Adeno-associated virus vectors

Abstract

The invention provides an isolated and purified DNA molecule comprising at least one DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability, persistence or abundance of the isolated DNA molecule in a host cell. The invention also provides a composition comprising at least two adeno-associated virus vectors.

1 . An isolated and purified DNA molecule comprising at least one DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability, persistence or abundance of the isolated DNA molecule in a host cell. 
     
     
         2 . The DNA molecule of  claim 1  in which the DNA segment comprises at least a portion of a 5′ inverted terminal repeat of adeno-associated virus. 
     
     
         3 . The DNA molecule of  claim 1  in which the DNA segment comprises at least a portion of a 3′-inverted terminal repeat of adeno-associated virus. 
     
     
         4 . The DNA molecule of  claim 1  which further comprises a marker or selectable gene. 
     
     
         5 . A plasmid comprising the DNA molecule of  claim 1 . 
     
     
         6 . A gene transfer vector, comprising:
 a) at least one first DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability or integration of the vector in a host cell; and   b) a second DNA segment comprising a gene.   
     
     
         7 . The vector of  claim 6  in which the first DNA segment comprises at least about 550 bp of adeno-associated virus sequence. 
     
     
         8 . The vector of  claim 6  in which the first DNA segment comprises at least about 400 bp of adeno-associated virus sequence. 
     
     
         9 . A therapeutic gene transfer vector, comprising:
 a) at least one first DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability or integration of the vector in a host cell; and   b) a second DNA segment comprising a gene encoding a therapeutically effective polypeptide.   
     
     
         10 . A method of delivering a gene to a cell comprising: contacting the cell with the vector of  claim 6  or  9 . 
     
     
         11 . A composition for delivering a gene to a cell, comprising: the vector of  claim 6  or  9  and a delivery vehicle. 
     
     
         12 . The composition of  claim 11  wherein the delivery vehicle is a pharmaceutically acceptable carrier. 
     
     
         13 . The composition of  claim 11  wherein the delivery vehicle is a liposome. 
     
     
         14 . The DNA molecule of  claim 1  which comprises concatamers of the circular intermediate. 
     
     
         15 . The DNA molecule of  claim 1  in which the stability, persistence or abundance of the DNA in cells is enhanced by a DNA binding protein. 
     
     
         16 . The DNA molecule of  claim 15  wherein the DNA binding protein is adenovirus E2a. 
     
     
         17 . A host cell comprising the vector of  claim 6  or  9 . 
     
     
         18 . A host cell comprising the DNA molecule of  claim 1 . 
     
     
         19 . An animal comprising the vector of  claim 6  or  9 . 
     
     
         20 . The animal of  claim 19  which is not a human. 
     
     
         21 . A method of expressing a gene product in the muscle tissue of an animal, which comprises: administering the vector of  claim 6  or  9  to the muscle tissue of said animal in an amount effective to express the gene. 
     
     
         22 . The method of  claim 21  wherein the vector is administered dissolved or suspended in a liquid pharmaceutically acceptable carrier. 
     
     
         23 . The method of  claim 22  wherein said liquid carrier comprises an aqueous solution. 
     
     
         24 . The method of  claim 21  wherein said gene comprises a DNA segment encoding a protein operably linked to a promoter operable in said muscle tissue. 
     
     
         25 . The method of  claim 21  wherein said administering is by intramuscular injection. 
     
     
         26 . The method of  claim 21  wherein said administering is by transdermal transport. 
     
     
         27 . The method of  claim 21  wherein said animal is a bird or mammal. 
     
     
         28 . The method of  claim 1  wherein said animal is a human. 
     
     
         29 . A method of expressing a gene in a eukaryotic cell, comprising:
 a) transfecting a eukaryotic host cell susceptible to adenovirus infection with the vector of  claim 6  or  9  and a recombinant adenovirus helper vector so as to form packaged viral particles; and   b) infecting a eukaryotic host cell with the viral particles in an amount effective to detect expression of the gene.   
     
     
         30 . A composition comprising:
 a) a first adeno-associated virus vector comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of adeno-associated virus; 
 ii) a second DNA segment comprising at least a portion of an open reading frame operably linked to a promoter, wherein the DNA segment does not comprise the entire open reading frame; 
 iii) a third DNA segment comprising a splice donor site; and 
 iv) a fourth DNA segment comprising a 3′-inverted terminal repeat of adeno-associated virus; and 
   b) a second adeno-associated virus vector comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of adeno-associated virus; 
 ii) a second DNA segment comprising a splice acceptor site; 
 iii) a third DNA segment comprising at least a portion of an open reading frame which together with the DNA segment of (a)(ii) encodes a full-length polypeptide; and 
 iv) a fourth DNA segment comprising a 3′-inverted terminal repeat of adeno-associated virus. 
   
     
     
         31 . The composition of  claim 30  further comprising a delivery vehicle. 
     
     
         32 . A method to transfer and express a polypeptide in a host cell comprising contacting the host cell with the composition of  claim 30 . 
     
     
         33 . A method to transfer and express a polypeptide in a host cell comprising contacting the host cell with a first adeno-associated virus vector comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of adeno-associated virus;   ii) a second DNA segment comprising at least a portion of an open reading frame operably linked to a promoter, wherein the DNA segment does not comprise the entire open reading frame;   iii) a third DNA segment comprising a splice donor site; and   iv) a fourth DNA segment comprising a 3′-inverted terminal repeat of adeno-associated virus.   
     
     
         34 . The method of  claim 33  wherein the host cell is further contacted with a second adeno-associated virus vector comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of adeno-associated virus;   ii) a second DNA segment comprising a splice acceptor site;   iii) a third DNA segment comprising at least a portion of an open reading frame which together with the DNA segment of (a)(ii) encodes a full-length polypeptide; and   iv) a fourth DNA segment comprising a 3′-inverted terminal repeat of adeno-associated virus.   
     
     
         35 . The method of  claim 32  or  33  wherein the host cell is a lung epithelial cell, a muscle cell or a neuron. 
     
     
         36 . The method of  claim 32  or  34  wherein the polypeptide is the CFTR polypeptide. 
     
     
         37 . A method of expressing a gene product in the muscle tissue of an animal, comprising contacting the muscle tissue with the composition of  claim 30  in an amount effective to express the polypeptide. 
     
     
         38 . A method of expressing a gene product in the airway epithelia of an animal, comprising contacting the airway epithelia with the composition of  claim 30  in an amount effective to express the polypeptide. 
     
     
         39 . A method of expressing a gene product in the neurons of an animal, comprising contacting the neurons with the composition of  claim 30  in an amount effective to express the polypeptide.