Medical publications - Biomapilot Viscchini Enzymes

Transcription

1 II uii IIi US B2 (12) United States Patent Pavlakis (10) Patent No.: US 7,608,422 B2 (45) Date of Patent: Oct. 27, 2009 (54) SIMIAN IMMUNODEFICIENCY VIRUS (SIV) MOLECULAR CLONE ENCODING MUTANT GAG GENE LACKING INHIBITORY/INSTABILITY REGIONS (75) Inventor: George N. Pavlakis, Rockville, MD (US) (73) Assignee: The United States of America as represented by the Secretary of the Department of Health and Human Services, Washington, DC (US) (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 962 days. (21) Appl. No.: 10/644,027 (22) Filed: Aug. 19, 2003 (65) Prior Publication Data US 2004/ Al Apr. 22, 2004 Related U.S. Application Data (60) Division of application No. 09/872,733, filed on Jun. 1, 2001, now Pat. No. 6,656,706, which is a continuationin-part of application No. PCT/US00/34985, filed on Dec. 22, (60) Provisional application No. 60/173,036, filed on Dec. 23, (51) Int. Cl. C12P 21/06 ( ) C07H 21/04 ( ) C12N 15/00 ( ) C12N 5/00 ( ) (52) U.S. Cl /69.1; 435/325; 435/455; 536/23.72; 514/44 (58) Field of Classification Search... None See application file for complete search history. (56) References Cited U.S. PATENT DOCUMENTS 5,786,464 A 7/1998 Seed et al. 5,795,737 A 8/1998 Seed 5,965,726 A * 10/1999 Pavlakis et al / ,972,596 A 10/1999 Pavlakis et al. 6,114,148 A 9/2000 Seed et al. 6,174,666 B1 1/2001 Pavlakis et al. OTHER PUBLICATIONS Myers, G., et al., Human Retroviruses and AIDS 1995: A compilation and analysis of nucleic acid and amino acid sequences, Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, section IB (HIV-2/SIV gag nucleotide sequence alignment).* Benson, J., et al., 1998, Recombinant vaccine-induced protection against the highly pathogenic simian immunodeficiency virus SlVmac251: dependence on route of challenge exposure, J. Virol. 72(5): * * cited by examiner Primary Examiner Jeffrey S. Parkin (74) Attorney, Agent, or Firm Townsend and Townsend and Crew LLP (57) ABSTRACT Nucleic acid constructs containing HIV-1 gag/pol and SIV gag or SIV env genes which have been mutated to remove or reduce inhibitory/instability sequences are disclosed. Viral particles and host cells containing these constructs and/or viral particles are also disclosed. The exemplified constructs and viral particles of the invention may be useful in gene therapy for numerous disorders, including HIV infection, or as a vaccine for HIV- 1 immunotherapy and immunoprophylaxis. 8 Claims, 45 Drawing Sheets

2 U.S. Patent Oct. 27, 2009 Sheet 1 of 45 US 7,608,422 B2 ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAUUU%AT TCGGTTAAGGCCAGGGGGAAAGAAGAAGTACAAGCTAAAGCACATCGTATGGGCAA GCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGC TGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAḠGAGCT TCGATCACTATACAACACAG TAG CAACCCTC TAT TGTGTGCACCAGCGGATCGAGA TCAAGGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAGTCCAAG AAGAAGGCCCAGCAGGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAA TTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTA CAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTG ATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAGGACCTGAACACGAT GTTGAACACCGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCA ATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCA CCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCT TCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAGATCT P.CAAGAGGTGGATAATCCTGGGATTGAACAAGATCGTGAGGATGTATAGCCCTACC AGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTA-GACCG GTTCTATAAAACTCTAAGAGCTGAGCAAGCTTCACAGGAGGTAAA.AAATTGGATGA CAGAAACCTTGTTGGTCCAAAATGCGAA-CCCAGATTGTAAGACCATCCTGAAGGCT CTCGGCCCAGCGGCTACACTAGAAGAAATGATGACAGCATGTCAGGGAGTAGGAGG ACCCGGCCATAAGGCAAGAGTTTTGGCCGAGGCGATGAGCCAGGTGACGAACTCGG CGP_CCATAATGATGCAGAGAGGCAACTTCCGGAACCAGCGCAAGATCGTCAAGTGC TTCAATTGTGGCAAAGAAGGGCACACCGCCAGGAACTGCCGGGCCCCGCGGAT AA GGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGAC FIG. IA

3 U.S. Patent Oct. 27, 2009 Sheet 2 of 45 US 7,608,422 B2 AGGCTAATTTTTTAGGGAAGATCTGGCCTTCCTACAAGGGAAGGCCAGGGAATTTT CTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTCTGGGGT AGAGACAACAACTCCCCCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTT TAACTTCCCTCAGATCACTCTTTGGCAACGACCCCTCGTCACAGTAAGGATCGGGG GGCAACTCAAGGAAGCGCTGCTC GATACAGGAGCAGATGATACAGTATTAGAAGAA ATGAGTTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGGATCGCGGGCTTCAT CAAGGTGAGGCAGTACGACCAGATACTCATAGAAATCTCTGGACATAAAGCTATAG GTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACC CAGGATCGGCTGCACCTTGAACTTCCCCATCAGCCCTATTGAGACGGTGCCCGTGAA GTTGAAGCCGGGGATGGACGGCCCCAAGGTCAAGCAATGGCCATTGACGAAAGAGA AGATCAAGGCCTTAGTCGAAATCTGTACAGAGATGGAGAAGGAAGGGAAGATCAGC AAGATCGGGCCTGAGAACCCCTACAACACTCCAGTCTTCGCAATCAAGAAGAAGGA CAGTACCAAGTGGAGAAAGCTGGTGGACTTCAGAGAGCTGAACAAGAGAACTCAGG ACTTCTGGGAAGTTCAGCTGGGCATCCCACATCCCGCTGGGTTGAAGAAGAAGAAG TCAGTGACAGTGCTGGATGTGGGTGATGCCTACTTCTCCGTTCCCTTGGACGAGGA CTTCAGGAAGTACACTGCCTTCACGATACCTAGCATCAACAACGAGACACCAGGCA TCCGCTACCAGTACAACGTGCTGCCACAGGGATGGAAGGGATCA-CCAGCCATCTTT CAAAGCAGCATGACCAAGATCCTGGAGCCCTTCCGCAAGCAAAACCCAGACATCGT GATCTATCAGTACATGGGACGACCTCTAGGTAGGAAGTGACCTGGAGATCGGGCAGC ACAGGACCAAGATCGAGGAGCTGAGACAGCATCTGTTGAGGTGGGGACTGACCACA CCAGACAAGAAGCACCAGAAGGAACCTCCCTTCCTGTGG4TGGGCTACGAACTGCA TCCTGACAAGTGGACAGTGCAGCCCAT-CGTCTGCCTGAGAAGGACAGCTGGACTG TG..ACGACATACAGAAGCTCGTGGGCAAGTTGAACTCiGGCAAGCCAGATCTACCCA GGCATCAAAGTTAGGCAGCTGTGCAAGCTGCTTCGAGGAACCAAGGCACTGACAGA FIG. IB

4 U.S. Patent Oct. 27, 2009 Sheet 3 of 45 US 7,608,422 B2 AGTGATCCCACTGACAGAGGAAGCAGAGCTAGAACTGGCAGAGAACCGA,GAGATCC TGAAGGAGCCAGTACATGGAGTGTACTACGACCCAAGCAAGGACCTGATCGCAGAG ATCCAGAAGCAGGGGCAAGGCCAATGGACCTACCAAATCTACCAGGAGCCCTTCAA GAACCTGAAGACAGGCAAGTACGCAAGGATGAGGGGTGCCCACACCAACGATGTGA AGCAGCTGACAGAGGCAGTGCAGAAGATCACCACAGAGAGCATCGTGATCTGGG^GC AAGACTCCCAAGTTCAAGCTGCCCATACAGAA GGAGACATGGGAGACATGGTGGAC CGAGTACTGGCAAGCCACCTGGATCCCTGAGTGGGAGTTCGTGAACACCCCTCCCT TGGTGAAACTGTGGTATCAGCTGGAGAAGGAACCCATCGTGGGAGCAGAGACCTTC TACGTGGATGGGGCAGCCAACAGGGAGACCAAGCTGGGCAAGGCAGGCTACGTGAC CAACCGAGGACGACAGAAAGTGGTGACCCTGACTGACACCACCAACCAGAAGACTG AGCTGCAAGCCATCTACCTAGCTCTGCAAGACAGCGGACTGGAAGTGAACATCGTG ACAGACTCACAGTACGCACTG-GGCATCATCCAAGCACAACCAGACCAATCCGAGTC AGAGCTGGTGAACCAGATCATC-GAGCAGCTGATCAAGAAGGAGAAAGTGTACCTGG CATGGGTACCAGCACACAAAGGAATTGGA'GGAAATGAACAAGTAGATAAATTAGTC AGTGCTGGGATCCGGAAGGTGCTGTTCCTGGACGGGATCGATAAGGCCCAAGATGA ACATCAGAAGTACCACTCCAACTGGCGCGCTATGGCCAGCGACTTCAACCTGCCAC CTGTAGTAGCAAAA,GAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAA GCCATGCATGGACAAGTAGACTGTAGTCCAGGAATATGGCAGCTGGACTGCACGCA CCTGGAGGGGAAGGTGATCCTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAG CAGAAGTTATCCCTGCTGAAACTGGGCAGGAAACAGCATATTTTCTTTTAAAATTA GCAGGAAGATGGCCAGTAAAAACAATACACACGGACAACGGAAGCAACTTCACTGG TGCTACGGTTAAGGCCGCCTGTTGGTGGGCCGGGAATCAAGCAGGAATTTGGAATTC CCTACAATCCCCAATCGCAAGGAGTCGTGGAGAGCATGAACAAGGAGCTGAAGAAG ATCATCGGACAAGTGAGGGATCAGGCTGAGCA,CCTGAAGACAGCAGTGCAGAT'GGC FIG. IC

5 U.S. Patent Oct. 27, 2009 Sheet 4 of 45 US 7,608,422 B2 AGTGTTCATCCAGAACTTCAAAAGAAAAGGGGGGATTGGG-GGGTACAGTGCAGGGG AAAGGATCGTGGACATCATCGCCAC-CGACATC-CAAACCAAG,GAGCTGCAGAAGCAG ATCACCAA GATCGAGAACTTCCGGGTGTACTACCGCGACA GCCGCAACCCACTGTG GAAGGGACCAGCAAAGCTCCTCTGGAAGGGAGAGGGGGCAGTGGTGATCCAGGACA ACA-GTGACATCAAAGTG-GTGCCAAGGCGCAAGGCCAAGATCATCCGC-GACTATGGA AAA,CAGATGGCAGGTGATGATTGTGTGGCAAGTA,GACAGGATGAGGATTAGAACCT GGAAGAGCCTGGTGAAGCACCATATG (SEQUENCE ID NO:1) FIG. ID

6 U.S. Patent Oct. 27, 2009 Sheet 5 of 45 US 7,608,422 B2 >wildtype >mutated TGTACAGAGA TGGAAAAGGA AGGGAAAATT TCAAAAATTG TGTACAGAGA TGGAGAAGGA AGGGAAGATC AGCAAGATCG # >wildtype >mutated GGCCTGAAAA TCCATACAAT ACTCCAGTAT TTGCCATAAA GGCCTGAGAA CCCCTACAAC ACTCCAGTCT TCGCAATCAA #41... >wildtype >mutated GAW.. AGAC AGTACTAAAT GGAGAAA-ATT AGTAGATTTC GAAGAAGGAC AGTACCAAGT GGA3AA0.GCT GGTGGACTTC #el... >wildtype >mutated AGAGAACTTA ATAAGAGAAC TCAAGACTTC TGGGAAGTTC AGAGAGCTGA ACAAGAGAAC TCAGGACTTC TGGGAPtGTTC # a... >wildtype >mutated AATTAGGAAT ACCACATCCC-GCAGGGTTAA AAAAGA N AGCTGGGCAT CCCACATCCC GCT.GGGTTGA AGAAGAA'GAA # >wildtype >mutated ATCAGTAACA GTACTGGATG TGGGTGATGC ATATTTT'TCA GTCAGTGACA GT'GCTGGATG TGGGTGATGC CTACTTCTCC # >wildtype >rnutated GTTCCCTTAG ATGAAGACTT CAGGAAATAT ACTGCATTTA GTTCCCTTGG ACGAGGACTT CAGGAAGTAC ACTGCCTTCA # >wildtype >mutated CCATACCTAG TATAAACAAT GAGACACCAG GGATTAGATA CGATACCTAG CATCAACAAC GAGACACCAG GCATCCGCTA # >wildtype >mutated TCAGTACAAT GTGCTTCCAC AGGGATGGAA AGGATCACCA CCAGTACAAC GTGCTGCCAC AGGGATGGAA GGGATCACCA # >wildtype >mutated GCAATATTCC AAAGTAGCAT GACAAAAATC TTAGAGCCTT GCCATCTTTC AAAGCAGCAT GAGCAAfsATC CTGGAGCCCT # r.. r r *4 * >wildtype >mutated TTAGAT~AACA AAATCCAGAC ATAGTTATCT ATCAATACAT TCCGCAAGCA AAACCCAGAC ATCGTGATCT ATCAGTACAT # FIG. 2A

7 U.S. Patent Oct. 27, 2009 Sheet 6 of 45 US 7,608,422 B2 >wildtype GGATGATTTG TATGTAGGAT CT-ACTTAGA AATAGGGCAG >mutated GGACGACCTC TACGTAGGAA GTGACCTGGA GATZGGGCAG # >wildtype CATAGAACAA AAATAGAGGA ractgagacaa CATCTGTTGA >mutated CACAGGACCA AGATCGAGGA GCTGAGACAG CATCTGTTGA # >wildtype GGTGGGGACT TACCACACCA GACAAAM.AC ATCAGAAAGA >mutated GGTGGGGACT GACCACACCA GACAAGAAGC ACCAGAAGGA # >wildtype ACCTCCATTC CTTTGGATGG GTTATGAACT CCATCCTGAT >mutated ACCTCCCTTC CTGTGGATGG GCTACGAA-CT GCATCCTGAC # >wildtype AAATGGACAG TACAGCCTAT AGTGCTGCCA GAAAAAGACA >mutated AAGTGGACAG TGCAGCCCAT CGTGCTGCCT GAGAAGGACA # >wildtype GCTGGACTGT CAAT-GACATA CAGAAGTTAG TGGGGAAATT >mutated GCTGGACTGT GAACGACATA CAGAAGCTCG TGGGCAAGTT # >wildtype GAATTGGGCA AGTCAGATTT ACCCAGGGAT TAAAGTAAGG >mutated GAACTGGGCA AGCCAGATCT ACCCAGGCAT CAAAGTTAGG # >wildtype CAATTATGTA AACTCCTTAG AGGAACCAAA GCACTAACAG >mutated CAGCTGTGCA AGCTG'CTTCCG AGGAACCAAG GCACTGACAG # >wildtype AAGTAATACC ACTAACAGAA GAAGCAGAGC TAGAACTGGC >mutated AAGTGATCCC ACTGACAGAG GAAGCAGAGC TAGAACTGGC # >wildtype AGAAAACAGA GAGATTCTAA AAGAACCAGT ACATGGAGTG >mutated AGAGAACCGA GAGATCCTGA AGGAGCCAGT ACATGGAGTG # >wildtype TATTATGACC CATCAAAAGA CTTAATAGCA GAAATACAGA >mutated TACTACGACC CAAGCAAGGA CCTGATCGCA GAGATCCAGA #

8 U.S. Patent Oct. 27, 2009 Sheet 7 of 45 US 7,608,422 B2 >wildtype >mutated AGCAGGGGCA AGGCCAATGG ACATATCAAATTTATCAAGA AGCAGGGGCA AGGCCAATGG ACCTACCAAA TCTACCAGGA # >wildtype >rmutated GC CATTTAAA AATCTGAAAA CAGGAAAATA TGCAAGAATG GOCCCTTCAAG AACCTGAAGA CAGGCAPCTA CGCAAGGATG # >wildtype AGGGGTGCCC ACACTAATGA TGTAAAACAA TTAACAGAGG >mnutated AGGGGTGCCC ACACCAACGA TGTGAAGCAG CTGACAGAGG >wildtype >mutated CAGTGCAAAA AATAACCACA GAAAGCATAG TAATATGGGG CAGTGCAGAA GATCACCACA GAGAGCATCG TGATCTGGGG # >wildtype AAAGACTCCT AAATTTAAAC TGCCCATACA AAAGGAAACA >mutated CAAGACTCCC AAGTTCAAGC TGCCCATACA GAAGGAGACA # >wildtype >mutated 4 a. r TGGGAAACAT GGTGGACAGA GTATTGGCAA GCCACCTGGA TGGGAGACAT GGTGGACCGA GTACTGGCAA GCCACCTGGA # >wildtype >mutated TTCCTGAGTG GGAGTTTGTT AATACCCCTC CTTTAGTGAA TCCCTGAGTG GGAGTTCGTG AACACCCCTC CCTTGGTGAA # r t t >wildtype >mutated ATTATGGTAC CAGTTAGAGA AAGAACCCAT AGTAGGAGCA ACTGTGGTAT CAGCTGGAGA AGGAACCCAT CGTGGGAGCA # >wildtype GAAACCTTCT ATGTAGATGG GGCAGCTAAC AGGGAGACTA >mutated GAGACCTTCT ACGTGGATGG GGCAGCCAAC AGGGAGACCA # >wildtype >mutated AATTAGGAAA GGCAGGATAT GTTACTAATA GAGGAAGACA AGCTGGGCAA GGCAGGCTAC GTGACCAACC GAGGACGACA # >wildtype >mutated AAAAGTTGTC ACCCTAACTG ACACAACAAA TCAGAAGACT GAAAGTGGTG ACCCTGACTG ACACCACCAA CCAGAAGACT # FIG. 2C

9 U.S. Patent Oct. 27, 2009 Sheet 8 of 45 US 7,608,422 B2 >wildtype GAGTTACAAG CAATTTATCT AGCTTTGCAG GATTCGGGAT >mutated GAGCTGCAAG CCATCTACCT AGCTCTGCAA GACAGCGGAC # a a a a t aaa a >wildtype TAGAAGTAAA CATAGTAACA GACTCACAAT ATGCATTAGG >mutated TGGAAGTGAA CATCGTGACA GACTCACAGT ACGCACTGGG # a. a a * a a >wildtype AATCATTCAA GCACAACCAG ATCAAAGTGA ATCAGAGTTA >mutated CA TCATCCAA GCACAACCAG ACCAATCCGA GTCAGAGCTG # <... a a aaa a a s >wiidtype GTCAATCAAA TAATAGAGCA GTTAATAAAA AAGGAAAAGG >mutated GTGAA.CCAGA TCATCGAGCA GCT-GATCAAG AAGGAGAAAG # a a a a r a a a a t >wildtype TCTATCT.GGC ATGGGTACCA GCACACAAAG GAATTGGAGG >mutated TGTACCTGGC ATGGGTACCA. GCACACAAAG GAATTGGAGG # a >wildtype AAATGAACAA GTAGATAAAT TAGTCAGTGC TGGAATCAGG >mutated AAATGAACAA GTAGATAAAT TAGTCAGTGC TGGGATCCGG a a >wildtype AAAGTACTAT TTTTAGATGG AATAGATAAG GCCCAAGATG >mutated AAGGTGCTGT TCCTGGACGG GATCGATAPG GCCCPAGATG X a... a. a a a >wildtype AACATGAGAA ATATCACAGT AATTGGAGAG CAATGGCTAG >mutated AACATGAGAA GTACCCACTCC AACTGGCGCG CTATGGCCAG # a aaa a *4 a >wildtype TGATTTTAPLC CTGCCACCTG.TAGTAGCAAA AGAAATAGTA >mutated CGACTTCAAC CTGCCACCTG TAGTAGCAAA AGAAATAGTA # a a a >wildtype GCCAGCTGTG ATAAATGTCA GCTAAAAG P. xuacatgc >mutated GCCAGCTGTG ATAAATGTCA GCTAAAAGGA GAAGCCATGC # >wildtype ATGGACAAGT AGAZTGTAGT CCAGGAATAT GGCAACTAGA >mutated ATGGACAAGT AGACTGTAGT CCAGGAATAT GGCAGCTGGh # FIG. 2D

10 U.S. Patent Oct. 27, 2009 Sheet 9 of 45 US 7,608,422 B2 >wildtype >mutated TTGTACACAT TTAGAAGGAA AAGTTATZCT GGTAGCAGTT CTGCACGCAC CTGGAGGGGA AGGTGATCCT GGTAGCAGTT # >wildtype >mutated CATGTAGCCA GTGG4TATAT AGAAGCAGAA GTTATTCCAG CATGTAGCCA GTGGATATAT AGAAGCAGAA GTTATcCCTG # >wildtype CAGAAACAGG GCAGGAAACA GCATATTTTC TTTTAAAATT >mutated CTGAAACTGG GCAGGAAACA GCATATTTTC TTTTAAAATT r >wildtype >mutated AGCAGGAAGA TGGCCAGTAA AAACAATACA TACAGACAAT AGCAGGAAGA TGGCCAGTAA AAACAATACA CACGGACAAC # >wildtype GGCAGCAATT TCA CCAGTGC TACGGTTAAG GCCGCCTGTT >mutated GGAAGCAACT TCACTGGTGC TACGGTTAAG GCCGCCTGTT a...i.... >wildtype >mutated GGTGGGCGGG AATCAAGCAG GAATTTGGAA TTCCCTACAA GGTGGGCGGG AATCAAGCAG GAATTTGGAA TTCCCTACAA # >wildtype TCCCCAAAGT CAAGGAGTAG TAGAATCTAT GAATAAAGAA >mutated TCCCCAATCG CAAGGAGTCG TGGAGAGCAT GAACAAGGAG # I >wildtype TTAAAGAAAA TTATAGGACA GGTRAGAGAT CAGGCTGAAC mutated CTGAAGAAGA TCATCGGACA AGTGAGGGAT CAGGCTGAGC # r a a + >wildtype ATCTTAAGAC AGCAGTACAA ATGGCAGTAT TCATCCACAA >mutated ACCTGAAGAC AGCAGTGCAG ATGGCAGTGT TCATCCACAA # >wildtype TTTTAAAAGA AAAGGGGGGA TTGGGGGGTA CAGTGCAGGG >mutated CTTCAAAAGA AAAGGGGGGA TTGGGGGGTA CTGCPGGG # >wildtype GAAAGAATAG TAGACATAAT AGCAACAGAC ATACAAACTA >mutated GRRAGGATCG TGGACATCAT cgccaccgac ATCCAAACCA # r a * t * t FIG. 2E

11 U.S. Patent Oct. 27, 2009 Sheet 10 of 45 US 7,608,422 B2 >wildtype >mutated AAGAATTACA AAAACAAATT ACAAAAATTC AAAATTTTCG AGGAGCTGCA GAAGCAGATC ACCAAGATCC AGAACTTCCG # >wildtype GGGTTTATTAC AGGGACA-G-CA GAAATCCACT TTGGAAAGGA >mutated GGTGTACTAC CGCGACAGCC GGCAACCCACT GTGGAAGGGA # «>wildtype CCAGCAAAGC TCCTCTGGAA AGGTGAAGGG GCAGTAGTAA >mutated CCAGCAAAGC TCCTCTGGAA GGGAGAGGGG rgcagtggtga # a >wildtype TACAAGATAA TAGTGACATA AAAGTAGTGC CAAGAAGAAA >mutated TCCAGGACAA CAGTGACATC AAAGTGGTGC CAAGGCGCAA # *44 >wildtype >mutated AGCAAAGATC ATTAGGGATT ATGGAAAACA GATGGCAGGT GGCCAAGATC ATCCG-CGACT ATGGAAAACA GATGGCAGGT # >wildtype GATGATTGTG TGGCAAGTAG ACAGGATGAG GATTAGAACA >mutated GATGATTGTG TGGCAAGTAG ACA GGATGAG GATTAGAACC # >wildtype TGGAAAAGTT TAGTAAAACA GCATATG >mutated TGGAAGAGCC TGGTGAAGCA CCATATG # FIG. 2F

12 U.S. Patent Oct. 27, 2009 Sheet 11 of 45 US 7,608,422 B2 ATGGGCGTGAGAAACTCCGTCTTGTCAGGGAAGAAAGCAGATGAATTAG AAAAAATTAGGCTACGACCCAACGGAAAGAAAAAGTACATGTTGAAGC ATGTAGTATGGGCAGCAAATGAATTAGATAGATTTGGATTAGCAGAAAG CCTGTTGGAGAACAAAGAAGGATGTCAAAAAATACTTTCGGTCTTAGCT CCATTAGTGCCAACAGGCTCAGAAAATTTAAAAAGCCTTTATAATACTG TCTGCGTCATCTGGTGCATTCACGCAGAAGAGAAAGTGAAACACACTGA GGAAGCAAAACAGATAGTGCAGAGACACCTAGTGGTGGAAACAGGAAC CACCGAAACCATGCCGAAGACCTCTCGACCAACAGCACCATCTAGCGGC AGAGGAGGAAACTACCCAGTACAGCAGATCGGTGGCAACTACGTCCAC CTGCCACTGTCCCCGAGAACCCTGAACGCTTGGGTCAAGCTGATCGAGG AGAAGAAGTTCGGAGCAGAAGTAGTGCCAGGATTCCAGGCACTGTCAG AAGGTTGCACCCCCTACGACATCAACCAGATGCTGAACTGCGTTGGAGA CCATCAGGCGGCTATGCAGATCATCCGTGACATCATCAACGAGGAGGCT GCAGATTGGGACTTGCAGCACCCACAACCAGCTCCACAACAAGGACAA CTTAGGGAGCCGTCAGGATCAGACATCGCAGGAACCACCTCCTCAGTTG ACGAACAGATCCAGTGGATGTACCGTCAGCAGAACCCGATCCCAGTAGG CAACATCTACCGTCGATGGATCCAGCTGGGTCTGCAGAAATGCGTCCGT ATGTACAACCCGACCAACATTCTAGATGTAAAACAAGGGCCAAAAGAG CCATTTCAGAGCTATGTAGACAGGTTCTACAAAAGTTTAAGAG CAGAAC AGACAGATGCAGCAGTAAAGAATTGGATGACTCAAACACTGCTGATTCA AAATGCTAACCCAGATTGCAAGCTAGTGCTGAAGGGGCTGGGTGTGAAT CCCACCCTAGAAGAAATGCTGACGGCTTGTCAAGGAGTAGGGGGGCCG GGACAGAAGGCTAGATTAATGGCAGAAGCCCTGAAAGAGGCCCTCGCA CCAGTGCCAATCCCTITTGCAGCAGCCCAACAGAGGGGACCAAGAAAGC CAATTAAGTGTTGGAATTGTGGGAAAGAGGGACACTCTGCAAGGCAATG CAGAGCCCCAAGAAGACAGGGATGCTGGAAATGTGGAAAAATGGACCA TGTTATGGCCAAATGCCCAGACAGACAGGCGGGTTTTTTAGGCCTTGGT CCATGGGGAAAGAAGCCCCGCAATTTCCCCATGGCTCAAGTGCATCAGG GGCTGATGCCAACTGCTCCCCCAGAGGACCCAGCTGTGGATCTGCTAAA GAACTACATGCAGTTGGGCAAGCAGCAGAGAGAAAAGCAGAGAGAA.AG CAGAGAGAAGCCTTACAAGGAGGTGACAGAGGATTTGCTGCACCTCAAT TCTCTCTTTGGAGGAGACCAGTAG FIG. 3

13 U.S. Patent Oct. 27, 2009 Sheet 12 of 45 US 7,608,422 B2 SIV gag #1... ATGGGGCGTGAGAAACTCCGTCTTGTCAGGGAAGAAAGCAG SIV gag #41 SIV gag #81 SIV gag #121 SIV gag #161 SIV gag #201 sly gag #241 ATGAATTAGAAAAAATTAGGCTACGACCCAACGGAAAGAA... AAAGTACATGTTGAAGCATGTAGTATGGGCAGCAAATGAA... TTAGATAGATTTGGATTAGCAGAAAGCCTGTTGGAGAACA... AAGAAGGATGTCAAAAAATACTTTCGGTCTTAGCTCCATT... AGTGCCAACAGGCTCAGAAAATTTAAAAAGCCTTTATAAT ACTGTCTGCGTCATCTGGTGCATTCACGCAGARGAGAAAG SIV gag SIVgagDX.. # TGAAACACACTGAGGAAGCAAAACAGATAGTGCAGAGACA SIVgag A--A-----T-----A--A SlVgagDX C--C-----C-----G--G # CCTAGTGGGTGGAAACAGGAACMACMGAAACYATGCCRAAR SIV gag --AAG-A sivgagdx..--ctc-c # ACMWSTMGACCAACAGCACCATCTAGCGGCAGAGGAGGAA FIG. 4A

14 U.S. Patent Oct. 27, 2009 Sheet 13 of 45 US 7,608,422 B2 SIV gag -T A--A--A T SIVgagDX..-C G--G--CC C # AYTACCCAGTACARCARATMG3GTGGTAACTAYGTCCACCT SIV gag ----T-AAG AT-A--T--C-----A--AT-- SlVgagDX..----C-GTC CC-G--C--T-----C--GC-- # GCCAYTRWSCCCGAGAACMYTRAAYGCYTGGGTMAARYTG SIV gag --A-----A SlVgagDX..--C-----G-----G--C # ATMGAGGARAAGAARTTYGGACCAGAAGTAGTCCAGGAT SIVgag -T T-----T-- SlVgagDX..-C C-----C-- # TYCAGGCACTGTCAGAAGGTTGCACCCCCTAYGACATYAA SIV gag T------T-A--T--T--G A SIVgagDX..C------C-G--C--C--T G YCAGATGYTRAAYTGYGTKGGAGACCATCARGCGGCTATG SIV gag -----T---A-A--T--T--A SIVgagDX C---C-T--C--C--C # CAGATYATCMGWGAYATYATMAACGAGGAGGCTGCAGATT SIV gag SIVgagDX..-- ft GGGACTTGCAGCACCCACAACCAGCTCCACAACAAGGACA SIV gag T--T A--T SIVgagDX C--C C--~C # ACT TAGGGAGCCGTCAGGATCAGAYATYGCACGAACMACY SIV gag AGT-----A--T-----A A-A--A- SlVgagDX..TCC-----T--C-----.G C-T--G- # WSYTCAGTWGAYGAACARATCCAGTGGATGTACMGWCARC FIG. 4B

15 U.S. Patent Oct. 27, 2009 Sheet 14 of 45 US 7,608,422 B2 SIV gag C--A T---A-GA SIVgagDX G--C C---C-TC # AGAACCCSATMCCAGTAGGCAACATYTACMGI GATGGAT SIV gag ---A-----GT----A--A--T--CA-A-----T-----A SlVgagDX..---G-----TC----G--G--C--TC-T-----C-----G # CCARCTGGGKYTGCARAARTGYGTYMGWATGTAYAACCCR SIV gag --A SlVgagDX..--C # ACMAACATTCTAGATGTAAA.CCAAGGGCCAAAAGAGCCAT SIV gag # TTCAGAGCTATGTAGACAGGTTCTACAAAAGTTTAAGAGC SIV gag # AGAACAGACAGATGCAGCAGTAAAGAATTGGATGACTCAA SIV gag # ACACT{GCTGATTCAAAATGCTAACCCPGATTGCAAGCTAG SIV gag # TGCTGAAGGGGCTGGGTGTGAATCCCACCCTAGAAGAAAT SIV gag # GCTGACGGCTTGTCAAGGAGTAGGGGGGCCGGGACAGAAG SIV gag # GCTAGATTAAT-GGCAGAAGCCCT-GAAAGAGGCCCT-CGCAC SIV gag #1121 CAGTGCCAATCGCTTTTGCAGCAGCCCAACAGAGGGGACC SIV gag # AAGAAAGCCAATTAA`GTGTTGGAATTGTGGGAAAGAGGGA FIG. 4C

16 U.S. Patent Oct. 27, 2009 Sheet 15 of 45 US 7,608,422 B2 SIV gag CACTCTGCAAGGCAATGCAGAGCCCCAAGAAGACArGGAT SIV gag * GCTGGAAATGTGGAAA ATGGACCATGTTATGGCCAAATG SIV gag CCCAGACAGACAGGGCGGGGTTTTTTAGGCCTTGGTCCATGG SIV gag # GGAAAGAAGCCCCGCAATTTCCCCAT-GGCTCAAGTGCATC SIV gag #1361 AGGiCGCI'GAI'GCCAACTGCTCCCCCAGAGGACCCP.GCTGGT SIV gag # :... GGATCTGCTAAAGAACTACATGCAGTDGGGCAAGCAGCAG SIV gag # AGAGAAAAGCAGAGAGAAAGGCAGAGAGAAGCCTTACAAGG SIV gag # AGGTGACAGAGGATTTGCTGCACiCTCAATTCTCT,CTTTGG SIV gag # AGGAGACCAGTAG FIG. 4D

17 Packaging construct CMV G g BGH poly(a) b pcmvgagpolbn Pro RT Int >y Transfer construct 1 LTR 1r1 G0 I CMV luciferase Fj LTR 1 pmbcwcnluci msd /i Q Transfer construct 2 LTR mg0 I CMV luciferose 'fi LTR pmbcwcnluci msd i/ Transfer construct 3 CMV R' 0 I CMV luciferase }1 I LTR Envelope expression CMV VS/ G poly(a) vector phcmv G intron msd MV1 LTR Gog LTR iat Pro RT lnt msd i/i FIG. 5 MV2 R RELTR

18 U.S. Patent Oct. 27, 2009 Sheet 17 of 45 US 7,608,422 B2 Soil (758) CMV promoter konamycin resistance gene pcmvgagpolbnkon (8366bp) BGH polya mgogpol Nhel (5117) BstBI (5111) FIG. 6

19 U.S. Patent Oct. 27, 2009 Sheet 18 of 45 US 7,608,422 B2 U3, PBS BssHll (711) Clal (830) Accl (959) mbc GAGmAUG Nsi 1050 CMV promoter pmbcwcnici (8937 bp) luciferose PTT U3 U5~ Clol (3259) Xhol (3548) Apo l (3557) Kpnl (3563) FIG. 7

20 U.S. Patent Oct. 27, 2009 Sheet 19 of 45 US 7,608,422 B2 BssHII (711) U3 R U5 12:11 mbc mgagmaug Nsi Clol (830) Accl (959) 1050 CMV promoter pmbcwcnici (8937 bp) luciferose PTT Clol (3259) U3 U5 Xhol (3548) Apol (3557) Kpnl (3563) FIG. 8

21 U.S. Patent Oct. 27, 2009 Sheet 20 of 45 US 7,608,422 B2 1 CCTGGCCATT GCATACGTTG TATCCATATC ATAATATGTA CATTTATATT CGCTCATCTC CAACATTACC 71 GCCATGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA TCAATTACCG GGTCATTACT TCATAGCCCA 141 TATATCCAGT TCCGCGTTAC ATAACTTACG CTAAATCGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC 211 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC CTCAATCGGT 281 GGACTATTTA CGGTAAACTG CCCACTTGGC ACTACATCAA GTCTATCATA TGCCAACTAC CCCCCCTATT 351 GACGTCAATG ACGGTAAATG GCCCGCCTGC CATTATGCCC AGTACATGAC CTTATCGCAC TTTCCTACTT 421 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT CATGCGGTTT TGCCAGTACA TCAATGGGCG 491 TCGATAGCGG TTTGACTCAC GCGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG 561 CACCAAAATC AACGCGACTT TCCAAAATCT CGTAACAACT CCGCCCCATT GACGCAAATG GGCGCTAGGC 631 GTCTACGGTG CGAGCTCTAT ATAAGCAGAG CTCGTTTAGT GAACCGTCAG ATCGCCTCGA CACGCCATCC Sall. (758) 70L ACCCTGTTTT GACCTCCATA GAAGACACCC GGACCCATCC AGCCTCCGCG GGCGCGCGTC CACAGAGACA 771 TGGGTGCCAG AGCGTCAGTA TTAAGCGGGG GAGAATTAGA TCGATGCCAA AAAATTCGGT TAAGGCCAGG 841 GGGAAACAAG AAGTACAAGC TAAAGCACAT CGTATGGGCA AGCAGGGAGC TAGAACGATT CCCAGTTAAT 911 CCTGGCCTGT TAGAAACATC ACAAGGCTGT AGACAAATAC TGGGACACCT ACAACCATCC CTTCAGACAG 981 CATCAGACGA GCTTCGATCA CTATACAACA CAGTAGCAAC CCTCTATTGT GTGCACCAGC CCATCGAGAT 1051 CAAGGACACC AAGGAAGCTT TAGACAAGAT AGAGGAAGAG CAAAACAAGT CCAAGAAGAA GGCCCAGCAC 1121 GCAGCAGCTG ACACAGGACA CACCAATCAG GTCAGCCAAA ATTACCCTAT AGTGCAGAAC ATCCAGGGGC 1191 AAATGGTACA TCAGGCCATA TCACCTACAA CTTTAAATGC ATGGGTAAAA GTAGTACAAG AGAAGGCTTT 1261 CAGCCCAGAA GTGATACCCA TCTTTTCAGC ATTATCAGAA GGAGCCACCC CACAGGACCT GAACACCATG 1331 TTCAACACCG TCGGGGGACA TCAAGCAGCC ATGCAAATCT TAAAAGAGAC CATCAATGAG GAACCTGCAG 1401 AATGGGATAG AGTGCATCCA CTGCATGCAG GGCCTATTGC ACCAGGCCAG ATGAGAGAAC CAAGGGGAAG 1471 TCACATAGCA GGAACTACTA.GTACCCTTCA GGAACAAATA GCATCGATGA CAAATAATCC-ACCTATCCCA 1541 GTACGAGAGA TCTACAAGAG GTGGATAATC CTCGCATTGA ACAACATCGT GAGGATGTAT AGCCCTACCA 1611 GCATTCTGGA CATAAGACAA GGACCAAAGG AACCCTTTAG AGACTATGTA CACCGGTTCT ATAAAACTCT 1681 AAGAGCTCAG CAACCTTCAC AGGAGGTAAA AAATTGGATC ACAGAAACCT TGTTGGTCCA AAATGCCAAC 1751 CCACATTCTA AGACCATCCT GAAGGCTCTC GGCCCAGCCC CTACACTAGA ACAAATGATG ACACCATCTC 1821 ACGGAGTAGG AGGACCCGGC CATAAGGCAA CACTTTTCCC CGACCCGATG AGCCAGGTGA CGAACTCCGC FIG. 9A

22 U.S. Patent Oct. 27, 2009 Sheet 21 of 45 US 7,608,422 B GACCATAATC ATCCAGACAC GCAACTTCCG CAACCAGCGC AACATCCTCA AGTGCTTCAA TTGTOGCAAA 1961 CAAGGGCACA CCCCCAGGAA CTGCCGGGCC CCCCCCAACA ACCCCTGTTG GAAATCTGGA AACCAAGCAC 2031 ACCAAATCAA AGATTCTACT GACACACAGG CTAATTTTTT ACGGAAGATC TCCCCTTCCf ACAAGGGAAG 2101 GCCACCCAAT TTTCTTCACA GCAGACCACA CCCAACACCC CCACCAGAAG ACACCTTCAG CTCTGGGGTA 2171 GAGACAACAA CTCCCCCTCA GAACCACCAG CCGATAGACA AGCAACTCTA TCCTTTAACT TCCCTCAGAT 2241 CACTCTTTCG CAACGACCCC TCCTCACACT AAGGATCGGG GGGCAACTCA AGGAACCGCT CCTCCATACA 2311 GGACCAGATC ATACAGTATT ACAAGAAATG AGTTIGCCAC GAACATCGAA ACCAAAAATC ATACGGCGGA 2381 TCGGGGCCTT CATCAACCTG ACGCACTACG ACCACATACT CATACAAATC TCTGGACATA AAGCTATAGG 2451 TACAGTATTA-CTAGCACCTA CACCTCTCAA CATAATTGCA AGAAATCTGT TGACCCAGAT CGCCTGCACC 2521 TTCAACTTCC CCATCACCCC TATTCAGACC CTCCCCCTCA AGTTGAACCC GGGGATCCAC GCCCCCAACG 2591 TCAAGCAATC CCCATTCACC AAAGAGAAGA TCAAGGCCTT AGTCGAAATC TCTACAGAGA TCGAGAAGGA 2661 AGGGAACATC ACCAAGATCC CCCCTGAGAA CCCCTACAAC ACTCCACTCT TCGCAATCAA CAACAACCAC 2731 AGTACCAACT GCAGAAAGCT GGTGCACTTC AGAGAGCTGA ACAAGAGAAC TCAGGACTrC TGGGAAGTTC 2801 AGCTGGGCAT CCCACATCCC CCTCCGTTCA ACAACAACAA CTCAGTCACA CTCCTCGATG TCGGTCATCC 2871 CTACTTCTCC GTTCCCTTGC ACCACGACTT CAGGAAGTAC ACTCCCTTCA CGATACCTAC CATCAACAAC 2941 GAGACACCAC CCATCCCCTA CCACTACAAC GTGCTGCCAC ACGCATGGAA CGGATCACCA GCCATCTTTC 3011 AAAGCACCAT CACCAAGATC CTGGAGCCCT TCCCCAAGCA AAACCCAGAC ATCCTCATCT ATCACTACAT 3081 GGACGACCTC TACCTAGGAA GTGACCTGGA GATCGGCCAC CACAGGACCA ACATCGAGGA GCTGAGACAG 3151 CATCTGTTGA GGTGGGGACT CACCACACCA GACAAGAAGC ACCACAACGA ACCTCCCTTC CTGTGCATGG 3221 GCTACCAACT CCATCCTGAC AACTGGACAG TCCACCCCAT CGTGCTGCCT GAGAACCACA GCTGGACTGT 3291 CAACGACATA CACAACCTCC TCGCCAACTT CAACTCCCCA AGCCAGATCT ACCCACGCAT CAAAGTTACC 3361 CAGCTGTGCA AGCTCCTTCG AGGAACCAAC CCACTCACAG AAGTGATCCC ACTCACACAC CAACCACAGC 3431 TACAACTGCC ACAGAACCCA CACATCCTCA ACCAGCCACT ACATCGACTG TACTACCACC CAACCAACGA 3501 CCTGATCGCA CAGATCCACA ACCAGGGGCA AGGCCAATGG ACCTACCAAA TCTACCAGGA CCCCTTCAAG 3571 AACCTGAAGA CACGCAAGTA CCCAAGGATG AGCCCTCCCC ACACCAACGA TGTGAAGCAG CTGACAGACC 3641 CAGTCCACAA CATCACCACA GAGAGCATCC TGATCTCCCG CAACACTCCC AACTTCAAGC TCCCCATACA 3711 GAAGGAGACA TCCCACACAT GGTGGACCCA GTACTGGCAA GCCACCTGGA TCCCTGAGTC CCAGTTCCTG FIG. 9B

23 U.S. Patent Oct. 27, 2009 Sheet 22 of 45 US 7,608,422 B AACACCCCTC CCTTCCTGAA ACTGTGGTAT CAGCTCCACA AGCAACCCAT CGTCCGAGCA GAGACCTTCT 3851 ACCTCCATCG GGCAGCCAAC ACGCAGACCA ACCTCCCCAA GGCAGGCTAC CTCACCAACC CAGGACGACA 3921 CAAAGTGGTC ACCCTCACTG ACACCACCAA CCAGAACACT CAGCTGCAAG CCATCTACCT AGCTCTGCAA 3991 GACAGCGGAC TGGAACTCAA CATCCTCACA CACTCACACT ACCCACTGCG CATCATCCAA CCACAACCAG 4061 ACCAATCCGA GTCACAGCTG GTCAACCACA TCATCGACCA GCTCATCAAC AAGCAGAAAG TCTACCTGGC 4131 ATCGGTACCA CCACACAAAG CAATTGGACG AAATCAACAA GTAGATAAAT TACTCAGTCC TCGCATCCCG 4201 AACCTCCTGT TCCTCGACGC CATCCATAAC CCCCAACATG AACATGACAA CTACCACTCC AACTCGCGCG 4271 CTATGGCCAC CGACTTCAAC CTGCCACCTC TAGTAGCAAA AGAAATACTA CCCAGCTCTG ATAAATGTCA 4341 CCTAAAACCA GAACCCATGC ATGGACAAGT AGACTGTAGT CCAGGAATAT CGCACCTGGA CTCCACCCAC 4411 CTGGAGGCGA AGGTCATCCT CCTAGCAGTT CATGTAGCCA CTCCATATAT AGAACCAGAA GTTATCCCTG 4481 CTCAAACTGG CCACGAAACA GCATATTTTC TTTTAAAATT ACCAGCAACA TGGCCACTAA AAACAATACA 4551 CACGGACAAC GGAAGCAACT TCACTCCTGC TACGCTTAAG GCCGCCTCTT GCTCGGCCGG AATCAAGCAG 4621 CAATTTGCAA TTCCCTACAA TCCCCAATCC CAAGCAGTCG TCGAGACCAT CAACAAGGAG CTCAAGAAGA 4691 TCATCGGACA AGTCAGGGAT CACCCTCACC ACCTCAACAC ACCAGTGCAG ATCCCACTCT TCATCCACAA 4761 CTTCAAAAGA AAACCCCCCA TTGGGGCGTA CAGTCCAGGC CAAAGGATCC TGGACATCAT CCCCACCGAC 4831 ATCCAAACCA AGGAGCTCCA CAAGCACATC ACCAACATCC AGAACTTCCG CGTGTACTAC CCCGACAGCC 4901 CCAACCCACT CTGGAACGGA CCAGCAAACC TCCTCTGGAA GCGACAGCCC CCACTCGTGA TCCACCACAA 4971 CAGTCACATC AAAGTGCTGC CAACCCGCAA GGCCAAGATC ATCCGCGACT ATCGAAAACA GATGGCACGT 5041 CATCATTGTC TCGCAACTAC ACACGATCAG CATTAGAACC TGGAAGACCC TGCTCAACCA CCATATGGCG NheI (5117) BstBI (5111) 5111 TTCCAAGCTA GCCTCGAGAT CCAGATCTGC TGTCCCTTCT AGTTGCCAGC CATCTGTTCT TTCCCCCTCC 5181 CCCGTCCCTT CCTTCACCCT GCAACGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG GAAATTGCAT 5251 CGCATTCTCT GACTACCTCT CATTCTATTC TGGGGGGTGG GCTGCGCCAG CACACCAACC CGGACCATTC 5321 CGAACACAAT AGCACGCATC CTCCCCATGC CCTGGGCTCT ATGCCTACCC ACCTCCTCAA GAATTGACCC 5391 CCTTCCTCCT CCCCCACAAA GAACCAGGCA CATCCCCTTC TCTCTGACAC ACCCTCTCCA CGCCCCTGGT 5461 TCTTAGTTCC AGCCCCACTC ATACCACACT CATAGCTCAG CACGCCTCCG CCTTCAATCC CACCCGCTAA 5531 AGTACTTGCA CCGCTCTCTC CCTCCCTCAT CACCCCACCA AACCAAACCT AGCCTCCAAG ACTGGGAAGA FIG. 9C

24 U.S. Patent Oct. 27, 2009 Sheet 23 of 45 US 7,608,422 B AATTAARGCA AGATAGGCTA TTAAGTGCAC ACGGACAGAA AATCCCTCCA ACATGTCAGG AAGTAATGAG 5671 AGAAATCATA GAATTTCTTC CGCTTCCTCC CTCACTGACT CGCTCCGCTC CGTCGTTCGG CTCCGGCGAC 5741 CCCTATCACC TCACTCAAAG GCGCTAATAC 5811 GTCAGCAAAA CGCCAGCAAA AGCCCAGCAA 5881 CGCCCCCCTG ACCACCATCA CAAAAATCCA 5951 GATACCAGCC GTTTCCCCCT CGAAGCTCCC 6021 CCTCTCCCCC TTrCTCCCTT CCGGAACCCT 6091 GTCTAGCTCC TTCCCTCCAA CCTCCCCTGT 6161 CCGCTAACTA TCGTCTTGAC TCCAACCCGG 6231 CAGCATTAGC AGAGCGAGGT ATCTAGCCGG 6301 ACTACAAGCA CAGTATTTCC TATCTCCCCT 6371 CTTGATCCGG CAAACAAACC ACCGCTCCTA 6441 AAAAAAAGCA TCTCAAGAAG ATCCTITCAT 6511 CGTTAAGGCA TTTTGCTCAT CAGATTATCA 6581 GTTTTAAATC AATCTAAACT ATATATGAGT 6651 ACCTATCTCA GCCATCTCTC TATTTCCTTC 6721 TCTCCCTCGT CAACAAGGTG TTGCTGACTC 6791 AGCGACCCAC GGTTCATCAC ACCTTTCTTG 6861 CGGAACCCTC TCCGTTCTCG CCAACATGCG 6931 ACAAAGCCGC CCTCCCCTCA AGTCACCGTA 7001 ACAAAAACTC ATCGACCATC AAATGAAACT 271 IPhePheGlu AspLeu4etL euhizpheg AAAAAGCCGT TTCTGTAATC AAGCAGAAAA PheLeuArgL ysclnleuse rproserphe 7141 CCGTCTGCCA TTCCCACTCC TCCAACATCA rgaspalall eg1yva1arg G1yValAspI 7211 CAACTGACAA ATCACCATCA GTCACCACTC pleuserphe AspClyHisT hrvalvalse 7281 GACTTCTTCA ACACCCCAGC CATTACGCTC 1781 Va1CInG1uV a1protrpc1 yasnargglu 7351 CGTGATTGCG CCTGACCGAC ACGAAATACG 1544 rgserglna1 aglnalaleu ArgPheValA 7421 GCAACCCGCC CAGGAACACT GCCACCCCAT sleuargarg LeuPheValA laleualaas 7491 CTCGAATGCT GTTTTCCCCG CGATCCCACT 1084 GlnPheAlaT hrlysclypr ollealathr 7561 TTCATGGTCG GAAGACCCAT AAATTCCCTC 84 4 ysllethrpr oleupromet PheGluThrL 7631 CAACGCTACC TTTGCCATGT TTCAGAAACA 61 4 avalsergly LysGlyHisL ysleuphele 7701 CGCACCTGAT TGCCCGACAT TATCGCCACC 38 4 A1aGIySerG 1nGlyValAs naspargala 7771 AATCGCCCCC TCCACCAAGA CCTTTCCCCT 14 4 euargproar gsercysser ThrGluArgG 7841 AACCAGACAG TTTTATTCTT CATCATGATA 7911 CACAACCTGG CTTTCCCCCC CCCCCCATTA 7981 ATATTTGAAT CTATTTAGAA AAATAAACAA 8051 ACGTCTAAGA AACCAITATT ATCATCACAT 8121 CGCGCGTTTC GGTGATGACG CTGAAAACCT 8191 TAACCGCATG CCCCCAGCAG ACAACCCCGT 8261 TTAACTATCC GGCATCAGAC CACATTCTAC 8331 CGTAAGGACA AAATACCGCA TCACATTCGC CCTTATCCAC ACAATCAGCG CATAACGCAC CAAAGAACAT CCGTAAAAAC GCCCCGTTGC TGGCCTTTTT CCATACCCTC CCCTCAAGTC ACAGGTGCCG AAACCCCACA GCACTATAAA TCGTCCCGCTC TCCTGTTCCC ACCCTGCCGC TTACCGGATA GGCCCTTTCT CAATGCTCAC GCTCTAGGTA TCTCACTTCG GTGCACCAAC CCCCCCTTCA GCCCCACCCC TCCCCCTTAT TAACACACCA CTTATCCCCA CTCGCACCAC CCACTGGTAA TGCTACAGAG TTCTTGAAGT GCTCGCCTAA CTACCCCTAC CTGCTCAAGC CACTTACCTT CGCAAAAAGA GTTGCTAGCT GCGGTGGTTT TTTTCTTTGC AACCAGCAGA TTACCCCCAC CTTTTCTACG GCCTCTCACG CTCACTGGAA CGAAMCTCA AAAACGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA AAACTTGCfC TGACACTTAC CAATCCTTAA TCACTCAGCC ATCCATACTT GCCTCACTCC GGCCGCCGGG GCCGCTCACG ATACCACGCC TGAATCGCCC CATCATCCAG CCAGAAACTG TACGTCGACC AGTTCGTGAT TTTCAACT2T TGCTTTGCCA TGATCTGATC CTTCAACTCA GCAAAAGTTC CATTTATTCA ATCCTCTGCC AGTGTTACAA CCAATTAACC AATTCTCATT CCAATTTATT CATATCAGCA TTATCAATAC CT-j,TTTG nleulysasn MetAspProA snasplieci ytyrlyscln CTCACCGACC CAGTTCCATA GGATCCCAAC ATCCTGGTAT G1uGlyLeuC ysasntrple ullealaleu AspGlnTyrA ATACAACCTA TTAATTTCCC CTCGTCAAAA ATAAGGTTAT lecyaclyll eleulyacly C1uAspPheI leleuasnas AATCCCCTCA CAATGGCAAA ACCTTATCCA TTTCTTTCCA raspproser PheProLeuL eulyshisme tglulystrp CTCATCAAAA TCACTCCCAT CAACCAAACC GTTATTCATT AspAspPheA spseralaas pvaileugly AsnAsnMetA CCATCGCTCT TAAAAGGACA ATTACAAACA GGAATCCAAT rgaspseras npheprocys AsnCysValP roileserki CAACAATATT TTCACCTGAA TCAGGATATT CTTCTAATAC pvallleasn GluGlySerA spprotyrgi ugluleuva1 GCTCACTAAC CATCCATCAT CACGAGTACG CATAAAATGC ThrLeuLeuT rpalaaspas pprothrarg IlePheHisL ACCCACTTTA GTCTGACCAT CTCATCTG.TA ACATCATTGG eutrpasnle uargvalmet G1uAspThrV alaspasnal ACTCTGGCGC ATCGGGCTTC CCATACAATC_G(,TACATTGT ucluproala AspProLysC lytyrleuar gtyrllethr CCATTTATAC CCATATAAAT CAGCATCCAT CTTCGAAYTT TrpLysTyrC lytyrleuas palaaspmet AsnSerAsnL TGAATATGGC TCATAACACC CCTTGTATTA CTCTTTATCT 1nIleRisSe rmet TATTTTTATC TTGTGCAATG TAACATCAGA GATTTTGAGA TTGAAGCATT TATCACGGTT ATTGTCTCAT GAGCGGATAC ATAGCGGTTC CGCGCACATT TCCCCCAAAA GTGCCACCTC TAACCTATAA AAATAGGCCT ATCACGAGGC CCITTCCTCT CTGACACATG CACCTCCCGG AGACGGTCAC ACCTTGTCTC CAGGCCCCGT CACCGGGTGT TGGCGCCTCT CCGCGCTGGC CTAGACTGCA CCATATGCGG TCTGAAATAC CCCACAGATC TATTGG (SEQUENCE ID NO: 6) FIG. 9D

25 U.S. Patent Oct. 27, 2009 Sheet 24 of 45 US 7,608,422 B2 1 TGCAAGGCCT AATTTCGTCC CAAAAAACAC AACACATCCT TCATCTGTGG ATCTACCACA CACAAGGCTA 71 CTTCCCTCAT TGGCACAACT ACACACCACG GCCACCGATC ACATATCCAC TCACCTTTGG ATCCTGCTTC 141 AACTTACTAC CACTTGAACC ACACCAACTA CAACAGCCCA AATAAGCAGA GAAGAACACC TTGTTACACC 211 CTATGAGCCA CCATGCCATC CAGGACCCCG ACGCACAACT ATTACTCTCG AACTTTCACA GCCTCCTACC 281 ATTTCGTCAC ATGGCCCCAC ACCTGCATCC GCACTACTAC AAACACTGCT CACATCCAGC TTTCTACAAC 351 CCACTTTCCC CTGCCGACTT TCCAGGGAGG TGTGGCCTCG GCGGCACTGG GGACTCGCCA GCCCTCACAT 421 CCTACATATA AGCAGCTGCT TTTTCCCTCT ACTGCCTCTC TCTGGTTACA CCAGATCTCA CCCTGGGACC 491 TCTCTGGCTA ACTACCCAAC CCACTGCTTA AGCCTCAATA AAGCTTGCCT TGAGTGCTCA AACTACTCTG 561 TCCCCCTCTG TTCTGTGACT CTGGTAACTA CACATCCCTC AGACCCTTTT AGTCAGTCTG GAAAATCTCT 631 ACCACTCCCC CCCCAACAGG GACTTGAAAG CGAAAGTAAA GCCAGAGGAG ATCTCTCGAC GCAGGACTCG BssHII (711) 701 CCTTGCTGAA GCGCCCacgg caagaggcga ggggcggcgc ctgacgagga cgccaaaaat tttgactagc C1aI (830) 771 ggaggctaga aggagagagc TCGCTCCCAG ACCGTCAGTA TCAAGCGGGG CAGAATTACA TCCATGGCAA 841 AAAATTCGCT TAAGCCCAGG GGGAAAGAAA AAATATAAAT TAAAACATAT AGTATGGGCA ACCAGGGAGC AccI (959) 911 TAGAACGATT CGCACTTAAT CCTGGCCTCT TACAAACATC AGAAGGCTGT ACACAAATAC TGGGACAGCT 981 ACAACCATCC CTTCAGACAC CATCAGAACA ACTTAGATCA TTATATAATA CACTAGCAAC CCTCTATTGT 1051 CTCCATCAAA CCATACAGAT AAAAGACACC AACGAAGCTT TACACAAGAT AGAGGAAGAG CAAAACAAAA FIG. 10A

26 U.S. Patent Oct. 27, 2009 Sheet 25 of 45 US 7,608,422 B GTAAGAAAAA AGCACACCAA GCAGCACCTG ACACAGGACA CACCAATCAG GTCAGCCAAA ATTACCCTAT 1191 AGTGCAGAAC ATCCAGGCGC AAATGGTACA TCAGGCCATA TCACCTAGAA CTTTAAACGA TP.AGCTTGGG 1261 AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGC CTCACCGCCC AACGACCCCC GCCCATTGAC 1331 GTCAATAATG ACGTATCTTC CCATACTAAC GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGGAGTAT 1401 TTACGGTAAA CTGCCCACTT GGCAGTACAT CAACTCTATC ATATGCCAAG TACGCCCCCT ATTGACGTCA 1471 ATGACGGTAA ATCCCCCGCC TGGCATTATG CCCAGTACAT GACCTTATCG GACTTTCCTA CTTGGCAGTA 1541 CATCTACGTA TTAGTCATCG CTATIACCAT GGTGATCCGG TTITGGCAGT ACATCAATGG GCGTCGATAG 1611 CGGTTTGACT CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTIT TGGCACCAAA 1681 ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA GGCGTGTACG 1751 GTGGGAGGTC TATATAAGCA CAGCTCCTTT AGTGAACCGT CAGATCGCCT GGAGACCCCA TCCACGCTCT 1821 TTTGACCTCC ATACAACACA CCGACTCTAG AGgatccATC TAAGTAAGCT TGGCATTCCG GTACTGTTGG 1891 TAAAATGGAA CACGCCAAAA ACATAAAGAA AGGCCCGGCG CCATTCTATC CTCTAGAGCA TGGAACCGCT 1961 GGAGAGCAAC TGCATAAGGC TATGAAGAGA TACGCCCTGC TTCCTGGAAC AATTGCTTTT ACACATCCAC 2031 ATATCGAGGT GAACATCACG TACGCGGAAT ACTTCGAAAT GTCCGTTCGG TTGGCAGAAG CTATGAAACG 2101 ATATGGGCTG AATACAAATC ACAGAATCGT CCTATGCAGT GAAAACTCTC TTCAATTCTT TATGCCGCTG 2171 TTGGGCCCGT TATTTATCCG AGTTGCAGTT GCGCCCGCCA ACGACA:TIA TAATGAACGT GAATTGCTCA 2241 ACAGTATCAA CATTTCGCAG CCTACCGTAG TCTTTGTTTC CAAAAAGGGG TTCCAAAAAA TTTTGAACGT 2311 GCAAAAAAAA TTACCAATAA TCCACAAAAT TATTATCATG CATTCTAAAA CGGATTACCA GGGATTTCAG FIG. 10B

27 U.S. Patent Oct. 27, 2009 Sheet 26 of 45 US 7,608,422 B TCGATCTACA CGTTCGTCAC ATCTCATCTA CCTCCCGCTT TTAATGAATA CGATTTTCTA CCACACTCCT 2451 TTCATCGTGA CAAAACAATT GCACTGATAA TCAATTCCTC TCCATCTACT GGGTTACCTA AGGGTCTCGC 2521 CCTTCCGCAT AGAACTCCCT GCGTCAGATT CTCCCATGCC ACAGATCCTA TTTTTGGCAA TCAAATCATT 2591 CCCGATACTC CCATITTAAG TGTTGTTCCA TTCCATCACG CTTTTGGAAT GTTTACTACA CTCGGATATT 2661 TCATATGTGG ATTTCGAGTC GTCTTAATCT ATAGATTTGA ACAAGAGCTG TTTTTACGAT CCCTTCAGCA 2731 TTACAAAATT CAAACTCCGT TGCTAGTACC AACCCTATTT TCATTCTTCG CCAAAAGCAC TCTGATTGAC 2801 AAATACGATT TATCTAATTT ACACGAAATT GCTTCTGGGG GCGCACCTCT TTCGAAAGAA CTCCGGGAAG 2871 CGGTTGCAAA ACCCTTCCAT CTTCCAGGCA TACGACAACG ATATCGCCTC ACTCAGACTA CATCAGCTAT 2941 TCTGATTACA CCCGAGGGGG ATGATAAACC CGCCCCCGTC GGTAAAGTTG TTCCATTTTT TGAAGCCAAG 3011 GTTGTGGATC TGGATACCGG GAAAACCCTG GGCGTTAATC AGAGAGGCGA ATTATGTGTC AGAGGACCTA 3081 TGATTATGTC CCGTTATGTA AACAATCCGG AAGCCACCAA CCCCTTGATT GACAAGGATC GATCGCTACA 3151 TTCTGGACAC ATAGCTTACT GGGACGAAGA CGAACACTTC TTCATAGTTG ACCGCTTGAA GTCTTTAATT Clal (3259) 3221 AAATACAAAG CATATCAGGT GGCCCCCCCT GAATTGGAAT CGATATTCTT ACAACACCCC AACATCTTCG 3291 ACCCGGGCGT GGCAGGTCTT CCCGACGATC ACGCCCGTGA ACTTCCCGCC GCCGTTCTTG TTTTGGAGCA 3361 CGGAAAGACG ATGACGGAAA AAGAGATCGT GGATTACGTC GCCAGTCAAG TAACAACCCC GAAAAACTTG 3431 CGCCGAGGAG TTGTCTTTGT GGACGAAGTA CCGAAAGGTC TTACCGGAAA ACTCCACGCA AGAAAAATCA Apal (3557) Xhol (3548) Kpnl(3563) 3501 CACACATCCT CATAAAGGCC AAGAAGGGCG GAAAGTCCAA ATTGTAAcTC GAGGGGCGCC CCGCTACCTT FIG. 10C

28 U.S. Patent Oct. 27, 2009 Sheet 27 of 45 US 7,608,422 B TAAGACCAAT GACTTACAAG GCAGCTGTAG ATCTTAGCCA CTTTTTAAAA GAAAACGGGG CACTGGAAGG 3641 CCTAATTCAC TCCCAAAGAA GACAACATAT CCTTCATCTG TCGATCTACC ACACACAACC CTACTTCCCT 3711 CATTCGCACA ACTACACACC AGGGCCAGGG GTCAGATATC CACTGACCTT TGGATGGTGC TACAAGCTAG 3781 TACCAGTTCA CCCACATAAG GTAGAAGAGG CCAATAAAGG ACACAACACC ACCTTCTTAC ACCCTCTCAC 3851 CCTGCATGGA ATGGATGACC CTCAGAGAGA ACTCTTAGAG TGGAGGTTTG ACAGCCGCCT AGCATTTCAT 3921 CACGTGGCCC CAGAGCTGCA TCCGGAGTAC TTCAAGAACT GCTGACATCG AGCTTGCTAC AAGGGACTTT 3991 CCGCTGGGGA CTTTCCAGGG AGGCGTGGCC TGGCCGGGAC TGGGGAGTGG CGAGCCCTCA GATGCTGCAT 4061 ATAAGCAGCT GCTTTTTGCC TGTACTCGGT CTCTCTGGTT AGACCAGATC TGAGCCTGGG ACCTCTCTGG 4131 CTAACTAGGG AACCCACTGC TTAAGCCTCA ATAAAGCTTG CCTTGAGTGC TTCAACTAGf GTGTCCCCGT 4201 CTGTTGTGTC ACTCTCGTAA CfAGAGATCC CTCAGACCCT TTTAGTCACT GTCGAAAATC TCTAGCACCC 4271 CCCAGGAGGT AGAGGTTGCA GTGAGCCAAG ATCCCGCCAC TGCATTCCAG CCTGGGCAAG AAAACAAGAC 4341 TGTCTAAAAT AATAATAATA AGTTAAGGGT ATTAAATATA TTTATACATG GACCTCATAA AAATATATAT 4411 ATTTGCGCTG CGCGCAGTGC CTCACACCTG CCCCCGGCCC TTTGGGAGGC CGAGCCACCT GGATCACCTG 4481 AGTTTGCGAG TTCCACACCA CCCTGACCAA CATGGAGAAA CCCCTTCTCT CTGTATTTTT ATGAGATTTT 4551 ATTTTATCTG TATTTTATTC ACAGGTATTT CTGGAAAACT GAAACTGTTT TTCCTCTACT CTGATACCAC 4621 AAGAATCATC ACCACAGAGG AAGACTTCTG TGATCAAATG TGGTGGGAGA GGGAGCTTTT CACCAGCACA 4691 TGACCAGTCA GTTCTGCCGC ACACTCGGCC CCTCTCCTTC CGTTCAGTTC CAACACCGCC TGCCTGCAGA 4761 GAGGTCAGAC CACAGCGTGA GGGCTCAGTC CCCAACACAT AAACACCCAA CACATAAACA CCCAACAGGT 4831 CCACCCCGCC TGCTGCCCAG GCAGAGCCGA TTCACCAAGA CGGGAATTAG GATAGAGAAA GAGTAAGTCA 4901 CACACACCCG GCTGTCCCGG AGAACGGAGT TCTATTATGA CTCAAATCAC TCTCCCCAAC CATTCGGGGA 4971 TCAGAGTTTT TAACGATAAC TTACTGTGTA GGGCGCCAGT GACTTGGAGA TGAAAGCGTA GGGAGTCGAA 5041 GGTGTCCTTT TGCGCCGAGT CAGTTCCTGG GTGGGGGCCA CAAGATCGGA TGACCCACTT TATCAATCCG 5111 GGGGTGCCAG CTCATCCATG GAGTGCACGG TCTCCAAAAT ATCTCAAGCA CTGATTGATC TTAGGTT'[TA 5181 CAATAGTGAT GTTACCCCAG GAACAATTTG GGGAAGGTCA GAATCTTGTA GCCTGTAGCT GCATGACTCC 5251 TAAACCATAA TTTCITTITT GTTTT TTCIT TTTTATTTTT GAGACAGGGT CTCACTCTCT CACCTAGCCT 5321 GGAGTGCAGT GCTGCAATCA CAGCTCACTC CAGCCCCTAG AGCGGCCGCC ACCGCCCTCG ACCTCCAATT 5391 CGCCCTATAC TCACTCGTAT TACAATTCAC TGGCCGTCCT TTTACAACGT CCTGACTGGG AAAACCCTGC 5461 CGTTACCCAA CTTAATCGCC TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGCCCCCC 5531 ACCCATCGCC CTTCCCAACA CTTGCCCACC CTGAATGGCG AATGGCGCGA AATTCTAAAC CTTAATATTT 5601 TGITAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT TTTTAACCAA TAGGCCGAAA TCGGCAAAAT 5671 CCCTTATAAA TCAAAAGAAT AGACCGACAT ACCCTTGAGT GTTCTTCCAC TTTCGAACAA GAGTCCACTA 5741 TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG TCTATCAGGG CGATGGCCCA CTACGTGAAC 5811 CATCACCCTA ATCAAGTTTT TTGGGGTCGA GGTGCCCTAA AGCACTAAAT CCCAACCCTA AAGCGAGCCC 5881 CCGATTTAGA GCTTGACGGC GAAAGCCGGC GAACGTGGCC ACAAAGGAAG GGAACAAAGC CAAAGGACCG FIG. 10D

29 U.S. Patent Oct. 27, 2009 Sheet 28 of 45 US 7,608,422 B GGCGCTACGG CCCTCGCAAG TCTAGCGGTC ACGCTGCGCG TAACCACCAC ACCCGCCCCG CTTAATGCGC 6021 CGCTACAGGG CGCGTCCCAG GTGGCACTTT TCCGGCAAAT CTCCGCGGAA CCCCTATTTG TTTATTTTTC 6091 TAAATACATT CAAATATGTA TCCGCTCATG ACACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 6161 GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT CCGGCATTTT GCCTTCCTCT 6231 TTTTGCTCAC CCAGAAACGC TGCTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCACG AGTGGGTTAC 6301 ATCCAACTGG ATCTCAACAC CGCTAAGATC CTTCACACTT TTCGCCCCGA AGAACGTTTT CCAATGATGA 6371 GCACTTITAA AGTTCTCCTA TGTGGCGCGG TATTATCCCG TATTGACCCC GGGCAACAGC AACTCCGTCG 6441 CCGCATACAC TATTCTCAGA ATCACTTCCT TGACTACTCA CCAGTCACAC AAAAGCATCT TACCGATGGC 6511 ATGACACTAA GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGCCCAAC TTACTTCTGA 6581 CAACGATCGG ACGACCCAAG GAGCTAACCC CTTTTTTGCA CAACATGGGG GATCATCTAA CTCGCCTTGA 6651 TCCTTGGGAA CCGGAGCTGA ATCAACCCAT ACCAAACGAC CACCGTCACA CCACGATCCC TGTAGCAATG 6721 CCAACAACCT TGCCCAAACT ATTAACTGGC CAACTACTTA CTCTACCTTC CCGGCAACAA TTAATACACT 6791 GGATGGAGGC CGATAAAGTT GCAGGACCAC TTCTCCGCTC CGCCCTTCCC CCTGGCTGGT TTATTCCTGA 6861 TAAATCTGGA CCCGGTGACC GTGGCTCTCG CGGTATCATT GCACCACTGG CGCCAGATGG TAACCCCTCC 6931 CGTATCGTAG TTATCTACAC CACGGCCAGT CAGGCAACTA TGCATCAACG AAATAGACAG ATCGCTGAGA 7001 TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT AGATTGATTT 7071 AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTCAAGATC CTTTTLGATA ACTTCATCAC CAAAATCCCT 7141 TAACGTGACT TTTCCTTCCA CTGACCGTCA CACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT 7211 TTTTTCTGCG CGTAATCTGC TGCTTCCAAA CAAAAAAACC ACCGCTACCA GCCGTGGTTT GTTTCCCGGA 7281 TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGACCGC AGATACCAAA TACTGTCCTT 7351 CTAGTGTAGC CGTAGTTAGC CCACCACTTC AAGAACTCTG TACCACCGCC TACATACCTC GCTCTGCTAA 7421 TCCTGTTACC AGTGGCTGCT GCCAGTGCCC ATAACTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT 7491 ACCGGATAAG CCCCAGCGGT CGGGCTGAAC GGGGGGTTCG TCCACACAGC CCAGCTTGGA GCGAACGACC 7561 TACACCGAAC TGAGATACCT ACAGCGTCAG CTATGACAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG 7631 ACAGGTATCC CGTAAGCGCC AGGGTCGGAA CAGGACACCG CACGAGGCAG CTTCCAGGGC GAAACGCCTG 7701 GTATCTTTAT ACTCCTGTCG.GcTTTCGCCA CCTCTCACTT GAGCGTCGAT '1'TTTCTGATG CTCCTCAGGG 7771 GGGCGGAGCC TATGGAAAAA CGCCACCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG 7841 CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTCTGGA TAACCCTATT ACCGCCTTTG ACTGAGCTGA 7911 TACCGCTCGC CGCAGCCCAA CGACCGAGCC CAGCGACTCA CTGAGCCAGG AAGCCCAAGA GCGCCCAATA 7981 CGCAAACCGC CTCTCCCCGC GCGTTCCCCG ATTCATTAAT GCAGCTCGCA CGACAGCTTT CCCGACTGGA 8051 AAGCGGCCAG TGAGCCCAAC GCAATTAATC TGAGTTAGCT CACTCATTAG GCACCCCAGG CTITACACTT 8121 TATGCTTCCG GCTCCTATGT TGTGTCCAAT TCTGACCGGA TAACAATTTC ACACACCAAA CAGCTATGAC 8191 CATGATTACC CCAACCTCCG AATTAACCCT CACTAAAGCG AACAAAAGCT GCTGCAGGCT CCCTAACTGC 8261 CAACCCCCAC ACTGTCCCCT CAGGCTGCCC CTTCCTTCTA GCGCCTGCCC CCACTCCGCT TTCCTCTCCC 8331 TACTTTCACT TACTTCCGTT CAGCCAAGGT CTGAAACTAG GTGCGCACAG AGCGCTAAGA CTCCGAGAGA 8401 AAGAGACCAG CTTTACACGG GGTTTATCAC ACTGCACCCT GACAGTCGTC ACCCTCACAG CGGGTTTATC 8471 ACATTCCACC CTGACACTCG TCACCCTCAC AGGGGGTTTA TCACAGTGCA CCCTTACAAT CATTCCATTT 8541 GATTCACAAT YTTTTTAGTC TCTACTCTGC CIAACTTGTA ACTTAAATTT CATCAGAGGT GTGTTCGCAC 8611 AGGCGAAAAC ACTATATACA GGGTTCACTA CTATCGCATT TCACGCCTCC ACCTGGCTCT TGGAATGTGT 8661 CCCCCGAGGG CTGATGACTA CCTCAGTTCG ATCTCCACAG CTCACACTGA CACAACATAA CCAAGACACC 8751 TCCCAAGGCT ACCACAATGG GCCCCCCTCC ACGTGCACAT GCCCGGAGGA ACTGCCATGT CGGAGGTGCA 8821 AGCACACCTC CGCATCAGAG TCCTTGGTGT GGACGGAGGG ACCACCCCAC CTTCCAGCCA TCCACCTCAT 8891 GAACAGAACC TACCGAAACC CCCAGTTCTA CTTACACCAG GAAAGGC (SEQUENCE ID NO: 8) FIG. 1 of

30 U.S. Patent Oct. 27, 2009 Sheet 29 of 45 US 7,608,422 B2 1 TGGAAGGGCT AATTTGGTCC CAAAAAAGAC AAGAGATCCT TGATCTGTCG ATCTACCACA CACAAGGCTA 71 CTTCCCTCAT TGGCAGAACT ACACACCAGG GCCAGGGATC ACATATCCAC TGACCTTTGG ATGGTGCTTC 141 AAGTTACTAC CAGTTGAACC AGAGCAACTA CAAGAGGCCA AATAAGGACA CAAGAACAGC TTGTTACACC 211 CTATGACCCA GCATGGGATG GAGCACCCGG AGGGAGAAGT ATTAGTGTGG AAGTTTCACA GCCTCCTAGC 281 ATTTCGTCAC ATGGCCCGAG AGCTCCATCC GGACTACTAC AAACACTCCT GACATCGAGC TTTCTACAAG 351 GGACTTTCCG CTGGGGACTT TCCAGGGAGG TCTGGCCTGG GCGGGACTGC GCAGTGGCGA GCCCTCACAT 421 GCTACATATA AGCAGCTGCT TTTTGCCTCT ACTGGGTCTC TCTGCTTAGA CCAGATCTGA GCCTGGCACC 491 TCTCTGCCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA AACCTTGCCT TGAGTGCTCA AAGTACTGTG 561 TGCCCGTCTG TTGTCTGACT CTGGTAACTA GAGATCCCTC AGACCCTTTT AGTCAGTGTG GAAAATCTCT 631 AGCAGTGGCG CCCGAACAGG GACTTGAAAC CCAAACTAAA GCCAGAGGAG ATCTCTCGAC GCAGGACTCG BssHII (711) 701 GCTTGCTGAA CCGCGCacgg caagaggcga ggggcggcgc ctgacgagga cgccaaaaat tttgactagc ClaI (830) 771 ggaggctaga aggagagagc TCGGTGCGAG AGCGTCAGTA TTAAGCCGGC GAGAATTAGA TCCATGCCAA 841 AAAATTCGGT TAAGGCCAGG GGGAAAGAAG AAGTACAAGC TAAACCACAT CGTATGGCCA AGCAGGGAGC Accl (959) 911 TAGAACGATT CGCAGTTAAT CCTGGCCTGT TAGAAACATC AGAAGCCTCT AGACAAATAC TGGGACAGCT 981 ACAACCATCC CTTCAGACAG GATCAGAGGA GCTTCGATCA CTATACAACA CACTAGCAAC CCTCTATTGT 1051 GTGCACCAGC CGATCGACAT CAAGGACACC AACGAACCTT TAGACAAGAT ACAGCAAGAC CAAAACAACT 1121 CCAAGAAGAA GGCCCAGCAG CCACCAGCTG ACACAGGACA CACCAATCAG CTCAGCCAAA ATTACCCTAT FIG. 11 A

31 U.S. Patent Oct. 27, 2009 Sheet 30 of 45 US 7,608,422 B AGTGCAGAAC ATCCAGGGGC AAATGGTACA TCACGCCATA TCAGCTAGAA CTTTAAACGA TAAGCTTGGG 1261 AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC AACGACCCCC GCCCATTGAC 1331 GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGCACTAT L401 TTACCGTAAA CTGCCCACST GGCA!GTACAT CAAGTGTATC ATATGCCAAG TACCCCCCCT ATTCACCTCA 1471 ATGACGCTAA ATGGCCCCCC TGGCATTATG CCCAGTACAT GACCTTATCC CACTTTCCTA CTTGGCAGTA 1541 CATCTACGTA TTAGTCATCG CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG 1611 CCGTTTGACT CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT TGGCACCAAA 1681 ATCAACGGGA CTTTCCAAAA TGTCCTAACA ACTCCCCCCC ATTGACCCAA ATGGGCCGTA GGCGTGTACG 1751 GTGGGAGGTC TATATAACCA GAGCTCGTTT AGTGAACCGT CAGATCCCCT GGAGACGCCA TCCACGCTCT 1821 TTTGACCTCC ATAGAAGACA CCGACTCTAG AGgatccATC TAAGTAAGCT TGGCATTCCG GTACTGTTGG 1891 TAAAATGCAA GACGCCAAAA ACATAAAGAA AGCCCCGGCG CCATTCTATC CTCTAGAGGA TGGAACCGCT 1961 GGAGAGCAAC TGCATAAGGC TATCAACAGA TACCCCCTGG TTCCTGGAAC AATTGCTTTT ACACATGCAC 2031 ATATCGAGCT GAACATCACG TACGCGGAAT ACTTCGAAAT CTCCCTTCGG TTGGCAGAAC CTATGAAACG 2101 ATATGGGCTG AATACAAATC ACACAATCGT CGTATGCAGT GAAAACTCTC TTCAATTCTT TATGCCGCTC 2171 TTGGGCCCGT TATTTATCCG AGTTGCAGTT GCGCCCGCGA ACGACATTTA TAATGAACGT CAATTGCTCA 2241 ACAGTATGAA CATTTCGCAG CCTACCGTAG TCTTtGTITC CAAAAAGGGG TTGCAAAAAA TTTTGAACGT 2311 GCAAAAAAAA TTACCAATAA TCCACAAAAT TATTATCATG CATTCTAAAA CCGATTACCA GCGATTTCAG 2381 TCGATGTACA CGTTCGTCAC ATCTCATCTA CCTCCCGGTT TTAATCAATA CGATTTTCTA CCAGACTCCT FIG. 11B

32 U.S. Patent Oct. 27, 2009 Sheet 31 of 45 US 7,608,422 B TTGATCGTGA CAAAACAATT CCACTCATAA TGAATTCCTC TGGATCTACT GGGTTACCTA AGCGTCTGGC 2521 CCTTCCGCAT AGAACTCCCT GCGTCACATT CTCGCATGCC ACAGATCCTA TTTTTGGCAA TCAAATCATT 2591 CCGGATACTG CGATTTTAAG TGTTGTTCCA TTCCATCACG GTTTTGGAAT CTTTACTACA CTCGCATATT 2661 TGATATGTGG ATTTCGAGTC GTCTTAATGT ATAGATTTGA AGAAGAGCTG TTTTTACCAT CCCTTCACCA 2731 TTACAAAATT CAAAGTGCGT TGCTAGTACC AACCCTATTT TCATTCTTCG CCAAAAGCAC TCTCATTGAC 2801 AAATACGATT TATCTAATTT ACACGAAATT GCTTCTGGGG GCGCACCTCT TTCGAAAGAA CTCGCGGAAG 2871 CGCTTGCAAA ACGCTTCCAT CTTCCAGGCA TACGACAAGC ATATGGGCTC ACTGAGACTA CATCAGCTAT' 2941 TCTGATTACA CCCGAGCGGG ATGATAAACC GGGCGCGGTC GGTAAAGTTG TTCCATTTTT TGAAGCGAAG 3011 GTTCTGCATC TGGATACCGG GAAAACGCTG GCCGTTAATC AGAGAGCCCA ATTATGTGTC AGAGGACCTA 3081 TGATTATGTC CCGTTATGTA AACAATCCCC AACCGACCAA CGCCTTGATT CACAACCATG GATGGCTACA 3151 TTCTGGAGAC ATAGCTTACT GGGACGAACA CGAACACTTC TTCATAGTTG ACCGCTTCAA CTCTTTAATT Clal (3259) 3221 AAATACAAAG CATATCAGGT CGCCCCCGCT CAATTGGAAT CGATATTCTT ACAACACCCC AACATCTTCG 3291 ACGCCGGCGT GGCAGCTCTT CCCGACGATG ACGCCGCTCA ACTTCCCCCC GCCGTTGTTG TTTTGCAGCA 3361 CGGAAAGACG ATGACGGAAA AAGAGATCGT CGATTACCTC CCCAGTCAAC TAACAACCCC GAAAAAGTTG 3431 CGCCGAGGAC TTGTCTTTGT GGACGAAGTA CCGAAAGGTC TTACCGGAAA ACTCGACCCA ACAAAAATCA Apal (3557) Xhol (3548) Kpnl(3563) 3501 GAGAGATCCT CATAAAGCCC AAGAACCGCC GAAAGTCCAA ATTGTAAcTC GAGGCCCGGC CCGGTACCTT 3571 TAACACCAAT CACTTACAAG CCAGCTGTAG ATCTTAGCCA CTTTTTAAAA GAAAAGGGGG CACTGGAAGG FIG. 110

33 U.S. Patent Oct. 27, 2009 Sheet 32 of 45 US 7,608,422 B GCTAATTCAC TCCCAAAGAA GACAACATAT CCTTCATCTG TGGATCTACC ACACACAAGG CTACTTCCCT 3711 GATTGGCAGA ACTACACACC ACGCCCAGCG GTCAGATATC CACTCACCTT TCGATGGTCC TACAAGCTAG 3781 TACCAGTTGA GCCAGATAAC CTAGAAGAGG CCAATAAAGG AGAGAACACC AGCTTGTTAC ACCCTGTGAG 3851 CCTGCATGGA ATGGATGACC CTGAGACAGA AGTGTTACAC TGGAGGTTTG ACAGCCGCCT AGCATTTCAT 3921 CACGTGGCCC GAGAGCTGCA TCCGCAGTAC TTCAACAACT GCTGACATCG AGCTTGCTAC AAGGGACTTT 3991 CCGCTGGGGA CTTTCCAGGG AGGCGTGGCC TGGCCGGGAC TGGGGAGTGG CGACCCCTCA GATGCTGCAT 4061 ATAAGCAGCT GCTTTTTGCC TGTACTGGGT CTCTCTGGTT AGACCAGATC TCAGCCTGGG AGCTCTCTGG 4131 CTAACTAGGG AACCCACTGC TTAAGCCTCA ATAAAGCTTG CCTTGAGTGC TTCpAGTAGT GTGTGCCCGT 4201 CTGTTGTGTG ACTCTGGTAA CTAGAGATCC CTCACACCCT.TTTAGTCAGT CTGGAAAATC TCTAGCACCC 4271 CCCAGGAGGT AGAGGTTGCA GTGAGCCAAG ATCGCGCCAC TGCATTCCAG CCTGGGCAAG AAAACAAGAC 4341 TGTCTAAAAT AATAATAATA ACTTAAGGGT ATTAAATATA TTTATACATG CAGGTCATAA AAATATATAT 4411 ATTTGGGCTG GGCGCAGTGG CTCACACCTG CGCCCGGCCC TTTGGGACGC CGAGGCACGT GGATCACCTG 4481 AGTTTGGGAG TTCCAGACCA GCCTGACCAA CATGGAGAAA CCCCTTCTCT GTGTATTTTT AGTAGATTTT 4551 ATTTTATGTG TATTTTATTC ACAGGTATTT CTGGAAAACT GAAACTGTTT TTCCTCTACT CTCATACCAC 4621 AAGAATCATC AGCACAGAGG AAGACTTCTG TGATCAAATG TGGTGGGAGA GGGAGGTTTT CACCAGCACA 4691 TGAGCAGTCA CTTCTCCCGC AGACTCCGCG CGTGTCCTTC GCTTCAGTTC CAACACCCCC TGCCTGGAGA 4761 GAGGTCAGAC CACAGGGTGA GGGCTCACTC CCCAACACAT AAACACCCAA GACATAAACA CCCAACAGGT 4831 CCACCCCGCC TGCTGCCCAG GCAGAGCCGA TTCACCAAGA CGGGAATTAG GATAGAGAAA GAGTAAGTCA 4901 CACAGAGCCG GCTGTGCGGG AGAACGGACT TCTATTATGA CTCAAATCAG TCTCCCCAAG CATTCGGGGA 4971 TCAGAGTTTT TAAGGATAAC TTACTGTCTA GGGGGCCACT GAGTTGGAGA TGAAAGCCTA GGGACTCCAA 5041 GCTGTCCTTT TGCGCCGAGT CAGTTCCTGG GTGGGGGCCA CAAGATCGGA TGAGCCAGTT TATCAATCCG 5111 GGCGTGCCAC CTGATCCATG GAGTGCAGGG TCTGCAAAAT ATCTCAAGCA CTGATTGATC TTAGGTTIZ'A 5181 CAATAGTGAT CT[ACCCCAG GAACAATTTG GGGAAGGTCA GAATCTTCTA GCCTGTAGCT CCATGACTCC 5251 TAAACCATAA TTTCTTTTTT GTlTTTTTTT TTTTATITIT GAGACAGGGT CTCACTCTGT CACCTAGGCT 5321 GGACTGCAGT GCTGCAATCA CAGCTCACTG CAGCCCCTAG AGCGCCCGCC ACCGCGGTGG AGCTCCAATT 5391 CCCCCTATAG TGAGTCGTAT TACAATTCAC TGGCCCTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG 5461 CGTTACCCAA CTTAATCGCC TTCCAGCACA TCCCCCTTTC GCCAGCTCCC CTAATAGCCA AGAGGCCCCC 5531 ACCCATCGCC CTTCCCAACA CTTCCCCAGC CTGAATGCCG AATGGCCCCA AATTCTAAAC CTTAATATTT 5601 TGTTAAAATT CGCGTTAAAT TTTTCTTAAA TCACCTCATT TTTTAACCAA TAGGCCGAAA TCGGCAAAAT 5671 CCCTTATAAA TCAAAAGAAT AGACCCAGAT AGGGTTGACT GTTGTTCCAC TTTGGAACAA CACTCCACTA 5741 TTAAAGAACG TGGACTCCAA CGTCAAAGGG CCAAAAACCG TCTATCACGG CGATCCCCCA CTACGTGAAC 5811 CATCACCCTA ATCAAGTTTT TTCGGGTCCA GCTGCCCTAA AGCACTAAAT CGGAACCCTA AAGCCAGCCC 5881 CCGATTTAGA GCTTGACCGG GAAACCCGGC GAACCTGGCC AGAAAGGAAG GGAAGAAAGC GAAAGGAGCG 5951 GGCGCTAGGG CCCTGGCAAG TGTAGCGGTC ACGCTGCGCG TAACCACCAC ACCCGCCCCG CTTAATCCGC 6021 CCCTACAGGG CGCGTCCCAC GTGGCACTTT TCGCGGAAAT CTGCCCGGAA CCCCTATTTG TTTATTTTTC 6091 TAAATACATT CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTCAAAAA FIG. 11 D