprises a nonviral vector. In a pharmaceutical composition of the invention. Also provided another embodiment, the vector comprises a gold or tungsten is a method for treating or preventing a viral infection of a cell, particle and the coated particle is administered to the Subject the method comprising introducing a human TRIM5-Cyp A 65 using a gene gun. In another embodiment, the vector com fusion polypeptide into the cell. In one embodiment, the prises a liposome. In another embodiment, the vector com introducing comprises transfection, transduction, viral-medi prises a viral vector. In another embodiment, the vector is an US 8,835,617 B2 5 6 adenovirus vector, a lentiviral vector, a retroviral vector, oran 95% or 99% identity to SEQ ID NO: 13 or 15. In certain adeno-associated viral (AAV) vector. embodiments, variants include any of the variants described Provided herein is a method for delivering atherapeutically herein. In certain embodiments the variants of SEQID NO: effective amount of a human TRIM5-cyclophilin A fusion 13 or 15 have conservative substitutions, as described herein protein systemically to a subject, comprising transfecting or known in the art. In certain embodiments, the conservative cells of said subject with a vector of the invention, under variants of SEQ ID NO: 13 or 15 have at least about 85%, conditions that permit the expression of the nucleic acid and 90%. 95% or 99% identity to SEQID NO: 13 or 15. production of a human TRIM5-cyclophilin A fusion protein. In certain embodiments the invention provides polypeptide In one embodiment, the transfecting occurs ex vivo and the comprising SEQID NO: 10, 11, 12, or 17 or a variant of SEQ transfected cells are reintroduced into the subject. 10 ID NOS: 10, 11, 12, or 17 having about 55%, 60%, 70%, 75%, In certain embodiments the invention provides a fusion 80%, 85%, 90%, 95% or 99% identity to SEQID NO: 10, 11, polypeptide comprising the following consecutive amino 12, or 17. In certain embodiments, the variant have conser acids in N- to C-terminus orientation: (i) SEQ ID NO: 13, vative substations as described herein or known in the art. In which consists of amino acids at position 1-298 from certain embodiments the conservative variants have about hTRIM5C (SEQID NO:2) or a variant of SEQID NO: 13; (ii) 15 85%.90%, 95% or 99% identity to SEQID NO: 10, 11, 12, or a linker of 12 to 33 amino acids linking SEQID NO: 13 and 17. SEQ ID NO: 15 or the variants thereof; and (iii) SEQ ID In certain embodiments, the linker linking the TRIM5 and NO:15, which consists of amino acid at positions 2 to 166 Cyclophilin portions of the fusion polypeptide has SEQ ID from hCypA (SEQID NO: 4) or a variant of SEQID NO:15, NO: 20, 21, 22 or 23 or a variant of SEQID NO:20, 21, 22 or wherein the polypeptide has lentiviral restriction activity. 23 having about 55%, 60%, 70%, 75%, 80%, 85%, 90%.95% In certain embodiments the invention provides a fusion or 99% identity to SEQID NO: 20, 21, 22 or 23. In certain polypeptide comprising the following consecutive amino embodiments, the linker has SEQID NO: 20, 21, 22 or 23 or acids in N- to C-terminus orientation: (i) SEQ ID NO: 13, a conservative variant of SEQ ID NO: 20, 21, 22 or 23. In which consists of amino acids at position 1-298 from certain embodiments the conservative variants have about hTRIM5C (SEQID NO:2) or a variant of SEQID NO: 13; (ii) 25 85%, 90%. 95% or 99% identity to SEQID NO:20, 21, 22 or a linker of 11-33 amino acids linking SEQ ID NO: 13 and 23. SEQ ID NO: 15 or the variants thereof; and (iii) SEQ ID In certain aspects, the invention provides an isolated NO:15, which consists of amino acid at positions 2 to 166 nucleic acid encoding any of the polypeptides of the inven from hCypA (SEQID NO: 4) or a variant of SEQID NO:15, tion. In certain aspects, the invention provides a vector com wherein the polypeptide has lentiviral restriction activity. 30 prising any one of the nucleic acids of the invention. In certain In certain embodiments the invention provides a fusion aspects, the invention provides use of any of the inventive polypeptide comprising the following consecutive amino nucleic acids or variants thereof, and/or protein encoded by acids in N- to C-terminus orientation: (i) SEQ ID NO: 13, these nucleic acids or variants thereof, in any of the methods which consists of amino acids at position 1-298 from of the invention. hTRIM5C (SEQID NO:2) or a variant of SEQID NO:13; (ii) 35 In certain aspects the invention provides use of the nucleic a linker of 11, 12, 24 or 33 amino acids linking SEQID NO: acid and the polypeptides encoded by these nucleic acids in 13 and SEQID NO: 15 or the variants thereof; and (iii) SEQ any of the methods of the invention, including but not limited ID NO:15, which consists of amino acid at positions 2 to 166 to methods of treatment of lentiviral infection, methods of from hCypA (SEQID NO: 4) or a variant of SEQID NO:15, reducing viral loads, methods of restricting viral replication, wherein the polypeptide has lentiviral restriction activity. The 40 and so forth. linker of 11, 12, 24, or 33 amino acids may consist of any amino acid, or have the specific amino acid as described BRIEF DESCRIPTION OF THE FIGURES herein, including conservative variants of the specific linker as described herein. FIGS. 1a-1c. Identification of human T5Cyp proteins that In certain embodiments the invention provides a fusion 45 potently restrict HIV-1. (a) Amino acid sequence at the junc polypeptide comprising the following consecutive amino tion (SEQID NOs: 129-133) of the indicated T5Cyp fusion acids in N- to C-terminus orientation: (i) SEQ ID NO: 13, proteins and the corresponding anti-HIV-1 activity. (b) The which consists of amino acids at position 1-298 from sequence of hT5C. protein (SEQ ID NO: 134). Horizontal hTRIM5C (SEQ ID NO:2); (ii) a linker of 11, 12, 24 or 33 lines indicate the RING finger and B-box domains. The reg amino acids linking SEQID NO: 13 and SEQID NO: 15; and 50 ister of the predicted coiled-coils is indicated with lower case (iii) SEQID NO:15, which consists of amino acid at positions letters. PRYSPRY residues are boxed. Vertical lines indicate 2 to 166 from hCypA (SEQID NO: 4), wherein the polypep the site of fusion of CypA to T5. Numbers indicate the T5 tide has lentiviral restriction activity. amino acid to which CypA is fused and are color-coded for In certain embodiments, the lentiviral restriction activity is restriction : red, potently restrictive (S309, 5322, HIV-1 restriction activity. In other embodiments, the lentivi 55 A331); green, permissive (M244, W298, T302,5314, G357, ral restriction activity includes TRIM5 and Cyclophilin activ G398); orange, variably restrictive (M284, T369). (c) Jurkat ity as described herein and known in the art. T cells (left panel) and THP-1 monocytes (right panel) were In certain embodiments, the fusion polypeptides of the transduced with vectors encoding puromycin-resistance and invention are recombinant polypeptides. In certain embodi the indicated T5Cyp fusion proteins. Pools of puromycin ments, the fusion polypeptides are not naturally occurring. In 60 resistant cells were infected with increasing amounts of an certain embodiments, the fusion polypeptides of the inven HIV-1 vector encoding GFP (left to right on X-axis). The tion are isolated polypeptides. In certain embodiments, the percentage of GFP cells (Y-axis) was determined 48 hrs nucleic acids which encode the fusion polypeptide are iso later. lated. In certain embodiments, the nucleic acids which FIGS. 2a-2d. HIV-1 restriction activity correlates with encode the fusion polypeptides are not naturally occurring. 65 CypA fusion to the T5C. specificity determinant and the abil In certain embodiments the variants of SEQID NO: 13 or ity to form cytoplasmic bodies. (a) Expression of hT5-Cyp 15 have at least about 55%, 60%, 70%, 75%, 80%, 85%, 90%, fusion proteins. FLAG-hT5Cyp fusion proteins synthesized US 8,835,617 B2 7 8 in 293T cells were immunoprecipitated and immunoblotted challenged with HIV-1 as as in (e) except that the viral with anti-CypA antibodies. (b) HIV-1 CA binding activity of envelope was modified to be CCR5-tropic. (g) Selective hT5Cyp fusion proteins. FLAG-hT5Cyp and GST-CA fusion advantage of hT5Cyp-bearing CD4 T cells in the presence of proteins were co-expressed in 293T cells, pulled out on glu HIV-1. CD4 T cells were transduced with scALPS encoding tathione-sepharose beads in the presence or absence of 20 uM the indicated T5Cyp proteins. Cultures containing 23% GFP" CSA, and immunoblotted with anti-CypA and anti-p24-CA cells were challenged with 15 ng/10° cells HIV-1 was and antibodies. (c) Model of the PRYSPRY domain of human monitored for percentage GFP cells (left Y-axis, open sym T5C. based on crystal structures of PRYSPRY, GUSTAVUS bols). Supernatant RT activity was measured (right Y-axis, and TRIM21. Two ribbon representations showing the posi filled symbols). Cultures were re-stimulated using alloge tion of hCypA fusions (spheres) to the hT5C. PRYSPRY 10 domain. The grey transparent ribbon indicates regions of the neic-PBMC, IL-2, and PHA on day 26 post-infection (arrow). model that would be replaced by CypA inhT5-S331-Cyp. (d) FIGS. 6a-6e. ht5Cyp protects CD4 T cells from HIV-1 Restrictive T5Cyp fusion proteins form discrete puncta in the infection in vivo. (a) Experimental design for adoptive trans cytoplasm. Indirect immunofluorescence images of CRFK fer of transduced CD4 T cells and in vivo HIV-1 challenge in cells stably expressing the indicated T5Cyp fusions. Fixed 15 Rag2'Y' mice. Gy=Gray; i.p. intraperitoneal injection. samples were stained with anti-T5 antibody (green) and anti (b) hT5Cyp prolongs CD4 T cell-survival in vivo. 6-10 week tubulin antibody (red), followed by counter-stain with DAPI old Rag2'Y' mice were engrafted with GFP-sorted (blue) to visualize the nuclear DNA. For each color, one scALPS-transduced CD4 T cells expressing either hT5Cyp individual stack of 20 0.35um optical sections was acquired or hT5Cyph126Q. Mice were infected 5 days after adoptive and Subjected to maximum intensity projection along the CD4 T cell transfer with CXCR4-tropic HIV-1 and optical axis. Images represent three-color overlays. Bar: 5 analyzed for presence of hCD45" lymphocytes (Y-axis) at 4 um. All panels are color-coded for restriction phenotype: red, weeks post-infection. Individual mice are shown and fold restrictive; green, permissive; orange, variably restrictive. difference in mean peripheral blood CD45' lymphocyte FIGS. 3a-3e. T5Cyp restricts lentiviruses that encode engraftment (red bar) is indicated above the bracket. Capsid with CypA-binding activity. CRFK cells stably 25 (p=0.0037, Mann-Whitney test) (c, d, e) hT5Cyp protects expressing the indicated T5 proteins were transduced with CD4" from productive HIV-1 infection in lymphoid organs. increasing amounts (X-axis) of GFP vectors derived from Age-matched Rag2'Y' mice transplanted with CD4" T HIV-1 was (a), HIV-2 or (b), SIVoltan (c), FIVrez (d), or cells expressing either hT5Cyp or hT5Cyph126Q were SIV-251 (e). The percentage of GFP" (infected) cells was infected with CXCR4-tropic HIV-12 weeks after adop determined at 48 hrs (Y-axis). 30 tive transfer of CD4 T cells. Single-cell suspensions of thymi FIGS. 4a-4e. T5 and CypA components each make essen were analyzed 14 days post-infection for p24" (X-axis) CD3 tial contributions to T5Cyprestriction activity. (a-c) Disrup (Y-axis) T cells (c) or CD4-downregulation (Y-axis) (d). Rep tion of the CA-CypA interaction by the G89V mutation in resentative CD3- and p24-stained paraffin-embedded tissue HIV-1 CA (a), the H126Q mutation in CypA (b), or the sections of thymus are shown. While engraftment of CD3 competitive inhibitor cyclosporine (CSA) at 2.5 LM (c), 35 human T cells is similar in thymi of mice transplanted with blocks HIV-1 restriction activity in CRFK cells stably either hT5Cyp- (top panel) or hT5CypH126Q-expressing expressing the indicated T5 proteins or controls. Cells were CD4 T cells (bottom panel), rare and weaker p24 staining is challenged with increasing amounts (X-axis) of HIV-1 GFP observed in organs from mice transplanted with hT5Cyp vector bearing the G89V mutation in CA (a) or wild-type CD4 T cells as compared to those transplanted with HIV-1 GFP vector (b, c). (d) Introduction of a naturally 40 hT5CypH126Q-CD4 T cells (e), (c,d,e) Representative occurring quadruple mutation flanking the CA-CypAbinding results from three sets of experiments are shown. site in HIV-1 CA does notabrogatehT5Cyp-mediated restric FIGS. 7A-7C. hTRIMCyp Fusion proteins and superim tion of HIV-1. CRFK cells stably expressing T5Cyp or con position of site of fusion onto 3-D Model of TRIM5 trol were infected with increasing amounts (X-axis) of an PRYSPRY domain (SEQID NOs: 134-137). HIV-1 GFP vector containing the V86P/H87Q/I91V/M96I 45 FIGS. 8A-8D, hTRIMCypfusions and HIV-1 restriction in mutation in CA. (e) Disruption of T5 by ASO blocks HIV-1 multiple cell lines restriction activity by T5Cyp. CRFK cells stably expressing FIG. 9A-9C. hTRIMCyp fusions and restriction of mul the indicated T5Cyp were infected with HIV-1 GFP vector in tiple lentiviruses the presence of increasing amounts of ASO (X-axis). In each FIGS. 10A-10C. CA-binding by the CypA-moiety of case (a-e) the percentage of GFP cells was measured by flow 50 hTRIMCyp is required for HIV-1 restriction cytometry 48 hrs post-infection (Y-axis). FIG. 11. htRIMCyp restriction of NL4-3 in spreading FIGS. 5a-5g. ht5Cyp potently blocks replication-compe infection tent HIV-1. (a, b) Jurkat T cells expressing the indicated FIGS. 12A-12B. HIV-1 mouse model and transduction of proteins were infected with HIV-1 was creresya 15 ng hEHSCS p24/10° cells (a) or 1 ng p24/10° cells (b) and the percentage 55 FIG. 13. Human TRIM5alpha mRNA (SEQ ID NO: 1) of infected cells was recorded. (c) Design of bicistronic (AIG) (Homo sapiens tripartite motif-containing 5 (TRIM.5) and dual promoter (scALPS) lentiviral vectors. (d) GFP mRNA, transcript variant alpha). GenBank Accession No. expression by flow cytometry 48 hrs after transduction of NM 033034. Jurkat T cells (left) or primary CD4 T cells (right) with the FIG. 14. Human TRIM5alpha protein (SEQ ID NO:2) indicated vectors. (e) hT5Cyp inhibits HIV-1 in primary 60 (Homo sapiens tripartite motif protein TRIM5 isoform alpha human CD4 T cells. CD4 T cells transduced with ScALPS amino acid sequence). GenBankAccession No. NP 149023. encoding the indicated hT5Cyp proteins were sorted for GFP. FIG. 15. Human cyclophilin A mRNA (SEQ ID NO:3) infected with HIV-1, and Supernatant RT activity was (Homo sapiens peptidylprolyl isomerase A (PPIA; cyclophi measured. Triplicate spreading infection for one representa lin A) mRNA). GenBank Accession No. NM 021130. tive donor of four is shown. (f) hT5Cyp inhibits HIV-1 in 65 FIG. 16. Human cyclophilin A protein (SEQ ID NO:4) monocyte-derived macrophages. CD14" monocytes differen (Homo sapiens peptidylprolyl isomerase A). GenBank tiated with GM-CSF were transduced, sorted for GFP, and Accession No. NP 066953. US 8,835,617 B2 10 FIG. 17. Nucleotide sequence (SEQ ID NO:5) encoding tubulin antibody (red), followed by counter-stain with DAPI human hTSCyp fusion polypeptide (SEQID NO:10) where (blue) to visualize the nuclear DNA. For each color, one Cyp is fused to TRIM5atalanine 331 (underlined) of TRIM5 individual stack of 20 0.35um optical sections was acquired (hT5-A331-Cyp). and Subjected to maximum intensity projection along the FIG. 18. Nucleotide sequence (SEQ ID NO:6) encoding optical axis. Images represent three-color overlays. Bar: 5 human hTSCyp fusion polypeptide (SEQID NO:11) where um. All panels are color-coded for restriction phenotype: red, Cyp is fused to TRIM5 at serine 309 (underlined) of TRIM5 restrictive; green, permissive; orange, variably restrictive. (hT5-5309-Cyp). FIG. 26A-26E. T5 and CypA components each make FIG. 19. Nucleotide sequence (SEQ ID NO:7) encoding essential contributions to T5Cyp restriction activity. (A-C) human hT5Cyp fusion polypeptide (SEQ ID NO:12) where 10 Disruption of the CA-CypA interaction by the G89V muta Cyp is fused to TRIM5 at serine 322 (underlined) of TRIM5 tion in HIV-1 CA (A), the H126Q mutation in CypA (B), or (hT5-S322-Cyp). the competitive inhibitor cyclosporine (CSA) at 2.5 uM (C), FIGS. 20A-20C. Nucleotide sequence (SEQ ID NO:8) of blocks HIV-1 restriction activity in CRFK cells stably vector SCALPS-GFP. expressing the indicated T5 proteins or controls. Cells were FIGS. 21A-21D. Nucleotide sequence (SEQ ID NO:9) of 15 challenged with increasing amounts (X-axis) of HIV-1-GFP vector scALPS-GFP containing a nucleotide sequence single-cycle vector bearing the G89V mutation in CA (A) or encoding hT5Cyp. wild-type HIV-1 GFP vector (B, C). (D) Introduction of a FIGS.22A-22B. Comparison of anti-HIV-1 activity of pro naturally-occurring quadruple mutation flanking the teins as determined by single cycle HIV-1 infection in CRFK CA-CypA binding site in HIV-1 CA does not abrogate cells. Experimental data is displayed in a linear Scale (a) and hT5Cyp-mediated restriction of HIV-1. CRFK cells stably logarithmic scale (b). Note that hT5Cyp has superior anti expressing T5Cyp or control were infected with increasing HIV-1 activity compared to full-length rhesus monkey amounts (X-axis) of an HIV-1 GFP vector containing the TRIM5C. (RhT5C.) and a human/rhesus monkey chimera V86P/H87Q/I91V/M96I mutation in CA. (E) Disruption of TRIM5C. (hT5CR323-332). See Example 6. T5 by ASO blocks HIV-1 restriction activity by T5Cyp. FIGS. 23A-23B. Comparison the effect on spreading of 25 CRFK cells stably expressing the indicated T5Cyp were HIV-1 infection in Jurkat cells (24 days). Experimental data is infected with HIV-1 GFP vector in the presence of increasing displayed in a linear Scale (a) and logarithmic scale (b). Note amounts of ASOs (X-axis). In each case (A-E) the percentage that hT5Cyp has superior anti-HIV-1 activity compared to of GFP cells was measured by flow cytometry 48 hrs post full-length rhesus monkey TRIM5C. (RhT5C.) and a human/ infection (Y-axis). rhesus monkey chimera TRIM5C. (hT5CR323-332). See 30 FIG. 27A-27E. T5Cyp potently restricts lentiviruses that Example 6. encode Capsid with CypA-binding activity. CRFK cells sta FIG. 24A-24B. Identification of human TSCyp proteins bly expressing the indicated T5 proteins were transduced with that potently restrict HIV-1. (A) The sequence of hT5C. pro increasing amounts (X-axis) of single-cycle GFP vectors tein. Horizontal lines indicate the RING finger and B-box derived from HIV-1 (A), HIV-2 (B), SIV tan (C), domains. The register of the predicted coiled-coils is indi 35 FIV (D), or SIV-251 (E). The percentage of GFP" cated with lower case letters. PRYSPRY residues are boxed. positive cells (Y-axis) was determined 48 hrs later. Vertical lines indicate the site of fusion of CypA to T5. Num FIG. 28A-D. The anti-HIV-1 activity of hTSCyp is supe bers indicate the T5 amino acid to which CypA is fused and rior to that of rhT5C. CRFK (A) and Jurkat T cells (B) were are color-coded for restriction phenotype: red, potently transduced with lentiviral vectors encoding the indicated T5 restrictive; green, permissive; orange, variably restrictive. (B) 40 proteins and a puromycin-resistance cassette. Pools of puro Jurkat T cells (left panel) and THP-1 monocytes (right panel) mycin-resistant cells were infected with increasing amounts were transduced with vectors encoding puromycin-resistance of a single-cycle HIV-1-GFP vector (left to right on X-axis). and either AoT5Cyp or hT5Cyp (hT5-S222-Cyp) fusion pro The percentage of GFP" positive cells (Y-axis) was deter teins. Pools of puromycin-resistant cells were infected with mined 48 hrs later. (C and D) As in (A) and (B), Jurkat T cells increasing amounts of a single-cycle, HIV-1 vector encoding 45 were transduced with the indicated vectors and challenged GFP (left to right on X-axis). The percentage of GFP cells with HIV-1M4s-cretres ve. The percentage of infected (Y-axis) was determined 48 hrs later. cells was determined by flow cytometry. FIG. 25A-25D. HIV-1 restriction activity correlates with FIG. 29A-G. New lentiviral vectors bearing hTSCyp CypA fusion to the T5C. specificity determinant and the abil potently block replication-competent HIV-1 in primary cells. ity to form cytoplasmic bodies. (A) Expression of hT5-Cyp 50 (A) Design of conventional bicistronic (AIG) and new dual fusion proteins. FLAG-hT5Cyp fusion proteins synthesized promoter (scALPS) lentiviral vectors. (B) 48 hrs after trans in 293T cells were immunoprecipitated and immunoblotted duction with the indicated vectors, Jurkat T cells (left) or with anti-CypA antibodies. (B) HIV-1 CA binding activity of primary hCD4 T cells (right) were analyzed by flow cytom hT5Cyp fusion proteins. FLAG-hT5Cyp and GST-CA fusion etry for GFP. (C and D) Primary human CD4 T cells trans proteins were co-expressed in 293T cells, pulled out on glu 55 duced with scALPS encoding the indicated hT5Cyp proteins tathione-sepharose beads in the presence or absence of 20 uM were sorted for GFP and infected with lab clone HIV-1, CSA, and immunoblotted with anti-CypA and anti-p24-CA (C) or primary isolate HIV-1DH12 (D) and RT activity in the antibodies. (C) Model of the PRYSPRY domain of human Supernatant was measured. Triplicate spreading infection for T5C. based on crystal structures of PRYSPRY, GUSTAVUS one representative donor of four is shown. (E and F) Human and TRIM21. Two ribbon representations showing the posi 60 macrophages generated from GM-CSF-treated, CD14 tion of hCypA fusions (spheres) to the hT5C. PRYSPRY monocytes were transduced with the indicated vectors, Sorted domain. The grey transparent ribbon indicates regions of the for GFP, and challenged with modified HIV-1, bearing a model that would be replaced by CypA inhT5-S331-Cyp. (D) CCR5-tropic Env (E) or primary isolate HIV-1 (F). Infec Restrictive T5Cyp fusion proteins form discrete puncta in the tion was monitored as in (C) and (D). Error bars in (C) and (E) cytoplasm. Indirect immunofluorescence images of CRFK 65 represent +/- standard error of the mean, N=3. (G) hT5Cyp cells stably expressing the indicated T5Cyp fusions. Fixed bearing CD4 T cells exhibit a selective advantage after chal samples were stained with anti-T5 antibody (green) and anti lenge with HIV-1. CD4 T cells were transduced with US 8,835,617 B2 11 12 scALPS encoding the indicated T5Cyp proteins. Cultures HIV-1 vectors in Jurkat (A), CRFK (B), and 293T (C; left containing 23% GFP cells were challenged with 15 ng/10 panel linear Scale, right panel logarithmic scale) cells. (D) cells HIV-1 and monitored for percentage GFP cells hT5Cyprestriction activity in Jurkat T cells stably transduced (leftY-axis, open symbols). Supernatant RT activity was mea with lentiviral vectors coding for shRNAs targeting either sured (right Y-axis, filled symbols). Cultures were re-stimu cyclophilin A (CypA) or luciferase (Luc). In all cases, cells lated using allogeneic-PBMC, IL-2, and PHA on day 26 were transduced with vectors encoding puromycin-resistance post-infection (arrow). and the indicated T5Cypfusion proteins. Pools of puromycin FIG. 30A-30C. Adoptive transfer of hT5Cyp-transduced resistant cells were infected with increasing amounts of an hCD4 cells into Rag2'Y' mice. Human peripheral blood HIV-1 GFP vector (left to right on X-axis). The percentage of CD4 T cells transduced with lentiviral vector scALPS bear 10 ing either hT5Cyp or hT5CypH126Q were sorted for GFP GFP" positive cells (Y-axis) was determined 48 hrs later. and injected into 6-10 wk old Rag2Y, mice. The experi FIG.34A-34B. Transgene expression in T cells transduced mental design is shown schematically in (A). (B) Flow cyto with dual promoter Vectors. (A) Quantitative RT-PCR for metric analysis of the transduced, sorted cells prior to adop GFP and hT5Cyp wt or hT5CypH126Q mRNA expression tive transfer. The left panel shows that nearly all cells are 15 normalized by 18S RNA expression 48 hours after transduc hCD4". The x-axis of the right panel shows GFP fluorescence tion of primary CD4 T cells with scAPLS dual promoter in transduced cells (open histograms, scALPS) or in untrans vectors. N.D.—not detected (B) Schematic of dual promoter duced, donor-identical CD4 T cells (grey histogram, Mock). vectors (scAPLS and scAPLSdsRed) used to transduce Jurkat (C) 6 weeks post-transplant, single cell Suspensions of thy T cells (top panel). Transgene expression in transduced Jurkat mus were analyzed for human cell engraftment and transgene T cells (bottom panel). expression. The left panel shows recipient-derived CD45" FIG.35A-C. Transduction of primary human CD4+ T cells mouse cells (mCD45.2, Y-axis) and donor derived hCD45" using a dual promoter, lentiviral vector encoding T5Cyp and cells (X-axis). The right panel shows CD4 and GFP signals GFP does not affect basic T cell functions. (A) T5Cyp does for the hCD45" cells (right panels). In (A) and (C), the num not affect steady-state proliferation of CD4 T cells. CD4"T bers indicate the percentage of cells within the indicated 25 cells transduced with the indicated vector were sorted for gates. GFP expression and steady-state proliferation as compared to FIG. 31A-31E. hT5Cyp inhibits HIV-1 in a humanized un-transduced cells was measured using Thymidine incor mouse model. 6-10 week old Rag2'Y. mice were poration. Corrected counts per minute (ccpm, Y-axis) were engrafted with GFP-sorted scALPS-transduced CD4 T cells measured 24 hours after addition of Thymidine (for each expressing eitherhT5Cyp orhT5CypH126Q. (A)5 days after 30 condition, n=3). (B) IL-2 production in scALPS-transduced engraftment, mice were challenged with HIV-1. The CD4 T cells. IL-2 production (X-axis) in proliferating un percentage hCD4 T cells among all nucleated cells in the transduced CD4 T cells and CD4 T cells transduced with peripheral blood of individual mice is shown at 2 weeks indicated scALPS vectors was analyzed by flow cytometry post-infection. The fold-difference for the mean is indicated following stimulation with PMA and ionomycin. (C) Cell (red bar. ***p=0.0002, Mann-Whitney test). (B-E) 2 weeks 35 surface marker expression on scALPS-transduced CD4 T post-engraftment, mice were infected with HIV-1s. (B) cells. The expression of the indicated cell surface markers Plasma viral load for individual mice was determined 3 wks (X-axis) was assessed in proliferating un-transduced CD4"T post-infection. The fold-difference in the mean is indicated cells and CD4 T cells transduced with indicated scALPS (red bar. **p=0.005, Mann-Whitney test). (C) 3 wks post vectors by flow cytometry. infection, single-cell Suspensions from thymus were ana 40 FIG. 36A-36B. hT5Cyp enhances viability of CD4+ T lyzed by flow cytometry for p24 and hCD3 cells (C) orhGD45 cells in mixed cultures infected with HIV-1. (A) CD4+ T cells and hCD4 (D). The numbers indicate the percentage of cells were transduced with scAPLS encoding the indicated within the indicated gates. (E) Paraffin-embedded sections of hT5Cyp proteins. Cultures containing 23% GFP+ cells (same thymus are shown, stained with hematoxylin/eosin, anti-hu as in FIG. 29G) were challenged with 15 ng p24/106 cells man-CD3 or anti-p24, as indicated. (C, D, E) Representative 45 HIV-1NL4-3 and monitored for% viable cells by forward and results from three sets of experiments are shown. (E) all side scatter profiles (Y-axis) on the indicated day post-infec panels are at 40x magnification, except the two right panels tion (X-axis). Representative flow cytometry plots showing which are at 80x. viability (left panels) and% GFP+ cells (right panels) at three FIG. 32A-32E. HIV-1 restriction activity of different time points are shown in (B). hT5Cyp fusion proteins. (A) Design of a bicistronic lentiviral 50 FIG.37. In vivo GFP expression inhCD4+ T cells 6 weeks vector, FUPI, used to establish T5-expressing cell lines. posttransplant. 6-10 week old Rag2'Y' mice were Ubx-ubiquitin promoter, PuroR puromycin N-acetyltrans engrafted with GFP-sorted scAPLS-transduced CD4 T cells ferase, IRES internal ribosome entry site. (B) Total cellular (left panel). Indicated organs were analyzed for GFP expres DNA was purified from HIV-1 vector-infected Jurkat T cell sion 6 weeks post-transplant (right panel). Gates denote the lines. Late RT products were quantified by PCR. DNA was 55 percentage of GFP cells in the hCD4" population. Data from normalized to mitochondrial DNA content. (C, D) Compari one representative mouse are shown. son of restrictive T5Cyp fusions in CRFK (C) and Jurkat (D) FIG.38, hT5Cyp protects T cells from HIV-1 infection in cells. (E) Comparison of all designed fusions with reduced vivo. 6-10 week old Rag2'Y. mice were engrafted with anti-HIV-1 activity to restrictive T5Cyp and AoT5Cyp in GFP-sorted scAPLS-transduced CD4 T cells expressing Jurkat T cells. In all cases, cells were transduced with FUPI 60 either hT5Cyp or hT5CypH126Q. Mice were infected with encoding puromycin-resistance and the indicated T5Cyp HIV-1NL4-3 two weeks after transplant. Representative fusion proteins. Pools of puromycin-resistant cells were CD3- and p24-stained paraffin-embedded tissue sections of infected with increasing amounts of an HIV-1 GFP vector mesenteric lymph nodes are shown. While engraftment of (left to right on X-axis). The percentage of GFP+ positive CD3" human T cells is similar in lymph nodes of mice trans cells (Y-axis) was determined 48 hrs later. 65 planted with either hT5Cyp- (top panel) or hT5CypH126Q FIG. 33A-33D. HIV-1 restriction activity of different T5 expressing (bottom panel) T cells, rare and weaker p24 stain proteins. (A, B) Comparison of T5-mediated restriction of ing is observed in lymph nodes from mice transplanted with US 8,835,617 B2 13 14 hT5Cyp-CD4 T cells (top panel) as compared to those trans and the indicated T5Cypfusion proteins. Pools of puromycin planted with hT5CypH126Q-CD4 T cells (bottom panel). resistant cells were infected with increasing amounts of an FIG. 39A-39C. hT5Cyp reduces viral load in HIV-1 HIV-1 GFP (left to right on X-axis). The percentage of GFP" infected huRag2-/-yc-/- mice. (A) huRag2'Y' mice positive cells (Y-axis) was determined 48 hrs later. (D.) Total were generated using CD34" cells stably transduced with 5 cellular DNA was purified from Jurkat cell lines and late RT scAPLS-hT5Cyp or scAPLS-hT5CypH126Q.-12% of products were quantified by PCR. Values were normalized to CD34" cells were GFP'72 hours post-transduction in vitro mitochondrial DNA content. (left panels). Human cell engraftment in peripheral blood and FIGS. 46A-C. (A.) Staining of indicated cell-surface mark transgene expression in CD45" human lymphocytes, CD3"T ers in stable Jurkat T cell lines expressing the indicated T5. cells, and CD19 B cells was analyzed by flow cytometry at 10 (B.) Anti-HIV-1 activity of hT5Cyp in Jurkat T cell lines with eight weeks post transplant. (B) Viral load was determined in stable CypA knockdown. (C.) B- and N-MLV titration in plasma of infected mice at 24 days post-infection. (C) GFP Jurkat T cell lines stably expressing the indicated T5 or con expression of CD45" cells is shown in peripheral blood pre trol. For B and C increasing viral doses were used (X-axis) infection and 24 days post-infection of huRag2'Y. with and infected, GFP cells were measured (Y-axis) 48 hours HIV-1. (left Y-axis, black symbols=% GFP" of CD45" cells; 15 post-infection. right Y-axis, grey symbols=% CD45" of nucleated cells). FIG. 47A-D. (A.) FLAG-hT5Cyp fusion proteins synthe FIG. 40A-40B. (A.) Organization of HIV-1 provirus sized in 293T cells were immunoprecipitated and immunob genome (top panel), Gag polyprotein components (bottom lotted with anti-CypA antibodies. (B.) FLAG-hT5Cyp and panel). MA-matrix: CA capsid; NC-nucleocapsid. (B.) GST-CA fusion proteins were co-expressed in 293T cells, Simplified schematic of retroviral life cycle and site of action pulled down with glutathione-sepharose beads in the pres of HIV-1 restriction factors. ence or absence of 20 mM CSA, and immunoblotted. (C.) FIG. 41A-41B. (A.) Organization of functional domains Model of the hT5C. PRYSPRY domain based on crystal struc along the linear TRIM5 sequence: BB–B box, RF=RING tures of PYRIN, GUSTAVUS and TRIM21. Ribbon repre finger, E3=E3 ubiquitin ligase activity, CA capsid (B.) sentations show the position of hCypA fusions (spheres) to Alignment of amino acid sequences at the fusion junction 25 the hT5C. PRYSPRY domain. The grey ribbon indicates (SEQ ID NOs: 138-144); Ma Macacca (sequence identical regions of the model that would be replaced by CypA in for mulatta, nemestrina, and fascicularis), Ao Aotus, hT5-A331-Cyp. (D.) Indirect immunofluorescence images of h human, Rh rhesus (macacca mulatta); * indicates engi CRFK cells stably expressing the indicated T5Cyp fusions. neered not naturally-occurring protein. Color coded for Fixed samples were stained with anti-T5 antibody (green) domains: Blue=RBCC, Green-PRYSPRY. Red=Cyclophilin 30 and anti-tubulin antibody (red), followed by counter-stain A, underlined residues represent amino-acid changes with DAPI (blue) to visualize the nuclear DNA. Images rep between human and rhesus PRYSPRY domains, resent three-color overlays. Bar: 5um. All panels are color black Rhesus-inspired amino-acid changes in the human coded for restriction phenotype: red, restrictive; green, per TSC PRYSPRY domain. missive; orange, variably restrictive. FIG. 42. Alignment of AoT5Cyp (SEQ ID NO: 146) and 35 FIGS. 48A-E. T5Cyp restricts lentiviruses that encode hT5Cyp amino-acid sequences (SEQID NO: 145). Capsid with CypA-binding activity. CRFK cells stably FIGS. 43 A-C. (A.) Amino acid sequence at the junction of expressing the indicated T5 proteins were transduced with the indicated T5Cyp fusion proteins and the corresponding increasing amounts (X-axis) of GFP vectors derived from anti-HIV-1 activity. (B.) Schematic of lentiviral vector used HIV-1 was (A), HIV-2 (B), SIV tan (C). FIV. (D). for generation of stable cell lines: Ubx=Ubiquitin promoter, 40 or SIV-251 (E). The percentage of GFP" (infected) cells PuroR puromycin-N-acetyl-transferase, IRES=internal was determined at 48 hrs (Y-axis). ribosome entry site from encephalomyocarditis virus FIGS. 49A-F. (A-C) Disruption of the CA-CypA interac (ECMV) (C.) Jurkat T cells were transduced with vectors tion by the G89V mutation in HIV-1 CA (A), the H126Q encoding puromycin-resistance and the indicated T5Cyp mutation in CypA (B), or the competitive inhibitor cyclospo fusion proteins. Pools of puromycin-resistant cells were 45 rine (CSA) at 2.5uM (C), blocks HIV-1 restriction activity in infected with increasing amounts of an HIV-1 vector encod CRFK cells stably expressing the indicated T5 proteins or ing GFP (left to right on X-axis). The percentage of GFP" controls. Cells were challenged with increasing amounts cells (Y-axis) was determined 48 hrs later. (X-axis) of HIV-1 GFP vector bearing the G89V mutation in FIGS. 44A-B. (A.) The sequence of hT5C. protein. Hori CA (A) or wild-type HIV-1 GFP vector (B,C). (D) Introduc Zontal lines indicate the RING finger and B-box domains. The 50 tion of a naturally-occurring quadruple mutation flanking the register of the predicted coiled-coils is indicated with lower CA-CypA binding site in CA does not abrogate hT5Cyp case letters. PRYSPRY residues are boxed. Vertical lines indi mediated restriction of HIV-1 in Jurkat T cells (E) Disruption cate the site of fusion of CypA to T5. Numbers indicate the T5 of the CA-CypA interaction using the competitive, non-im amino acid to which CypA is fused and are color-coded for munosuppressive inhibitor DEBIO-025 blocks HIV-1 restric restriction phenotype: red, potently restrictive; green, permis 55 tion in Jurkat T cells. (F) Disruption of T5 by ASO blocks sive; orange, variably restrictive. (B.) Jurkat T cells (left HIV-1 restriction activity by TSCyp. CRFK cells stably panel) and THP-1 monocytes (right panel) were transduced expressing the indicated TSCyp were infected with HIV-1 with vectors encoding puromycin-resistance and the indi GFP vector in the presence of increasing amounts of ASOs cated T5Cyp fusion proteins. Pools of puromycin-resistant (X-axis). In each case (A-F) the percentage of GFP+ cells was cells were infected with increasing amounts of an HIV-1 60 measured by flow cytometry 48 hrs post-infection. vector encoding GFP (left to right on X-axis). The percentage FIGS. 50A-C. Comparison of T5 restriction activity in of GFP cells (Y-axis) was determined 48 hrs later. CRFK cells, (A.) 293T cells (B.), and Jurkat T cells (C.) In FIGS. 45A-D. Comparison of restrictive T5Cyp fusions in A-C, cells were transduced with vectors encoding puromy CRFK (A.) and Jurkat (B.) cells. (C.) Comparison of all cin-resistance and the indicated T5 proteins. Pools of puro designed fusions with reduced anti-HIV-1 activity to restric 65 mycin-resistant cells were infected with increasing amounts tive hT5Cyp and AoT5Cyp in Jurkat T cells. In all cases, cells of an HIV-1 GFP (left to right on X-axis). The percentage of were transduced with vectors encoding puromycin-resistance GFP" positive cells (Y-axis) was determined 48 hrs later. US 8,835,617 B2 15 16 FIGS. 51A-E.hT5Cyp potently blocks replication-compe with 15 ng/10° cells HIV-1 was and monitored for percentage tent HIV-1. (A,B,C) Jurkat T cells expressing the indicated GFP cells (left Y-axis, open symbols). Supernatant RT activ proteins were infected with cell-free HIV-1,4-scretres wea ity was measured (right Y-axis, filled symbols). Cultures were 15 ng p24/10° cells (A, C) or 1 ng p24/10° cells (B) and the re-stimulated using allogeneic-PBMC, IL-2, and PHA on day percentage of infected cells was recorded on indicated days. 26 post-infection (arrow). (B) '% viable cells (Y-axis) was (D) Indicated DiD-labelled target T cell lines were infected assessed as a function of time (X-axis) in the mixed cultures with HIV-1 a-infected Jurkat donor cells. The percentages described in (A). (C) Representative FACS plots showing of infected target cells were analyzed at the 4, 16, and 24 viability (left panels) and GFP expression (right panels) are hours post initiation of co-culture by intracellular p24 stain shown for three time points plotted in (B). ing. (E) Jurkat T cell lines were electroporated with HIV 10 FIGS. 58A-C. Transduction of primary human CD4" T 1M4s-cretres-ve? and the percentage of infected cells was cells using a dual-promoter, lentiviral vector encoding T5Cyp recorded on indicated days. A-E: time post-infection is and GFP does not affect basic T cell functions. (A) T5Cyp depicted on the X-axis, and % infected cells on the Y-axis. does not affect steady-state proliferation of CD4 T cells. FIGS. 52A-E. Indicated DiD-labelled target T cell lines CD4 T cells transduced with the indicated vector were sorted were infected with HIV-1M4s-or-tres ver-infected Jurkat 15 for GFP expression and steady-state proliferation as com donor cells. The percentages of infected target cells were pared to un-transduced cells was measured using Thymi analyzed at the indicated times post initiation of co-culture by dine incorporation. Corrected counts per minute (ccpm, flow cytometry for GFP expression. Puromycin was added at Y-axis) were measured 24 hours after addition of Thymi day 4 of co-culture to eliminate donor cells. Donor (D) and dine (for each condition, n=4). (B) IL-2 production in target (T) cells were mixed at a D:T ratio of (A) 2:1, (B) 1:1, scALPS-transduced CD4 T cells. IL-2 production (X-axis) (C) 1:10, (D) 1:100, (E) 1:1000. in proliferating un-transduced CD4 T cells and CD4 T cells FIGS. 53A-E. (A) Schematic of experimental layout for transduced with indicated scALPS vectors was analyzed by analysis of cell-to-cell spread in a single cycle of infection. flow cytometry following stimulation with PMA and iono (B) Jurkat T cells (target, left panel) and 293T cells (donor, mycin. (C) Cell-surface marker expression on scALPS-trans right panel) stably transduced with lentiviral vectors coding 25 duced CD4 T cells. The expression of the indicated cell for indicated T5Cyps or controls were challenged with Surface markers (X-axis) was assessed in proliferating un increasing doses of an HIV-1 GFP vectors (X-axis). The transduced CD4 T cells and CD4 T cells transduced with percentage of infected cells (Y-axis) was determined 48 hours indicated scALPS vectors by flow cytometry. later. (C) Donor 293T cell lines shown in FIG. 53B were FIGS.59A-C. (A) Invitro differentiated Th2 memory cells transfected with HIV-1 was crenes proviral DNA. 48 30 from wildtype and CypA mice were restimulated using hours post-transfection cells were washed and target DiD plate-bound C.CD3 and CCD28 antibodies. Supernatants labelled Jurkat T control or hT5Cyp-expressing cell lines were collected at given time points and assayed for IL-2 by shown in FIG. 53B were co-cultured with donor 293T cell ELISA. (B) The same cells as in (A) were assayed for IL-2 lines for 2 hours (left panel) or 12 hours. The percentage of mRNA content by quantitative RT-PCR at given time points. infected target Jurkat cells (Y-axis) was assessed by flow 35 Results as shown as IL-2/HPRT mRNA ratios. (C) Mice were cytometry 48 hours later. T5-expression in donor 293T cell injected intraperitoneally with CCD3 antibody and biotiny lines is indicated on the X-axis. (D) Prior to addition of target lated CIL-2 detection antibody. Serum IL-2 levels were cells, relative concentration of virus produced by donor 293T assayed by ELISA at given time points. cells was determined by RT assay of the supernatant. (E) FIGS. 60A-D. (A) Schematic of APM lentiviral vector Release of S-labelled Gag from indicated donor T cells 40 used to deliver shRNAs targeting CypA and T5 to hCD4 T (Y-axis) was measured at given time points (X-axis) follow cells. (B) CD4" T cells were stably transduced with APM ing addition of pulse. coding for CypA, T5, and luciferase (Luc) control shRNAs. FIGS. 54A-D. (A) Design of bicistronic (AIG) and dual Following puromycin selection, relative expression of CypA, promoter (scALPS) lentiviral vectors. (B) GFP expression by T5, and T5C. in transduced CD4 T cells was measured by flow cytometry 48 hrs after transduction of Jurkat T cells (left) 45 quantitative RT-PCR. (C) CD4"CrTh2" human memory Th2 or primary CD4 T cells (right) with the indicated vectors. (C. cells were stably transduced and selected as in (A). CypA D) Quantitative RT-PCR for GFP and hT5Cyp wt or H126Q expression in transduced Th2 cells was assessed by western mRNA expression in CD4 T cells stably transduced with blot. (D) CypA, T5, and Luc KD CD4 T cells were chal scAPLS vectors. lenged with increasing doses (X-axis) of HIV-1, HIV-2,

FIGS. 55A-B. (A) Schematic of dual-promoter lentiviral 50 SIV,239fic GFP vectors. The percentage of infected cells vectors (scALPS and scALPSdsRed) used to transduce Jurkat (Y-axis) was assessed 72 hours post-infection by flow cytom T cells. (B) Representative plots of GFP and dsRed expres etry. sion in scALPS-transduced Jurkat T cells. FIGS. 61A-E. (A) Proliferation in CypA KDT cells. CD4" FIGS. 56A-D. hT5Cyp inhibits HIV-1 in primary human Tand CD4"CrTh2 cells were stably transduced with CypA CD4 T cells. CD4 T cells transduced with scALPS encod 55 KD and Luc KD APM. Following puromycin selection, pro ing the indicated hT5Cyp proteins were sorted for GFP. liferation was measured using Thymidine incorporation. infected with HIV-1 (A) or the primary isolate HIV Corrected counts per minute (ccpm, Y-axis) were measured 1 (B), and Supernatant RT activity was measured. (C, D) 24 hours after addition of Thymidine (for each condition, hT5Cyp inhibits HIV-1 in monocyte-derived macrophages. n=4). (B) CypA (right panel) and Luc KD (left panel) CD4" (C) CD14" monocytes differentiated with GM-CSF were 60 T cells proliferating at steady state were stained with Pyro transduced, sorted for GFP, and challenged with HIV-1 nin-Y (Y-axis) for RNA content and 7-AAD (X-axis) for as in (A) except that the viral envelope was modified to be DNA content and analyzed by flow cytometry. (C) CypA CCR5-tropic or with the primary isolate HIV-1 (D). (open histogram) and Luc KD (grey histogram) CD4 T cells FIGS. 57A-C. Selective advantage of hT5Cyp-bearing proliferating at steady state were stained with CFSE and CD4" T cells in the presence of HIV-1. (A) CD4 T cells were 65 assayed by flow cytometry 48 hours later. (D) Steady state transduced with scALPS encoding the indicated T5Cyp pro CypA, T5, and Luc KD memory Th2 cells were assessed for teins. Cultures containing 23% GFP cells were challenged IL-2 production by ELISA. (E) IL-4, IFNY, and IL-2 (bottom US 8,835,617 B2 17 18 panel, X-axis) in proliferating CypA and Luc KD CD4 T FIGS. 67A-D. Establishment of a hCD4-Rag2'Y. cells was analyzed by flow cytometry following stimulation mouse model. (A) Experimental design for adoptive transfer with PMA and ionomycin. of PBMC or CD4 T cells into Rag2'Y' mice. Gy=Gray: FIGS. 62A-C. (A) Proliferation in T5 KDT cells. CD4"T i.p. intraperitoneal injection. (B) Titration of human PBMC cells were stably transduced with APM coding for T5 and Luc and CD4 T cells adoptively transferred to Rag2'Y'. shRNAs. Following puromycin selection, steady-state prolif Engraftment of CD45" cells (Y-axis) in the peripheral blood eration was measured using 'Thymidine incorporation. Cor was assessed by flow cytometry on indicated days post-trans rected counts per minute (ccpm, Y-axis) were measured 24 fer (X-axis). (C) Survival of Rag2'Y. mice after transfer hours after addition of 'Thymidine (for each condition, of human PBMC or CD4 T cells. Mice were sacrificed upon 10 the appearance of severe GvHD symptoms. (D) Left panel n=4). (B) T5 (right panel) and Luc KD (left panel) CD4 T shows a representative plot of input CD4 T cells following cells proliferating at steady state were stained with Pyronin-Y enrichment from PBMCs. Right panel shows engraftment of (Y-axis) for RNA content and 7-AAD (X-axis) for DNA human lymphocytes in peripheral blood 21 days following content and analyzed by flow cytometry. (C) T5 (open histo transfer of PBMCs or CD4 T cells to pre-conditioned mice. gram) and Luc KD (grey histogram) CD4 T cells proliferat 15 FIGS. 68A-C. (A) Experimental design for adoptive trans ing at steady state were stained with CFSE and assayed by fer of transduced CD4 T cells into Rag2'Y. mice. 6-10 flow cytometry 48 hours later. week old Rag2'Y. mice were engrafted with GFP-sorted FIGS. 63A-B. (A) Schematic describing generation of scALPS-transduced CD4 T cells expressing either hT5Cyp HHLS-Rag2'Y' mice. CD34" cells are enriched from or hT5Cyph126Q. (B) A representative plot of input cells is cord blood. Following optional transduction, >5x10 CD34" shown (left panel). Grey histograms represent GFP-expres cells are injected intrahepatically into newborn Rag2'Y. sion (X-axis) in un-transduced, donor identical CD" T cells, mice preconditioned with sublethal irradiation. Engraftment open histograms represent ScAPLS-transduced cells. (C) of hCD45" cells in peripheral blood is assessed 2-4 months Mice were sacrificed 6 weeks post-transplant and analyzed post-transplant, and if satisfactory, mice are infected with for human cell engraftment and transgene expression. Single HIV-1 by intraperitoneal (i.p.) injection. (B) GFP expression 25 cell Suspensions of thymus were analyzed for presence of (X-axis) in CD34" (top panel) and CD34"CD38 cells 48 recipient-derived CD45' mouse cells (mCD45.2, x-axis) and hours following optimized transduction of freshly-thawed donor derived hCD45" lymphocytes (X-axis). GFP expres CD34" cells with FCGW (hCypA promoter driving GFP sion (X-axis) in mCD45.2" cells (grey histogram) and expression). hCD45" (open histogram) cells is shown. FIGS. 64A-B. HIV-1 infection in HHLS-Rag2'Y' 30 FIGS. 69A-E, hT5Cyp protects CD4 T cells from HIV-1 mice. (A) HHLS-Rag2'Y. mice were generated and infection in vivo. (A) hT5Cyp prolongs CD4 T cell-survival mock-infected with HIV-1A (top panel) or infected with in vivo. 6-10 week old Rag2'Y' mice were engrafted with HIV-1, (bottom panel) 10 weeks post-transplant. Four GFP-sorted scALPS-transduced CD4 T cells expressing weeks post-infection, mice were sacrificed, and single cell either hT5Cyp or hT5Cyph126Q. Mice were infected 5 days Suspensions from indicated organs were analyzed for pres 35 after adoptive CD4 T cell transfer with CXCR4-tropic HIV ence of CD4" (Y-axis) and CD8" (X-axis) T cells. Viral load 1 and analyzed for presence of hCD4 T cells (Y-axis) at in plasma from these mice was determined. (B) Representa 2 weeks post-infection. Individual mice are shown and fold tive CD3- and p24-stained paraffin-embedded tissue sections difference in mean peripheral blood CD4" lymphocyte of thymus are shown. Engraftment of CD3 human T cells in engraftment (red bar) is indicated above the bracket. the thymus is robust and p24 staining is observed. 40 (***p=0.0002, Mann-Whitney test). (B) hT5Cyp reduces FIGS. 65A-C. (A) SIV-VLPs enhance CD34" HSC trans viral burden in vivo. Rag2'Y' mice were engrafted with duction. Donor-identical CD34" cells were transduced with transduced CD4 T cells were infected 2 weeks after adoptive FCGW following mock pretreatment (left panel) or pretreat CD4 T cell transfer with CXCR4-tropic HIV-1. Plasma ment with SIV-VLPs (right panel). The percentage of GFP" viral load was determined at 4 weeks post-infection. Indi (X-axis) cells was determined 72 hours post-transduction. 45 vidual mice are shown and fold-difference in viral load (red (B) HHLS-Rag2'Y' mice were generated using CD34" bar) is indicated above the bracket. (**p=0.005, Mann-Whit cells transduced with BID, a dual-promoter vector in which ney test). (C.D.E) hT5Cyp protects CD4" from productive the mGMV promoter drives GFP expression (see schematic). HIV-1 infection in lymphoid organs. Age-matched Rag2' Mice were sacrificed 12 weeks post-transplant and the per Y. mice transplanted with CD4 T cells expressing either centage of GFP cells in the thymus (left panel) and bone 50 hT5Cyp orhT5Cyph126Q were infected with CXCR4-tropic marrow (right panel) was determined by flow cytometry. (C) HIV-1 2 weeks after adoptive transfer of CD4 T cells. HHLS-Rag2'Y' mice were generated using CD34" cells Single-cell Suspensions of thymi were analyzed 14 days post transduced with FCGW, a lentiviral vector in which the infection for p24" (X-axis) CD3" (Y-axis) T cells (C) or hCypA promoter drives GFP expression (see schematic). CD4-downregulation (X-axis) (D). Representative CD3- and Mice were sacrificed after 12 weeks and analyzed as in (B). 55 p24-stained paraffin-embedded tissue sections of thymus are FIGS. 66A-C. hT5Cyp reduces viral loadin HIV-1 infected shown. While engraftment of CD3" human T cells is similar HHLS-Rag2'Y' mice. (A) HHLS-Rag2'Y' were gen in thymi of mice transplanted with either hT5Cyp- (top erated using CD34" cells stably transduced with scAPLS panel) or hT5CypH126Q-expressing CD4 T cells (bottom hT5Cyp or scAPLS-hT5CypH126Q.-12% of CD34" cells panel), rare and weaker p24 staining is observed in organs were GFP 72 hours post-transplant in vitro (left panels). 60 from mice transplanted with hT5Cyp-CD4 T cells as com Human cell engraftment in peripheral blood 8 weeks post pared to those transplanted with hT5CypH126Q-CD4 T transplant and transgene expression in CD45" human lym cells (E). phocytes, CD3' T cells, and CD19 B cells was analyzed by FIGS. 70A-D. hT5Cyp protects CD4 T cells from HIV-1 flow cytometry. (B) Viral load was determined in plasma of infection in vivo. (A) Lymphoid organs from Rag2'Y. infected mice at 24 days post-infection. (C) Change in trans 65 mice engrafted with transduced CD4 T cells are shown 4 gene (GFP) expression in CD45" cells was compared pre- and weeks after infection with CCR5-tropic HIV-1. T=thymus, post-infection by flow cytometry. S=spleen, MLN-mesenteric lymph node, PLN-peripheral US 8,835,617 B2 19 20 lymph node, A=axillary, I=inguinal, C-cervical, P-popliteal, tering is carried out via injection, oral administration, or B-brachial (B,C,D) hT5Cyp protects CD4" from productive topical administration. In another embodiment, administra HIV-1 infection in lymphoid organs. Single cell Suspensions tion is intramuscularly, intramucosally, intranasally, Subcuta from MLN and PLN of mice infected with CCR5-tropic neously, intradermally, transdermally, intravaginally, HIV-1 were analyzed for GFP transgene expression (B. left intrarectally, orally or intravenously. In one embodiment of panel, X-axis), CD4 downregulation (B, right panel, Y-axis), this invention, the Subject is a mammal, e.g., a mouse or a and presence of p24" (X-axis) CD3 (Y-axis) cells (C). Rep human. Preferably, the mammal is a human. In another resentative CD3- and p24-stained paraffin-embedded tissue embodiment of the invention, the “introducing or adminis sections of mesenteric lymph node are shown. While engraft tering is carried out by a means selected from the group ment of CD3" human T cells is similar in MLN of mice 10 consisting of transduction, viral-mediated introduction, transplanted with either hT5Cyp- (top panel) or adenovirus infection, liposome-mediated transfer, topical hT5CypH126Q-expressing CD4 T cells (bottom panel), rare application to the cell, and microinjection. In another and weaker p24 staining is observed in organs from mice embodiment of the invention, the carrier is an aqueous carrier, transplanted with hT5Cyp-CD4 T cells as compared to those a liposome, a vector, a viral vector, or a lipid carrier. transplanted with hT5CypH126Q-CD4" T cells (D). 15 As used herein"nucleic acid molecule' includes both DNA FIGS. 71A-D. (A) Experimental design for adoptive trans and RNA and, unless otherwise specified, includes both fer of transduced CD4 T cells and in vivo challenge with double-stranded and single-stranded nucleic acids. Also cell-associated HIV-1 in Rag2'Y. mice. Gy=Gray; included are hybrids such as DNA-RNA hybrids. Reference i.p. intraperitoneal injection. (B) hT5Cyp reduces viral bur to a nucleic acid sequence can also include modified bases as den in vivo. 6-10 week old Rag2'Y' mice were engrafted long as the modification does not significantly interfere either with GFP-sorted scALPS-transduced CD4 T cells express with binding of a ligand Such as a protein by the nucleic acid ing either hT5Cyp or hTSCyph126Q. Mice were infected 5 or Watson-Crick base pairing. days after adoptive CD4 T cell transfer with autologous, Two DNA or polypeptide sequences are “substantially untransduced, HIV-1 infected cells. Plasma viral load homologous' when at least about 80% (preferably at least (Y-axis) was assessed at indicated times post-infection. (C) 25 about 90%, and most preferably at least about 95%) of the hT5Cyp prolongs CD4 T cell-survival in vivo. CD4 T cell nucleotides or amino acids match over a defined length of the retention (Y-axis) was assessed as the percentage of CD4 T molecule. As used herein, “substantially homologous also cells in peripheral blood at 24 days compared to 11 days refers to sequences showing identity to the specified DNA or post-infection. (D) Single cell Suspensions of thymuses from polypeptide sequence. DNA sequences that are substantially mice that received hT5Cyp- or hTSCypH126Q-expressing 30 homologous can be identified in a Southern hybridization CD4 T cells were analyzed for p24-, CD3-, and GFP-expres experiment under, for example, stringent conditions, as sion by flow cytometry. defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., DETAILED DESCRIPTION Sambrook et al., Molecular Cloning. A Laboratory Manual. 35 Cold Springs Harbor Laboratory, 2nd ed., Cold Springs Har As used herein the term “lentiviral restriction activity” of a bor, N.Y. (1989). fusion protein or polypeptide is defined as binding and deg A DNA “coding sequence' or a “nucleotide sequence radation of a lentivirus, wherein the cyclophilin Apart of the encoding a particular protein, is a DNA sequence which is fusion protein specifically binds to the lentiviral capsid pro transcribed and translated into a polypeptide in Vivo or in tein, while the TRIM5 part of the fusion protein acts as 40 vitro when placed under the control of appropriate regulatory TRIM5 alone has been characterized, causing the degradation sequences. The boundaries of the coding sequence are deter of the substance bound to the cyclophilin A portion of the mined by a start codon at the 5'-(amino) terminus and a fusion protein. Thus, the fusion protein has coupled a specific translation stop codon at the 3'-(carboxy) terminus. A coding binding tropism, i.e., the binding of cyclophilin portion to sequence can include, but is not limited to, prokaryotic capsid of a lentivirus, with the degradation activity of TRIM5. 45 sequences, cDNA from eukaryotic mRNA genomic DNA The term “lentiviral restriction activity” of a fusion protein or sequences from eukaryotic (e.g., mammalian) sources, viral polypeptide may be used interchangeably with the term “both RNA or DNA, and even synthetic nucleotide sequences. A TRIM activity and cyclophilin activity.” transcription termination sequence will usually be located 3' In certain embodiments the term "lentiviral restriction to the coding sequence. activity” specifically includes the term “HIV-1 restriction 50 “Operably linked’ refers to an arrangement of nucleotide activity” of a fusion protein or polypeptide, which is defined sequence elements wherein the components so described are as binding and degradation of HIV-1, wherein the cyclophilin configured so as to perform their usual function. Thus, control A part of the fusion protein specifically binds to the HIV-1 sequences operably linked to a coding sequence are capable capsid protein once HIV-1 enters the cell, while the TRIM5 of effecting the expression of the coding sequence. The con part of the fusion protein acts as TRIM5 alone has been 55 trol sequences need not be contiguous with the coding characterized, causing the degradation of the Substance sequence, so long as they function to direct the expression bound to the cyclophilin A portion of the fusion protein. Thus, thereof. Thus, for example, intervening untranslated yet tran the fusion protein has coupled a specific binding tropism, i.e., scribed sequences can be present between a promoter the binding of cyclophilin portion to capsid of HIV-1, with the sequence and the coding sequence and the promoter sequence degradation activity of TRIM5. 60 can still be considered “operably linked to the coding As used herein, 'administration” means any of the standard Sequence. methods of administering a pharmaceutical composition A cell has been “transformed by exogenous DNA when known to those skilled in the art. Examples include, but are such exogenous DNA has been introduced inside the cell not limited to, intravenous, intraperitoneal or intramuscular membrane. Exogenous DNA may or may not be integrated administration, in vitrogene therapy via adenoviral vector or 65 (covalently linked) into chromosomal DNA making up the other vector (liposome), ex vivo gene therapy, oral, and inha genome of the cell. In prokaryotes and yeasts, for example, lation. In another embodiment of the invention, the adminis the exogenous DNA may be maintained on an episomal ele US 8,835,617 B2 21 22 ment, such as a plasmid. In eukaryotic cells, a stably trans protocol in Molecular Biology, Green publishing associates, formed cell is generally one in which the exogenous DNA has Inc., and John Wiley & Sons Inc., New York, at pages 6.3.1- become integrated into the so that it is inherited 6.3.6 and 2.10.3). Polynucleotides encoding these polypep by daughter cells through chromosome replication, or one tides are also encompassed by the invention. which includes stably maintained extrachromosomal plas By a polypeptide having an amino acid sequence at least, mids. This stability is demonstrated by the ability of the for example, 95% “identical to a query amino acid sequence, eukaryotic cell to establish cell lines or clones comprised of a it is intended that the amino acid sequence of the Subject population of daughter cells containing the exogenous DNA. polypeptide is identical to the query sequence except that the As used herein, a “variant, refers to a nucleic acid or a Subject/reference polypeptide sequence may in a non-limit protein of the invention differing in sequence from a “refer 10 ence' molecule, for example a sequence specifically ing example include up to five amino acid alterations per each described herewith, but retaining function, activity and/or 100 amino acids of the query amino acid sequence. In other therapeutic property of the inventive fusion proteins or inven words, to obtaina polypeptidehaving anamino acid sequence tive nucleic acids which encode the inventive fusion proteins at least 95% identical to a query amino acid sequence, up to with lentiviral restriction activity, which activity is described 15 5% of the amino acid residues in the Subject sequence may be elsewhere herein or otherwise known in the art. In certain inserted, deleted, or substituted with another amino acid. embodiments, the lentiviral restriction activity is HIV-1 These alterations of the reference sequence may occur at the restriction activity. Generally, variants are overall very simi amino- or carboxy-terminal positions of the reference amino lar, and, in many regions, identical to the amino acid sequence acid sequence or anywhere between those terminal positions, of the inventive proteins or the nucleic acid sequences encod interspersed either individually among residues in the refer ing the same. ence sequence or in one or more contiguous groups within the As used herein, “mutation' with reference to a polynucle reference sequence. otide or polypeptide, refers to a naturally-occurring, Syn As a practical matter, whether any particular polypeptide is thetic, recombinant, or chemical change or difference to the at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, primary, secondary, or tertiary structure of a polynucleotide 25 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or polypeptide, as compared to a reference polynucleotide or 99.5% or 100% identical to, for instance, the amino acid polypeptide, respectively (e.g., as compared to sequences of sequence of a fusion protein of the invention or a fragment polynucleotide or polypeptide as described herein). Muta thereof, can be determined conventionally using known com tions include Such changes as, for example, deletions, inser puter programs. A non-limiting example of a method for tions, or Substitutions. Polynucleotides and polypeptides hav 30 determining the best overall match between a query sequence ing Such mutations can be isolated or generated using (a sequence of the present invention) and a Subject sequence, methods well known in the art. also referred to as a global sequence alignment, can be deter As used herein, “deletion' is defined as a change in either mined using the FASTDB computer program based on the polynucleotide oramino acid sequences in which one or more algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 polynucleotides or amino acid residues, respectively, are 35 (1990)). In a sequence alignment the query and Subject absent. sequences are either both nucleotide sequences or both amino As used herein, “insertion” or “addition' is that change in acid sequences. The result of said global sequence alignment a polynucleotide or amino acid sequence which has resulted is expressed as percent identity. Parameters used in a in the addition of one or more polynucleotides or amino acid FASTDB amino acid alignment are: Matrix=PAM 0. residues, respectively, as compared to a reference polynucle 40 k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Ran otide or amino acid sequence. domization Group Length=0, Cutoff Score=1, Window As used herein, “substitution” results from the replacement Size=sequence length, Gap Penalty=5, Gap Size Pen of one or more polynucleotides or amino acids by different alty=0.05, Window Size=500 or the length of the subject polynucleotides or amino acids, respectively. amino acid sequence, whichever is shorter. The present invention is also directed to proteins which 45 If the Subject sequence is shorter than the query sequence comprise, or consist of, or consistessentially of anamino acid due to N- or C-terminal deletions, not because of internal sequence which is at least 80%, 81%, 82%, 83%, 84%, 85%, deletions, a manual correction must be made to the results. 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, This is because the FASTDB program does not account for N 97%, 98%, 99%, 99.5% or 100%, or which is 80%, 81%, and C-terminal truncations of the Subject sequence when 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 50 calculating global percent identity. For Subject sequences 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% truncated at the N- and C-termini, relative to the query identical to, for example, the amino acid and nucleic acids sequence, the percent identity is corrected by calculating the sequences of the invention. number of residues of the query sequence that are N- and Further polypeptides encompassed by the invention are C-terminal of the Subject sequence, which are not matched/ polypeptides encoded by polynucleotides which hybridize to 55 aligned with a corresponding Subject residue, as a percent of the complement of a nucleic acid molecule encoding a fusion the total bases of the query sequence. Whether a residue is protein of the invention under Stringent hybridization condi matched/aligned is determined by results of the FASTDB tions (e.g., hybridization to filter bound DNA in 6x Sodium sequence alignment. This percentage is then subtracted from chloride/Sodium citrate (SSC) at about 45 degrees Celsius, the percent identity, calculated by the above FASTDB pro followed by one or more washes in 0.2xSSC, 0.1% SDS at 60 gram using the specified parameters, to arrive at a final per about 50-65 degrees Celsius), under highly stringent condi cent identity score. This final percent identity score is what is tions (e.g., hybridization to filter bound DNA in 6x sodium used for the purposes of the present invention. Only residues chloride/Sodium citrate (SSC) at about 45 degrees Celsius, to the N- and C-termini of the subject sequence, which are not followed by one or more washes in 0.1xSSC, 0.2% SDS at matched/aligned with the query sequence, are considered for about 68 degrees Celsius), or under other stringent hybridiza 65 the purposes of manually adjusting the percent identity score. tion conditions which are known to those of skill in the art That is, only query residue positions outside the farthest N (see, for example, Ausubel, F. M. et al., eds., 1989 Current and C-terminal residues of the Subject sequence. US 8,835,617 B2 23 24 For example, a 90 amino acid residue Subject sequence is used in the art. Non-limiting examples of programs for aligned with a 100 residue query sequence to determine per sequence analysis include DNAStar R.LasergeneR) and cent identity. The deletion occurs at the N-terminus of the MacVector. subject sequence and therefore, the FASTDB alignment does The polynucleotide variants of the invention may contain not show a matching/alignment of the first 10 residues at the alterations in the coding regions, non-coding regions, or both. N-terminus. The 10 unpaired residues represent 10% of the The invention provides polynucleotide variants containing sequence (number of residues at the N- and C-termini not alterations which produce silent Substitutions, additions, or matched/total number of residues in the query sequence) so deletions, but do not alter the properties or activities of the 10% is subtracted from the percent identity score calculated encoded polypeptide. The invention provides nucleotide vari by the FASTDB program. If the remaining 90 residues were 10 ants produced by silent Substitutions due to the degeneracy of perfectly matched the final percent identity would be 90%. In the genetic code. Moreover, polypeptide variants in which another example, a 90 residue subject sequence is compared less than 50, less than 40, less than 30, less than 20, less than with a 100 residue query sequence. This time the deletions are 10, or 5-50, 5-25, 5-15, 5-10, 1-5, 1-3, 1-2 or 10,9,8,7,6, 5, internal deletions so there are no residues at the N- or C-ter 4, 3, 2, or 1 amino acids are substituted, deleted, or added in mini of the Subject sequence which are not matched/aligned 15 any combination are also preferred. Polynucleotide variants with the query. In this case the percent identity calculated by can be produced for a variety of reasons, e.g., to optimize FASTDB is not manually corrected. Once again, only residue codon expression for a particular host, for example but not positions outside the N- and C-terminal ends of the subject limited to yeast or mammalian cells. sequence, as displayed in the FASTDB alignment, which are In certain embodiments, the invention provides naturally not matched/aligned with the query sequence are manually occurring variants which are called “allelic variants.” and corrected for. In certain embodiments, no other manual cor refer to one of several alternate forms of a gene occupying a rections are to made for the purposes of the present invention. given locus on a chromosome of an organism. (Genes II, The variants of the invention will usually have at least 75%, Lewin, B., ed., John Wiley & Sons, New York (1985)). These 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, allelic variants can vary at either the polynucleotide and/or 91%, 92%, 93%, 94%, 95%, 96%,97%.98%, 99%, 99.5% or 25 polypeptide level and are included in the present invention. 100% or 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, Alternatively, non-naturally occurring variants may be pro 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, duced by mutagenesis techniques or by direct synthesis as 99%,99.5% or 100% sequence identity with any length or the described herein and known in the art. See Sambrooketal. In full length of any of the inventive sequences provided herein. certain embodiments, the invention contemplates allelic vari Homology or identity at the nucleotide or amino acid 30 ants of the TRIM5 and/or CyclophilinA portion of the nucleic sequence level maybe determined by BLAST (Basic Local acids which encode the fusion proteins of the invention. Alignment Search Tool) analysis using the algorithm Using known methods of protein engineering and recombi employed by the programs blastp, blastin, blastX, thlastn and nant DNA technology, variants may be generated to improve thlastx (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264 or alter the characteristics of the polypeptides of the present 2268 (1990) and Altschul, J. Mol. Evol. 36: 290-300 (1993), 35 invention. For instance, one or more amino acids can be fully incorporated by reference) which are tailored for deleted from the N-terminus or C-terminus of the polypeptide sequence similarity searching. of the present invention without substantial loss of biological The approach used by the BLAST program is to first con function. It is well known in the art that variants often retain sider similar segments between a query sequence and a data a biological activity similar to that of the reference protein. In base sequence, then to evaluate the statistical significance of 40 certain embodiments, variants of proteins of the invention or all matches that are identified and finally to summarize only nucleic acids encoding proteins of the invention retain HIV-1 those matches which satisfy a preselected threshold of sig restriction activity as described herein. The HIV-1 restriction nificance. For a discussion of basic issues in similarity search activity of variants may be comparable, increased or reduced ing of sequence databases, see Altschulet al., (Nature Genet compared to the HIV-1 restriction activity of the reference ics 6: 119-129 (1994)) which is fully incorporated by 45 polypeptides. Furthermore, even if deleting one or more reference. The search parameters for histogram, descriptions, amino acids from the N-terminus or C-terminus of a polypep alignments, expect (i.e., the statistical significance threshold tide results in modification or loss of one or more biological for reporting matches against database sequences), cutoff, activities, other biological activities may still be retained. For matrix and filter are at the default settings. The default scoring example, the ability of a variant to bind to HIV-1 may be matrix used by blastp, blastX, thlastin, and thlastx is the BLO 50 reduced compared to a reference polypeptide of the invention, SUM62 matrix (Henikoffet al., Proc. Natl. Acad. Sci. USA while the variant maintains the HIV-1 degradation activity, or 89: 10915-10919 (1992), fully incorporated by reference). vice versa, thus preserving the “HIV-1 restriction activity.” For blastin, the scoring matrix is set by the ratios of M (i.e., the which can readily be determined by methods described herein reward score for a pair of matching residues) to N (i.e., the and otherwise known in the art. penalty score for mismatching residues), wherein the default 55 In certain embodiments, the variants of the invention have values for M and N are 5 and -4, respectively. Four blastin conservative substitutions. The term “conservative substitu parameters may be adjusted as follows: Q=10 (gap creation tions' refers to substitution of an amino acid residue for a penalty); R=10 (gap extension penalty); wink-1 (generates different amino acid residue that has similar chemical prop word hits at every position along the query); and gapw-16 erties as known and understood by a person of ordinary skill (sets the window width within which gapped alignments are 60 in the art. Non-limiting examples are replacement of the generated). The equivalent Blastp parameter settings were aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; Q=9; R=2; wink=1; and gapw-32. A Bestfit comparison replacement of the hydroxyl residues Ser and Thr; replace between sequences, available in the GCG package version ment of the acidic residues Asp and Glu; replacement of the 10.0, uses DNA parameters GAP=50 (gap creation penalty) amide residues ASn and Gln, replacement of the basic resi and LEN=3 (gap extension penalty) and the equivalent set 65 dues Lys, Arg, and His; replacement of the aromatic residues tings in protein comparisons are GAP-8 and LEN=2. Other Phe, Tyr, and Trp, and replacement of the small-sized amino sequence analysis programs and algorithms are known and acids Ala, Ser, Thr, Met, and Gly. The term “nonconservative' US 8,835,617 B2 25 26 refers to substituting an amino acid residue for a different See Cunningham and Wells, Science 244: 1081-1085 (1989). amino acid residue that has different chemical properties. In The resulting mutant molecules can then be tested for bio certain embodiments the invention provides non-conserva logical activity. tive variants which have substantially the same HIV-1 restric As the authors state, these two strategies have revealed that tion activity compared to the reference polypeptides. The proteins are surprisingly tolerant of amino acid Substitutions. invention contemplates conservative and/or nonconservative The authors further indicate which amino acid changes are variants, so long as these variants retain HIV-1 restriction likely to be permissive at certain amino acid positions in the activity. protein. For example, most buried (within the tertiary struc In certain embodiments the invention provides variants ture of the protein) amino acid residues require nonpolar side 10 chains, whereas few features of Surface side chains are gen which modify the nucleic acids or amino acids of the linker erally conserved. Moreover, tolerated conservative amino between the TRIM5 and Cyclophilin portion of the fusion acid substitutions involve replacement of the aliphatic or proteins of the invention. Without being bound by theory, the hydrophobic amino acids Ala, Val, Leu and Ile; replacement linker between the TRIM5 and Cyclophilin portions of the of the hydroxyl residues Ser and Thr; replacement of the fusion proteins of the invention may tolerate a larger96 varia 15 acidic residues Asp and Glu, replacement of the amide resi tion in the number and/or types of modifications, including dues ASn and Gln, replacement of the basic residues Lys, Arg, different length of the linker, while maintaining the HIV-1 and His; replacement of the aromatic residues Phe, Tyr, and restriction activity. In certain embodiments, the invention Trp, and replacement of the Small-sized amino acids Ala, Ser, contemplates modifications of the linker which result in Thr, Met, and Gly. larger % variation in the linker sequence and thus lower % Besides conservative amino acid Substitution, variants of identity as compared to the % identity between the TRIM5 the present invention include (i) polypeptides containing Sub and/or Cyclophilin portions of the variants and the fusion stitutions of one or more of the non-conserved amino acid protein of the invention. A non-limiting example includes residues, where the Substituted amino acid residues may or variants wherein the identity between the TRIM5 and/or may not be one encoded by the genetic code, or (ii) polypep Cyclophilin portions of a variant and the fusion proteins of the 25 tides containing Substitutions of one or more of the amino invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, acid residues having a Substituent group, or (iii) polypeptides 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, which have been fused with or chemically conjugated to 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or another compound, such as a compound to increase the sta 100%, or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, bility and/or solubility of the polypeptide (for example, poly 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 30 ethylene glycol), (iv) polypeptide containing additional 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%, while the amino acids, such as, for example, an IgG Fc fusion region identity between the linker of the variant and the fusion pro peptide. Such variant polypeptides are deemed to be within teins of the invention is at least 10%, 15%, 20%, 25%, 30%, the scope of those skilled in the art from the teachings herein. 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, For example, polypeptide variants containing amino acid 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 35 Substitutions of charged amino acids with other charged or 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, neutral amino acids may produce proteins with improved 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, characteristics, such as less aggregation. Aggregation of phar 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, maceutical formulations both reduces activity and increases 98%, 99%, 99.5% or 100%, or 10%, 15%, 20%, 25%, 30%, clearance due to the aggregate’s immunogenic activity. See 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 40 Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Rob 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, bins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993). 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, In specific embodiments, the polypeptides of the invention 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, comprise, or alternatively, consist of, or consistessentially of 98%, 99%, 99.5% or 100%, or any combination there of 45 fragments or variants of the amino acid sequences of the Guidance concerning how to make phenotypically silent invention wherein the fragments or variants have 1, 2, 3, 4, 5, amino acid Substitutions is provided, for example, in Bowie et 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 1-5, 5-10, 5-15, 5-25, 5-50, al., “Deciphering the Message in Protein Sequences: Toler 10-50 or 50-150, amino acid residue additions, substitutions, ance to Amino Acid Substitutions. Science 247: 1306-1310 and/or deletions when compared to the reference amino acid (1990), wherein the authors indicate that there are two main 50 sequence. In preferred embodiments, the amino acid substi strategies for studying the tolerance of an amino acid tutions are conservative. Nucleic acids encoding these sequence to change. polypeptides are also encompassed by the invention. The first strategy exploits the tolerance of amino acid Sub The polypeptide of the present invention can be composed stitutions by natural selection during the process of evolution. of amino acids joined to each other by peptide bonds or By comparing amino acid sequences in different species, 55 modified peptide bonds, i.e., peptide isosteres, and may con conserved amino acids can be identified. These conserved tain amino acids other than the 20 gene-encoded amino acids. amino acids are likely important for protein function. In con The polypeptides may be modified by either natural pro trast, the amino acid positions where substitutions have been cesses, such as post-translational processing, or by chemical tolerated by natural selection indicates that these positions are modification techniques which are well known in the art. not critical for protein function. Thus, positions tolerating 60 Such modifications are well described in basic texts and in amino acid substitution could be modified while still main more detailed monographs, as well as in a Voluminous taining biological activity of the protein. The second strategy research literature. It will be appreciated that polypeptides uses genetic engineering to introduce amino acid changes at often contain amino acids other than the 20 amino acids specific positions of a cloned gene to identify regions critical commonly referred to as the 20 naturally occurring amino for protein function. For example, site directed mutagenesis 65 acids, and that many amino acids, including the terminal or alanine-scanning mutagenesis (introduction of single ala amino acids, may be modified in a given polypeptide, either nine mutations at every residue in the molecule) can be used. by natural processes Such as glycosylation and other post US 8,835,617 B2 27 28 translational modifications, or by chemical modification potently blocks N-tropic murine leukemia virus (N-MLV). In techniques which are well known in the art. Modifications can contrast, rhesus T5C. (RhT5C) inhibits HIV-1 but N-MLV occur anywhere in a polypeptide, including the peptide back only weakly. The specificity of retroviral restriction and the bone, the amino acid side-chains and the amino or carboxyl modular nature of the T5 components are further demon termini. It will be appreciated that the same type of modifi strated by the enhanced HIV-1 restriction activity that results cation may be present in the same or varying degrees at when the hT5C. PRYSPRY domain is replaced with that from several sites in a given polypeptide. Also, a given polypeptide RhT5C. (Perez-Caballero et al., 2005a; Sawyer et al., 2005; may contain many types of modifications. Polypeptides may Stremlau et al., 2005; Yap et al., 2005). be branched, for example, as a result of ubiquitination, and The T5 gene in the New World owl monkey (genus Aotus) they may be cyclic, with or without branching. Cyclic, 10 branched, and branched cyclic polypeptides may result from is unusual. It resulted from retrotransposition of the cyclophi posttranslation natural processes or may be made by synthetic lin A (CypA) cDNA into intron 7 (Nisole et al., 2004; Sayah methods. Modifications include acetylation, acylation, ADP et al., 2004). CypA is a CA-binding protein (Luban et al., ribosylation, amidation, covalent attachment of flavin, cova 1993) and the Aotus TRIM5Cyp fusion (AoT5Cyp) prevents lent attachment of a heme moiety, covalent attachment of a 15 HIV-1 infection (Nisole et al., 2004; Sayah et al., 2004). nucleotide or nucleotide derivative, covalent attachment of a AoT5Cyp has several properties that make it appealing for lipid or lipid derivative, covalent attachment of phosphoti gene therapy. It potently inhibits HIV-1 infection when dylinositol, cross-linking, cyclization, disulfide bond forma expressed in human cells (Nisole et al., 2004; Sayah et al., tion, demethylation, formation of covalent cross-links, for 2004), acting on the virus within minutes of entry (Diaz mation of cysteine, formation of pyroglutamate, formylation, Griffero et al., 2006b; Perez-Caballero et al., 2005a). Further gamma-carboxylation, glycosylation, GP1 anchor formation, more, AoT5Cyp is the only TRIM5 allele in 10 Aotus species hydroxylation, iodination, methylation, myristylation, oxida (Ribeiro et al., 2005), indicating that cells bearing AoT5Cyp tion, pegylation, proteolytic processing, phosphorylation, retain functionality. One drawback of AoT5Cyp is that it is prenylation, racemization, selenoylation, Sulfation, transfer not a human protein and if employed as gene therapy it might RNA mediated addition of amino acids to proteins such as 25 elicit an immune response (Riddell et al., 1996). Provided arginylation, and ubiquitination. (See, for instance, PRO herein is an engineered fully human TRIM5-cyclophilin A TEINS STRUCTURE AND MOLECULAR PROPER fusion proteins (hTSCyp) exhibiting HIV-1 restriction activ TIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, ity comparable to AoT5Cyp and possessing all the properties New York (1993); POST TRANSLATIONAL COVALENT desired of anti-HIV-1 gene therapy. MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Aca 30 In Old World primates, TRIM5-C. confers a potent block to demic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattanet al., Ann. N.Y. Acad. HIV-1 infection that acts after virus entry into cells. Cyp A Sci. 663:48-62 (1992)). binding to viral capsid protects HIV-1 from a similar activity The invention further provides a stem cell comprising a in human cells. Among New World primates, owl monkeys nucleic acid of the invention, wherein the nucleic acid is part 35 exhibit post-entry restriction of HIV-1. Paradoxically, the of the genome of the stem cell. As used herein a “stem cell barrier to HIV-1 in owl monkey cells is released by capsid refers to an undifferentiated cell in the bone marrow that has mutants or drugs that disrupt capsid interaction with CypA. the ability both to multiply and to differentiate into a specific, U.S. Patent Application Publication No. 2008/004.5454, specialized cell. Such as a blood cell. In one embodiment, incorporated herein by reference, describes a fusion protein human hematopoietic stem cells are mobilized from the bone 40 between TRIM5 and cyclophilinA which was discovered and marrow of an HIV-1 infected subject, the cells are transduced isolated from Owl monkey, which is a species of primate from in vitro with vectors expressing human TRIMCyp, and the South America that are resistant to HIV-1 infection. The cells are injected back into the patient as an auto-transplant. TRIM-cyclophilin fusion protein provides the Owl monkey As used in this specification and the appended claims, the with its resistance to HIV. The cyclophilin Apart of the fusion singular forms “a,” “an and “the include plural references 45 protein specifically binds to the HIV-1 capsid protein once unless the content clearly dictates otherwise. HIV-1 enters the cell. The TRIM5 part of the fusion protein Provided herein is a novel fusion between human acts as TRIM5 alone has been characterized, causing the TRIM5alpha (TRIM5C.: T5: TRIM) and human cyclophilin A degradation of the Substance bound to the cyclophilin A por (CypA: Cyp; Cyp A). A fully human nucleic acid fusion tion of the fusion protein, namely the degradation of HIV-1. sequence and polypeptide fusion sequence comprising a 50 Thus, the fusion protein has coupled a specific binding tro human TRIM5 sequence and a human CypA sequence are pism, i.e., the binding of cyclophilin to capsid of HIV-1, with provided. Such fusion molecules are referred to as human the degradation activity of TRIM5. The cyclophilin part of the TRIM5-CypA (or hT5-CypA or TRIM-Cyp or hTRIMCyp, fusion protein is highly conserved among different species. or similar variations). Also provided are methods of using the Cyclophilin is a protein that is present in yeast and in humans. fusion molecules. 55 It plays a role in protein-folding and in the biological response The discovery that certain TRIM5 (T5) orthologues inhibit to the immunosuppressive drug cyclosporine. The TRIM5 HIV-1 infection immediately after the virus enters otherwise part of the fusion protein is highly variable from one species susceptible cells (Diaz-Griffero et al., 2006b; Perez-Cabal to another. lero et al., 2005a; Sayah et al., 2004; Stremlau et al., 2004) The C-isoform of TRIM5 (T5C) contains a C-terminal raised the prospect that these host factors might be exploited 60 PRYSPRY domain that is required for T5C. binding to the in HIV-1 gene therapy. The C-isoform of TRIM5 (T5C.) con capsid (CA) of restriction-sensitive retroviruses''. The tains a C-terminal PRYSPRY domain that is required for T5C. specificity of the PRYSPRY-CA interaction determines binding to the capsid (CA) of restriction-sensitive retrovi which retrovirus a given T5C. orthologue inhibits. While ruses (Sebastian and Luban, 2005; Stremlau et al., 2006). The human T5C. (hT5C.) weakly blocks HIV-1, it potently blocks specificity of the PRYSPRY-CA interaction determines 65 N-tropic murine leukemia virus (N-MLV). In contrast, rhesus which retrovirus a given T5C. orthologue inhibits (Luban, T5C. (rhT5C) inhibits HIV-1 but not N-MLV. Restriction 2007). While human T5C. (hTSC) weakly blocks HIV-1, it specificity and the modular nature of the T5 components are US 8,835,617 B2 29 30 further demonstrated by the enhanced HIV-1 restriction activ ity was observed when hCypA was fused at the apex of the ity that results when the hT5C. PRYSPRY domain is replaced hT5C. PRYSPRY domain (See FIG. 47C). Analysis of non with that from rhT5C'''. synonymous mutations indicates that this region undergoes The potency of a human TRIM-Cyp fusion has been dem Some of the strongest selective pressure in the primate lineage onstrated in vitro and in vivo in a humanized mouse model of 5 and functional experiments pinpoint it as a specificity deter HIV-1. The use of TRIM-Cyp provides several benefits and minant for CA recognition (Sawyer et al., 2005; Song et al., improvements over current anti-HIV-1 technologies. Such as 2005b; Yap et al., 2005). CypA fusion to this site may have recombinant RevM10 protein, RNAi approaches, and disrup antiviral activity because this apical loop is uniquely situated tion of the CCR5 co-receptor. The high efficacy of human for coordinating CA recognition and effector domains of T5. TRIM-Cyp would enable its use as a monotherapy, which is a 10 Correlation between restriction activity and the ability of significant benefit over the use of combination therapies hT5Cyp fusion proteins to form cytoplasmic bodies suggests required by the current technologies. Also, the potency of that activity requires particular spatial constraints or associa human TRIM-Cyp minimizes the emergence of viral resis tion with cofactors. tance, a recurrent problem with current technologies. Human Optimal gene therapy candidates should provide high effi TRIMCyp acts early in the viral life cycle conferring true 15 cacy and low antigenicity. Hematopoietic progenitors trans protection and blocking the ligation of HIV-1 into host chro duced with rhesus macaque T5C. or human-rhesus T5C. chi mosomal DNA, thus minimizing viral persistence and poten meras differentiate into HIV-1-resistant macrophages and T tial mutagenic effects of HIV-1 infection. cells (Anderson and Akkina, 2005a, 2008). These proteins, Provided herein is an HIV-1 inhibitor modeled after however, are potentially antigenic, which could lead to the AoT5Cyp generated by fusing human CypA to hT5 elimination of modified cells in vivo (Riddellet al., 1996). By (hT5Cyp). As further described in Example 1, of 13 fusions, substituting only critical amino acids within the hT5C. three were as potent as AoT5Cyp. Antiviral activity of the PRYSPRY domain one can reduce the risk of antigenicity but human fusions required capsid (CA)-binding by hCypA, this also decreases anti-HIV-1 efficacy (hT5CR332P: See linkage of hCypA to the capsid-specificity determinant in FIG. 5, A and B). ht5Cyp potently inhibits HIV-1 and, being hT5, and the ability to form cytoplasmic bodies. CXCR4- and 25 composed of human components, is of low antigenic poten CCR5-tropic HIV-1 clones and primary isolates, a circulating tial. HIV-1 isolate previously reported as AoT5Cyp-resistant, HIV-1 rev, tat, and gag (Boden et al., 2007; Strayer et al., HIV-2, SIV tan, and FIV were all inhibited by hT5Cyp. 2005), as well as the HIV-1 co-receptor CCR5 (An et al., hT5Cyp blocked HIV-1 infection of primary CD4" T cells 2007; Perez et al., 2008), have been targeted by various means and macrophages, and conferred selective advantage after 30 including siRNAs (Boden et al., 2007: Strayer et al., 2005) HIV-1 infection, without selection for resistant virus or dis and zinc-finger endonucleases (Perez et al., 2008). With ruption of host cell function. Moreover, hT5Cyp preserved either approach there are concerns regarding toxicity, off hCD4 T cells after adoptive transfer to Rag2'Y' mice target effects, and viral resistance (Boden et al., 2007: and challenge with HIV-1; viremia and productive infection Strayer et al., 2005). For siRNA approaches, escape mutants in lymphoid organs were markedly reduced. Thus, hT5Cyp is 35 arise readily, since single point mutations are Sufficient to a robust candidate for HIV-1 gene therapy. escape the siRNA-mediated blocks to HIV-1 replication The more successful anti-HIV-1 gene therapy approaches (Boden et al., 2007). CCR5 disruption could select for CCR5 to date have followed two mainstrategies: either modification independent viruses (Taylor et al., 2008) and is not without of host-cell factors required for viral replication or inhibition consequence, as the CCR5A32 allele is a risk factor for symp ofessential viral elements (Strayer et al., 2005). As described 40 tomatic West Nile Virus infection (Glass et al., 2006). herein, a third approach was adopted: the exploitation of hT5Cyp is a broadly acting, anti-lentiviral agent that natural inhibitors that evolved over millions of years in pri blocks CCR5- and CXCR4-tropic HIV-1 clones and primary mates in response to retroviral attack (Li, 2006b; Sawyer, isolates, as well as some HIV-2, SIV, and FIV clones (See 2005; Stremlau, 2005:Yap, 2005). The potent block to HIV-1 FIGS. 48, 49, 56). T5 isoforms form hetero-multimers and observed with AoT5Cyp inspired the design of a human 45 hT5Cyp might interfere with endogenous hT5C. function equivalent, hT5Cyp, that robustly blocks HIV-1 in vitro and (Berthoux et al., 2005a). However, no significant decrease in in vivo. The strict structural requirements for engineering a hT5C.-mediated restriction of N-MLV, and no alteration in functional hT5Cyp emphasize the remarkable nature of the cell Surface markers or cytokine secretion, was observed in fusion gene that was generated by retrotransposition in New hT5Cyp-transduced cells (See FIG. 46). ht5Cyp-resistant World owl monkeys (Sayah et al., 2004). A distinct T5Cyp 50 viruses were not detected, perhaps due to the potency of the fusion gene was generated by an independent retrotransposi antiviral activity and the fact that hT5Cyp blocks HIV-1 prior tion event in Old World macaques. In this case, restriction to reverse transcription. ht5Cyp additionally blocked a natu activity was detected against HIV-2 and FIV, but not against ral HIV-1 CA variant reported to be resistant to AoT5Cyp HIV-1 (Stove and Yap, 2008). The convergent evolution of (Chatterji et al., 2005) (See FIG. 49D). These results suggest T5Cyp fusion proteins with distinct retroviral specificities 55 that, if hT5Cyp were utilized as anti-HIV-1 gene therapy in indicates a strong selection for these potent restriction factors. people, viral resistance would not emerge and host cell func While the specific force behind selection remains to be elu tion would remain intact. cidated for individual T5 orthologues, in each case the selec The evaluation of HIV-1 therapies is limited by a lack of tive pressure is likely to have been a retrovirus (Sawyer et al., adequate animal models for HIV-1 replication and pathogen 2005). 60 esis. The hCD4"-Rag2'Y' mouse described in Example 2 Since T5C. and T5Cyp are modular proteins, it was is a robust model for assessing inhibition of HIV-1 infection expected that the engineering of a restrictive hT5Cyp would and CD4 T cell protection by lentiviral transduction with be trivial. Surprisingly, only three of 13 hT5Cyp fusions hT5Cyp (See FIGS. 69, 70, 71). Autotransfusion of ex vivo inhibited HIV-1 comparably to AoT5Cyp. No correlation was expanded CD4 T cells is of clinical benefit to HIV-1-infected observed between protein levels and antiviral activity, and 65 people (Levine et al., 2002). Thus, given that hT5Cyp-trans CA-binding activity was not sufficient to restrict HIV-1 (See duced CD4 cells can be expanded in vitro and in vivo (See FIG. 47). Structural modeling showed that anti-HIV-1 activ FIGS. 54, 69), and that these cells exhibit a selective advan US 8,835,617 B2 31 32 tage during HIV-1 infection (See FIG. 57), an impressive that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, effect of hT5Cyp-transduction on autologous CD4 T cell or 99% identical to SEQ ID NO.4 and a sequence that is at gene therapy in the clinical setting is expected. CD34' least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hematopoietic stem cells transduced with state-of-the-art len identical to SEQID NO:2, or a sequence that is at least 60%, tiviral vectors (see FIGS. 65,66 (Amendola et al., 2005)) 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to achieve long-term engraftment of Rag2Y. mice, but SEQID NO:10, or a sequence that is at least 60%. 65%, 70%, transgene expression in the mature CD4 T cells that develop 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID within these animals has not yet been consistently detected. NO:11, or a sequence that is at least 60%. 65%, 70%, 75%, One could examine the effect of stem cell transduction with 80%, 85%, 90%, 95%, or 99% identical to SEQID NO:12. hT5Cyp on subsequent immune cell development and HIV-1 10 FIGS. 7A and 7C show amino acid residues depicted in red infection with a humanized mouse models currently under (308-311, 321-346, 366-371 and 381-393) indicating resi development (Goldstein, 2008). dues that comprise loops in the PRYSPRY domain that con Human TRIM5 and CypA sequences have been deposited tain S309, S322 and A331. Thus fusion of CypA to any in GenBank (accession numbers NM 033034 (human residue in these regions would be predicted to generate a TRIM5alpha mRNA: FIG. 13: SEQID NO:1), NP 149023 15 hTRIM-Cyp fusion that has HIV-1 restrictive activity. Addi (human TRIM5alpha protein: FIG. 14: SEQ ID NO:2), tionally, fusion of CypA at M285 generates a fusion protein NM 021130 (human CypA mRNA: FIG. 15: SEQID NO:3) that is variably restrictive, thus fusing to amino acid residues and NP 066953 (human CypA protein: FIG. 16: SEQ ID 280–290 would be predicted to have anti-HIV-1 activity. In NO:4)). one embodiment, the last (C-terminal) amino acid of SEQID The invention provides for a newly engineered nucleic NO:2, which is fused to CypA, is serine at position 309, serine acids and polynucleotides that comprise the nucleotide at position 322, or alanine at position 331. In one embodi sequence of SEQID NO:1, 3, 5, 6 or 7. The encoded polypep ment, the last amino acid of SEQID NO:2 is any one amino tide is a fusion between human TRIM and cyclophilin, i.e., acid residue from about residue 280 to about residue 435, or hTRIMCyp, which acts as an anti-HIV-1 factor. from about residue 280 to about residue 400, or from about The invention also provides for nucleic acid variants of any 25 residue 280 to about residue 290, or from about residue 308 to of SEQ ID NO: 1, 3, 5, 6 or 7 having at least about 50% about residue 311; or from about residue 321 to about residue identity to the SEQ ID NO, and encoding a polypeptide 346; or from about residue 366 to about residue 371; or from having both TRIM activity and cyclophilin activity. The vari about residue 381 to about residue 393. In one embodiment, ants may have at least about 55%, 60%, 70%, 75%, 80%, the last residue of SEQID NO:2 is residue 493, the end of the 85%, 90%, 95%, or 99% identity to the SEQ ID NO. Tech 30 TRIM5 PRYSPRY domain, thus generating a fusion protein niques for determining sequence identity are well known to where a sequence encoding cyclophilin A replaces the stop one skilled in the art, and include, for example, analysis with codon of the PRYSPRY domain of TRIM5. In one embodi a sequence comparison algorithm or FASTA version 3.0t78 ment, the last residue of SEQID NO:2 is 280,281,282,283, using default parameters. 284, 285, 286, 287, 288, 289, 290, 308, 309, 310, 311, 321, The invention also provides for an isolated nucleic acid 35 322,323,324, 325, 326, 327,328,329, 330, 331, 332,333, which comprises consecutive nucleotides having a sequence 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, complementary to the nucleic acids comprising the nucle 346,366, 367, 368, 369,370, 371,381,382,383,384,385, otide sequence of SEQID NO:1, 3, 5, 6 or 7 or variants of at 386,387,388,389, 390,391,392,393, or 493. The last amino least about 50% identity thereof. The invention also provides acid of SEQID NO:2 can be fused to Cyp A (for example, at for an isolated nucleic acid encoding a polypeptide compris 40 residue 2 of SEQID NO:4) by experimental methods known ing SEQID NO:4 (human Cyp A), and SEQID NO:2 (human in the art and described in the Examples. TRIM5), wherein the last amino acid of SEQ ID NO:2 is The polynucleotides provided by the invention may further alanine 331 (SEQ ID NO:5; FIG. 17), serine 309 (SEQ ID comprise a sequence encoding a linker located between the NO:6; FIG. 18), or serine 322 (SEQID NO:7: FIG. 19). Also TRIM sequence and the Cyp sequence. In one embodiment, provided by the invention is an isolated nucleic acid that 45 the linker comprises from about 10 to about 20 amino acids. hybridizes to a nucleic acid of the invention under conditions In other embodiments the linker comprises, consists of or of high Stringency, moderate stringency, or low stringency. consists essentially of from about 11 to about 20, from about The invention further provides for an isolated polypeptide 12 to about 20, from about 11 to about 24, from about 12 to encoded by an isolated nucleic acid of the invention. Purified about 24, 11 to about 33, from about 12 to about 33, 11 to polypeptides Substantially identical to the isolated polypep 50 about 43, from about 12 to about 43, 11 to about 59, from tide, as determined by analysis with a sequence comparison about 12 to about 59 amino acids. In other embodiments the algorithm or FASTA version3.0t78 using default parameters, linker comprises, consists of or consists essentially of from 11 are also included in the invention. to 20, from 12 to 20, from 11 to 24, from 12 to 24, 11 to 33, The invention also provides a human TRIM-cyclophilin from 12 to 33, 11 to 43, from 12 to 43, 11 to 59, from 12 to 59 fusion protein, and nucleic acids encoding Such fusion pro 55 amino acids. In other embodiments the linker comprises, teins, where the N-terminus of human CypA (SEQID NO:4) consists of or consists essentially of 11, 12, 13, 14, 15, 16, 17. is fused to the C-terminus of human TRIM5 (SEQID NO:5). 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, Fusion proteins with anti-HIV-1 activity include hT5-S309 35, 36, 37, 38, 39, 40, 59, 71, or up to 100 amino acids. In Cyp (SEQID NO:11), hT5-S322-Cyp (SEQID NO:12), hT5 another embodiment, the linker comprises the amino acid A331-Cyp (SEQ ID NO:10), hT5-M284-Cyp, hT5-T369 60 sequence SGGSGGSGGSGG (SEQID NO: 20) or a variant Cyp, where the indicated amino acid is the C-terminal residue thereof. In one embodiment, the linker consists essentially of of TRIM5 (SEQ ID NO:2) to which the N-terminal amino the amino acid sequence SGGSGGSGGSGG (SEQ ID NO: acid (for example, residue 2 of SEQ ID NO:4) of CypA is 20) or a variant thereof. In certain embodiments the linker directly attached or fused. Embodiments of the invention comprises or consists of a synthetic sequence. In other include a polypeptide comprising SEQID NO.4 and SEQID 65 embodiments the linker comprises, consists essentially of or NO:2, or a polypeptide comprising SEQID NO:10, 11, or 12. consists of sequences from hTRIM5, for example as The invention provides a polypeptide comprising a sequence described herein. In certain embodiments the fusion proteins,