Method of detecting and/or identifying adeno-associated virus (AAV) sequences and isolating novel sequences identified thereby

Abstract

A method for detecting and isolating AAV sequences in a sample of DNA obtained from tissue or cells is provided. The invention further provides AAV sequences identified by this method, and vectors constructed using these sequences.

What is claimed is:  
     
         1 . A method of detecting adeno-associated virus (AAV) sequences in a sample, said method comprising the steps of: 
 (a) subjecting a sample containing DNA to amplification via polymerase chain reaction (PCR) using a first set of primers which specifically amplify a first region of AAV nucleic acid sequences;    (b) optionally subjecting the DNA to further amplification using a second set of primers which specifically amplify a second region which comprises the first region of AAV sequences and sequences which are 5′ to the first region, such that AAV 5′ extension sequences which anneal to the 5′ end of the AAV sequences amplified by the primers for the first region are obtained;    (c) optionally subjecting the DNA to further amplification using a third set of primers which specifically amplify a third region which comprises the first region of AAV sequences and sequences which are 3′ to the first region, such that AAV 3′ extension sequences which anneal to the 3′ end of the AAV sequences amplified by the primers for the first region are obtained,    wherein each of said regions is predetermined based upon the alignment of the nucleic acid sequences of at least two AAV serotypes and each of said regions comprises nucleic acid sequences which are highly conserved over at least 18 base pairs at the 5′ end, optionally variable sequences in the middle, and sequences which are highly conserved over at least 18 base pairs at the 3′ end of the sequences of the region, relative to the sequences of the at least two aligned AAV serotypes; and    wherein each of the sets of primers consist of a 5′ primer and a 3′ primer;    wherein the presence of amplified sequences indicates the presence of an AAV in the sample.    
     
     
         2 . A method according to  claim 1 , wherein steps (b) and (c) are performed, and further comprising the step of: 
 (d) using the amplified sequences to construct a sequence comprising a partial and/or complete AAV gene, thereby isolating an AAV gene sequence from the sample.    
     
     
         3 . The method according to  claim 2 , wherein the first region is in the AAV capsid, the second region extends to the 3′ end of the rep genes, and the third region extends to 5′ of the AAV 3′ ITR, such that step (d) permits the construction of AAV gene sequences comprising the complete AAV capsid gene.  
     
     
         4 . The method according to  claim 2 , further comprising the step of subjecting the DNA to amplification with a fourth set of primers which specifically amplifies a fourth region which extend 5′ to the third region.  
     
     
         5 . The method according to  claim 4 , wherein the fourth region extends to 3′ of the AAV 5′ ITR, such that step (d) permits the construction of AAV gene sequences comprising the complete AAV rep and cap genes.  
     
     
         6 . The method according to  claim 1 , wherein said sample comprises cellular or genomic DNA.  
     
     
         7 . The method according to  claim 1 , wherein said DNA has been extracted from the group consisting of cells, cell culture, tissue, tissue culture, and biological fluids.  
     
     
         8 . The method according to  claim 1 , wherein the first region is about 250 base pairs in length.  
     
     
         9 . The method according to  claim 1 , wherein the first region is highly conserved over at least about 25 base pairs at the 5′ and/or 3′ end of the region.  
     
     
         10 . The method according to  claim 1 , wherein the first region is highly conserved over at least about 30 base pairs at the 5′ and/or 3′ end of the region.  
     
     
         11 . The method according to  claim 1 , wherein the highly conserved sequences of the first region have at least 80% identity among the aligned AAV serotypes at the 5′ and/or 3′ end of the region.  
     
     
         12 . The method according  claim 11 , wherein the highly conserved sequences of the first region have at least 90% identity among the aligned AAV serotypes at the 5′ and/or 3′ end of the region.  
     
     
         13 . The method according to  claim 1 , wherein the variable sequences in the middle of the first region have less than 70% identity among the aligned AAV serotypes.  
     
     
         14 . The method according to  claim 1 , wherein the first region spans about bp 2800 to about 3200 of AAV 1, SEQ ID NO:6, and corresponding base pairs in other AAV serotypes.  
     
     
         15 . The method according to  claim 14 , wherein the first region is 257 bp spanning bp 2886 to about 3143 bp of AAV 1, SEQ ID NO:6, and corresponding base pairs in other AAV serotypes.  
     
     
         16 . The method according to  claim 1 , wherein the primers are AV1ns and AV2cas.  
     
     
         17 . A method of identifying the serotype of adeno-associated virus (AAV) sequences in a sample as known or unknown, said method comprising the steps of: 
 (a) obtaining an enzymatic digestion analysis of a sample containing DNA molecules comprising AAV sequences which span all or a portion of bp 2886 to 3143 of AAV1, SEQ ID NO:1, and corresponding regions of other AAV serotypes; and    (b) comparing the enzymatic digestion analysis from the sample to enzymatic digestion analysis for corresponding regions of one or more AAV serotypes, thereby identifying the AAV sequences in the sample as being from one of the one or more AAV serotypes or from an unknown serotype.    
     
     
         18 . The method according to  claim 17 , wherein the AAV sequences were obtained by a method comprising the steps of: 
 (a) subjecting a sample containing DNA to amplification via polymerase chain reaction (PCR) using a first set of primers which specifically amplify a first region of AAV nucleic acid sequences;    (b) optionally subjecting the DNA to further amplification using a second set of primers which specifically amplify a second region which comprises the first region of AAV sequences and sequences which are 5′ to the first region, such that AAV 5′ extension sequences which anneal to the 5′ end of the AAV sequences amplified by the primers for the first region are obtained;    (c) optionally subjecting the DNA to further amplification using a third set of primers which specifically amplify a third region which comprises the first region of AAV sequences and sequences which are 3′ to the first region, such that AAV 3′ extension sequences which anneal to the 3′ end of the AAV sequences amplified by the primers for the first region are obtained,    wherein each of said regions is predetermined based upon the alignment of the nucleic acid sequences of at least two AAV serotypes and each of said regions comprises nucleic acid sequences which are highly conserved over at least 18 base pairs at the 5′ end, optionally variable sequences in the middle, and sequences which are highly conserved over at least 18 base pairs at the 3′ end of the sequences of the region, relative to the sequences of the at least two aligned AAV serotypes; and    wherein each of the sets of primers consist of a 5′ primer and a 3′ primer, and wherein the presence of amplified sequences indicates the presence of an AAV in the sample.    
     
     
         19 . A diagnostic kit for detecting the presence of an unknown adeno-associated virus (AAV) in a sample, said kit comprising: 
 (a) a first set of primers which specifically amplify a first region of AAV nucleic acid sequences;    (b) optionally a second set of primers specific for a second region of the AAV nucleic acid sequences which comprises the first region of AAV sequences and sequences which are 5′ to the first region, such that AAV 5′ extension sequences which anneal to the 5′ end of the AAV sequences amplified by the primers for the first region are obtained;    (c) optionally a third set of primers which specifically amplify a third region which comprises the first region of AAV sequences and sequences which are 3′ to the first region, such that AAV 3′ extension sequences which anneal to the 3′ end of the AAV sequences amplified by the primers for the first region are obtained,    wherein each of said regions is predetermined based upon the alignment of the nucleic acid sequences of at least two AAV serotypes and each of said regions comprises nucleic acid sequences which are highly conserved over at least 18 base pairs at the 5′ end, optionally variable sequences in the middle, and sequences which are highly conserved over at least 18 base pairs at the 3′ end of the sequences of the region, relative to the sequences of the at least two aligned AAV serotypes    wherein each of the sets of primers consist of a 5′ primer and a 3′ primer.    
     
     
         20 . A method for isolating novel adeno-associated viruses (AAV) from a cell, said method comprising the steps of: (a) infecting the cell with a virus which provides helper functions to said AAV; (b) isolating infectious clones containing AAV; (c) sequencing the isolated AAV; and (d) comparing the sequences of the isolated AAV to known AAV serotypes, whereby differences in the sequences of the isolated AAV and known AAV serotypes indicates the presence of a novel AAV.  
     
     
         21 . The method according to  claim 20 , wherein said virus is an adenovirus.  
     
     
         22 . The method according to  claim 21 , wherein said adenovirus is of human or non-human primate origin.  
     
     
         23 . A novel adeno-associated virus (AAV) serotype identified by a method according to  claim 2 .  
     
     
         24 . A novel adeno-associated virus (AAV) serotype identified by a method according to  claim 20 .  
     
     
         25 . An isolated adeno-associated virus (AAV) comprising an AAV capsid having an amino acid sequence selected from the group consisting of: 
 AAV7, amino acids 1 to 737 of SEQ ID NO:2; C1, SEQ ID NO:60; C2, SEQ ID NO:61; C5, SEQ ID NO:62; A3-3, SEQ ID NO:66; A3-7, SEQ ID NO:67; A3-4, SEQ ID NO:68; A3-5, SEQ ID NO: 69; 3.3b, SEQ ID NO: 62; 223.4, SEQ ID NO: 73; 223-5, SEQ ID NO:74; 223-10, SEQ ID NO:75; 223-2, SEQ ID NO:76; 223-7, SEQ ID NO: 77; 223-6, SEQ ID NO: 78; 44-1, SEQ ID NO: 79; 44-5, SEQ ID NO:80; 44-2, SEQ ID NO:81; 42-15, SEQ ID NO: 84; 42-8, SEQ ID NO: 85; 42-13, SEQ ID NO:86; 42-3A, SEQ ID NO:87; 42-4, SEQ ID NO:88; 42-5A, SEQ ID NO:89; 42-1B, SEQ ID NO:90; 42-5B, SEQ ID NO:91; 43-1, SEQ ID NO: 92; 43-12, SEQ ID NO: 93; 43-5, SEQ ID NO:94; 43-21, SEQ ID NO:96; 43-25, SEQ ID NO: 97; 43-20, SEQ ID NO:99; 24.1, SEQ ID NO: 101; 42.2, SEQ ID NO:102; 7.2, SEQ ID NO: 103; 27.3, SEQ ID NO: 104; 16.3, SEQ ID NO: 105; 42.10, SEQ ID NO: 106; 42-3B, SEQ ID NO: 107; 42-11, SEQ ID NO: 108; F1, SEQ ID NO: 109; F5, SEQ ID NO: 110; F3, SEQ ID NO:111; 42-6B, SEQ ID NO: 112; and 42-12, SEQ ID NO: 113.    
     
     
         26 . A host cell transfected with an adeno-associated virus according to  claim 25 .  
     
     
         27 . A composition comprising an AAV according to  claim 25  and a physiologically compatible carrier.  
     
     
         28 . A method of delivering a transgene to a cell, said method comprising the step of contacting the cell with an AAV according to  claim 25 , wherein said rAAV comprises the transgene.  
     
     
         29 . A protein comprising a fragment of an AAV capsid protein, said fragment selected from the group consisting of: 
 vp2 capsid protein, amino acids (aa) 138 to 737;    vp3 capsid protein, aa 203 to 737;    hypervariable region (HVR)1 through 12: aa 146 to 152; aa 182 to 187; aa 262 to 264; aa 263 to 266; aa 263 to 266; aa 381 to 383; aa 383 to 385; aa 450 to 474; aa 451 to 475; aa 490 to 495; aa 491 to 496; aa500 to 504; aa 501 to 505; aa 514 to 522; aa 533 to 554; aa 534 to 555; aa 581 to 594; aa 583 to 596; aa 658 to 667; aa 660 to 669; and aa 705 to 719; aa 707 to 772;    aa 24-42, aa 25-28; aa 81-85; aa133 to 165; aa 134-165; aa 137 to 143; aa 154 to 156; aa 194 to 208; aa 261 to 274; aa 262 to 274; aa 171 to 173; aa 413 to 417; aa 449to 478; aa 494to 525; aa 534to 571; aa 581 to 601; aa 660 to 671; aa 709 to 723; and    aa 1 to 184, aa 199 to 259; aa 274 to 446; aa 603 to 659; aa 670 to 706; aa 724 to 736; aa 185 to 198; aa 260 to 273; aa 447 to 477; aa 495 to 602; aa 660; and aa 707 to 723,    wherein the amino acid numbers are those of the AAV7 capsid, SEQ ID NO:2, and corresponding regions in the capsid of C1, SEQ ID NO:60; C2, SEQ ID NO:61; C5, SEQ ID NO:62; A3-3, SEQ ID NO:66; A3-7, SEQ ID NO:67; A3-4, SEQ ID NO:68; A3-5, SEQ ID NO: 69; 3.3b, SEQ ID NO: 62; 223.4, SEQ ID NO: 73; 223-5, SEQ ID NO:74; 223-10, SEQ ID NO:75; 223-2, SEQ ID NO:76; 223-7, SEQ ID NO: 77; 223-6, SEQ ID NO: 78; 44-1, SEQ ID NO: 79; 44-5, SEQ ID NO:80; 44-2, SEQ ID NO:81; 42-15, SEQ ID NO: 84; 42-8, SEQ ID NO: 85; 42-13, SEQ ID NO:86; 42-3A, SEQ ID NO:87; 42-4, SEQ ID NO:88; 42-5A, SEQ ID NO:89; 42-1B, SEQ ID NO:90; 42-5B, SEQ ID NO:91; 43-1, SEQ ID NO: 92; 43-12, SEQ ID NO: 93; 43-5, SEQ ID NO:94; 43-21, SEQ ID NO:96; 43-25, SEQ ID NO: 97; 43-20, SEQ ID NO:99; 24.1, SEQ ID NO: 101; 42.2, SEQ ID NO:102; 7.2, SEQ ID NO: 103; 27.3, SEQ ID NO: 104; 16.3, SEQ ID NO: 105; 42.10, SEQ ID NO: 106; 42-3B, SEQ ID NO: 107; 42-11, SEQ ID NO: 108; F1, SEQ ID NO: 109; F5, SEQ ID NO: 110; F3, SEQ ID NO:111; 42-6B, SEQ ID NO: 112; and 42-12, SEQ ID NO: 113.    
     
     
         30 . A molecule comprising a nucleic acid sequence encoding a protein according to  claim 29 .  
     
     
         31 . The molecule according to  claim 30 , wherein said molecule further comprises an AAV rep gene.  
     
     
         32 . A host cell transfected with a molecule according to  claim 30 .  
     
     
         33 . A composition comprising a molecule according to  claim 30  and a physiologically compatible carrier.  
     
     
         34 . A method of generating a recombinant adeno-associated virus (AAV) comprising an AAV serotype capsid comprising the steps of culturing a host cell containing: (a) a molecule according to  claim 30  which encodes an adeno-associated virus capsid; (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid protein.  
     
     
         35 . An artificial adeno-associated virus (AAV) capsid protein comprising one or more of the protein fragments according to  claim 29 .  
     
     
         36 . A molecule comprising a nucleic acid sequence encoding a protein according to  claim 29 .  
     
     
         37 . A recombinant adeno-associated virus (AAV) comprising an artificial capsid according to  claim 29 .  
     
     
         38 . A host cell transfected with an adeno-associated virus according to  claim 37 .  
     
     
         39 . A composition comprising an AAV according to  claim 37  and a physiologically compatible carrier.  
     
     
         40 . A method of delivering a transgene to a cell, said method comprising the step of contacting the cell with an AAV according to  claim 37 , wherein said rAAV comprises the transgene.  
     
     
         41 . The molecule according to  claim 36 , wherein said molecule is a plasmid.  
     
     
         42 . The molecule according to  claim 36 , wherein said molecule further comprises an AAV rep gene.  
     
     
         43 . A host cell transfected with a molecule according to  claim 36 .  
     
     
         44 . A composition comprising a molecule according to  claim 36  and a physiologically compatible carrier.  
     
     
         45 . A method of generating a recombinant adeno-associated virus (AAV) comprising an AAV serotype capsid comprising the steps of culturing a host cell containing: (a) a molecule according to  claim 36  which encodes an adeno-associated virus capsid; (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid protein.  
     
     
         46 . A molecule comprising a nucleic acid sequence encoding a novel adeno-associated virus (AAV) serotype capsid protein having an amino acid sequence selected from the group consisting of: AAV7, amino acids 1 to 737 of SEQ ID NO:2; C1, SEQ ID NO:60; C2, SEQ ID NO:61; C5, SEQ ID NO:62; A3-3, SEQ ID NO:66; A3-7, SEQ ID NO:67; A3-4, SEQ ID NO:68; A3-5, SEQ ID NO: 69; 3.3b, SEQ ID NO: 62; 223.4, SEQ ID NO: 73; 223-5, SEQ ID NO:74; 223-10, SEQ ID NO:75; 223-2, SEQ ID NO:76; 223-7, SEQ ID NO: 77; 223-6, SEQ ID NO: 78; 44-1, SEQ ID NO: 79; 44-5, SEQ ID NO:80; 44-2, SEQ ID NO:81; 42-15, SEQ ID NO: 84; 42-8, SEQ ID NO: 85; 42-13, SEQ ID NO:86; 42-3A, SEQ ID NO:87; 42-4, SEQ ID NO:88; 42-5A, SEQ ID NO:89; 42-1B, SEQ ID NO:90; 42-5B, SEQ ID NO:91; 43-1, SEQ ID NO: 92; 43-12, SEQ ID NO: 93; 43-5, SEQ ID NO:94; 43-21, SEQ ID NO:96; 43-25, SEQ ID NO: 97; 43-20, SEQ ID NO:99; 24.1, SEQ ID NO: 101; 42.2, SEQ ID NO:102; 7.2, SEQ ID NO: 103; 27.3, SEQ ID NO: 104; 16.3, SEQ ID NO: 105; 42.10, SEQ ID NO: 106; 42-3B, SEQ ID NO: 107; 42-11, SEQ ID NO: 108; F1, SEQ ID NO: 109; F5, SEQ ID NO: 110; F3, SEQ ID NO:111; 42-6B, SEQ ID NO: 112; and 42-12, SEQ ID NO: 113.  
     
     
         47 . The molecule according to  claim 46 , wherein said molecule is a plasmid.  
     
     
         48 . The molecule according to  claim 46 , wherein said molecule further comprises an AAV rep gene.  
     
     
         49 . The molecule according to  claim 46 , wherein said nucleic acid sequence is the AAV7 sequence, SEQ ID NO:1.  
     
     
         50 . A method of generating a recombinant adeno-associated virus (AAV) comprising an AAV serotype capsid comprising the steps of culturing a host cell containing: (a) a molecule according to  claim 46  which encodes an adeno-associated virus capsid; (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid protein.  
     
     
         51 . A host cell transfected with a molecule according to  claim 46 .  
     
     
         52 . A composition comprising a molecule according to  claim 46  and a physiologically compatible carrier.  
     
     
         53 . A molecule comprising, a nucleic acid sequence encoding a novel adeno-associated virus (AAV) serotype capsid protein, said nucleic acid sequence selected from the group consisting of: AAV5, SEQ ID NO:2; 42-2, SEQ ID NO:9; 42-8 ,SEQ ID NO:27; 42-15, SEQ ID NO:28; 42-5b ,SEQ ID NO: 29; 42-1b ,SEQ ID NO:30; 42-13, SEQ ID NO: 31; 42-3a, SEQ ID NO: 32; 42-4 ,SEQ ID NO:33; 42-5a, SEQ ID NO: 34; 42-10, SEQ ID NO:35; 42-3b, SEQ ID NO: 36; 42-11, SEQ ID NO: 37; 42-6b, SEQ ID NO:38; 43-1, SEQ ID NO: 39; 43-5, SEQ ID NO: 40; 43-12 ,SEQ ID NO:41; 43-20, SEQ ID NO:42; 43-21, SEQ ID NO: 43; 43-23, SEQ ID NO:44; 43-25 ,SEQ ID NO: 45; 44.1, SEQ ID NO:47; 44.5, SEQ ID NO:47; 223.10, SEQ ID NO:48; 223.2, SEQ ID NO:49; 223.4, SEQ ID NO:50; 223.5, SEQ ID NO: 51; 223.6, SEQ ID NO: 52; 223.7, SEQ ID NO: 53; A3.4, SEQ ID NO: 54; A3.5, SEQ ID NO:55; A3.7, SEQ ID NO: 56; A3.3 ,SEQ ID NO:57; 42.12, SEQ ID NO: 58; 44.2, SEQ ID NO: 59; AAV10, SEQ ID NO: 117; AAV11, SEQ ID NO: 118; AAV12 ,SEQ ID NO:119; A3.1, SEQ ID NO:120; and H6, SEQ ID NO: 25.  
     
     
         54 . The molecule according to  claim 53 , wherein said molecule is a plasmid.  
     
     
         55 . The molecule according to  claim 53 , wherein said molecule further comprises an AAV rep gene.  
     
     
         56 . A method of generating a recombinant adeno-associated virus (AAV) comprising an AAV serotype capsid comprising the steps of culturing a host cell containing: (a) a molecule according to  claim 53  which encodes an adeno-associated virus capsid; (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid protein.  
     
     
         57 . A host cell transfected with a molecule according to  claim 53 .  
     
     
         58 . A composition comprising a molecule according to  claim 53  and a physiologically compatible carrier.  
     
     
         59 . A molecule comprising a nucleic acid sequence encoding a fragment of an adeno-associated virus capsid protein, said nucleic acid sequence selected from the group consisting of: 
 vp1, nt 825 to 3049;    vp2, nt 1234 to 3049;    vp 3, nt 1434 to 3049;    nt 468 to 3090; and    nt 725 to 3090,    wherein the nucleotides numbers are of AAV7, SEQ ID NO:1 and correspond to sequences in 42-2, SEQ ID NO:9; 42-8 ,SEQ ID NO:27; 42-15 , SEQ ID NO:28; 42-5b ,SEQ ID NO: 29; 42-1b ,SEQ ID NO:30; 42-13, SEQ ID NO: 31; 42-3a, SEQ ID NO: 32; 42-4 ,SEQ ID NO:33; 42-5a, SEQ ID NO: 34; 42-10, SEQ ID NO:35; 42-3b, SEQ ID NO: 36; 42-11, SEQ ID NO: 37; 42-6b, SEQ ID NO:38; 43-1, SEQ ID NO: 39; 43-5, SEQ ID NO: 40; 43-12 ,SEQ ID NO:41; 43-20, SEQ ID NO:42; 43-21, SEQ ID NO: 43; 43-23, SEQ ID NO:44; 43-25 ,SEQ ID NO: 45; 44.1, SEQ ID NO:47; 44.5, SEQ ID NO:47; 223.10, SEQ ID NO:48; 223.2, SEQ ID NO:49; 223.4, SEQ ID NO:50; 223.5, SEQ ID NO: 51; 223.6, SEQ ID NO: 52; 223.7, SEQ ID NO: 53; A3.4, SEQ ID NO: 54; A3.5, SEQ ID NO:55; A3.7, SEQ ID NO: 56; A3.3 ,SEQ ID NO:57; 42.12, SEQ ID NO: 58; 44.2, SEQ ID NO: 59; AAV10, SEQ ID NO: 117; AAV11, SEQ ID NO: 118; AAV12, SEQ ID NO:119; A3.1, SEQ ID NO:120; and H6, SEQ ID NO: 25.    
     
     
         60 . The molecule according to  claim 59 , wherein said molecule is a plasmid.  
     
     
         61 . The molecule according to  claim 59 , wherein said molecule further comprises an AAV rep gene.  
     
     
         62 . A method of generating a recombinant adeno-associated virus (AAV) comprising an AAV serotype capsid comprising the steps of culturing a host cell containing: (a) a molecule according to  claim 59;  (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene; and (d) sufficient helper functions to permit packaging of the minigene into an AAV capsid protein.  
     
     
         63 . A host cell transfected with a molecule according to  claim 59 .  
     
     
         64 . A composition comprising a molecule according to  claim 59  and a physiologically compatible carrier.  
     
     
         65 . A molecule comprising a heterologous adeno-associated virus (AAV) serotype 7 nucleic acid sequence selected from the group consisting of: nucleotides (nt) 1 to 107of SEQ ID NO: 1; nt 107 to 2215 of SEQ ID NO:1; nt334to 2215 of SEQ ID NO:1; nt 2222 to 4435 of SEQ ID NO:1; nt 2633 to 4435 of SEQ ID NO:1; nt 2831 to 4435 of SEQ ID NO:1; and nt 4704 to 4721 of SEQ ID NO: 1.  
     
     
         66 . A host cell containing a molecule according to  claim 65 .  
     
     
         67 . A molecule encoding an adeno-associated virus (AAV) serotype 7 rep protein or a fragment thereof, said protein or fragment selected from the group consisting of: 
 amino acid (aa) 1 to 623, aa 1 to 171; aa 172 to 372, aa 373 to 444, and aa 445 to 623 of SEQ ID NO:3.    
     
     
         68 . A host cell containing a molecule according to  claim 67.