Adeno-associated viruses and uses thereof

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 . A composition comprising at least two recombinant adeno-associated viruses (rAAV), comprising:
 a) a first rAAV comprising a first recombinant DNA molecule comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV; 
 ii) a second DNA segment which comprises at least one heterologous enhancer and a heterologous promoter; and 
 iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV; and 
   b) a second rAAV comprising a second recombinant DNA molecule comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV; 
 ii) a second DNA segment which comprises an entire open reading frame for a therapeutic gene product; and 
 iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV, 
   wherein the recombinant DNA molecules of the two rAAVs, when contacted with a host cell, become linked so that the heterologous promoter initiates transcriptional expression of the gene product encoded by the entire open reading frame, and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame.   
     
     
         2 . The composition of  claim 1  further comprising a delivery vehicle. 
     
     
         3 . The composition of  claim 2  where the vehicle is a pharmaceutically acceptable carrier. 
     
     
         4 . The composition of  claim 1  wherein the first-recombinant DNA does not encode a protein. 
     
     
         5 . A first rAAV vector which comprises a cis-acting heterologous transcriptional regulatory including a heterologous enhancer and a heterologous promoter positioned in the vector so that the heterologous promoter is capable of regulating, in a host cell, transcriptional expression of a therapeutic gene product encoded by an entire open reading frame in a second rAAV vector, after sequences in the first and second rAAV vectors become linked in the host cell, and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame. 
     
     
         6 . The first AAV vector of  claim 5  wherein the heterologous promoter is not operably linked to an open reading frame encoding a protein in the first AAV vector. 
     
     
         7 . A plasmid comprising the vector of  claim 5 . 
     
     
         8 . A host cell contacted with at least two rAAV, wherein a first rAAV comprises a first recombinant DNA molecule comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV;   ii) a second DNA segment which comprises a heterologous enhancer and a heterologous promoter; and   iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV; and   
       wherein a second rAAV comprises a second recombinant DNA molecule comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV; 
 ii) a second DNA segment which comprises an entire open reading frame for a therapeutic gene product; and 
 iii) a third DNA segment comprising a 3′-inverted terminal repeat of 
 AAV, wherein the heterologous promoter initiates transcriptional expression of the gene product encoded by the open reading frame in the second rAAV in the host cell, and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame. 
 
     
     
         9 . The host cell of  claim 8  wherein the first recombinant DNA does not encode a protein. 
     
     
         10 . The host cell of  claim 8  wherein expression of the gene product in the host cell does not rely on splicing. 
     
     
         11 . A method to transfer recombinant DNAs to a host cell, comprising:
 contacting the host cell with at least two rAAV,   wherein a first rAAV comprises a first recombinant DNA molecule comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV; 
 ii) a second DNA segment which comprises a heterologous enhancer and a heterologous promoter; and 
 iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV; and 
   wherein a second rAAV comprises a second recombinant DNA molecule comprising linked:
 i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV; 
 ii) a second DNA segment which comprises an entire open reading frame for a therapeutic gene product; and 
 iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV, wherein the heterologous promoter initiates transcriptional expression of the gene product encoded by the open reading frame in the second rAAV in the host cell, and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame. 
   
     
     
         12 . A method to transfer and express a polypeptide in a host cell comprising contacting the host cell with the composition of  claim 1 . 
     
     
         13 . The method of  claim 11  or  12  wherein the first recombinant DNA does not encode a protein. 
     
     
         14 . A method to enhance the expression of a polynucleotide in a host cell, comprising: contacting a host cell comprising a recombinant AAV vector comprising a polynucleotide segment which encodes a polypeptide, with a composition comprising a further recombinant AAV vector corresponding to the vector of  claim 5  in an amount which enhances expression of the polynucleotide. 
     
     
         15 . A method to enhance the expression of a polynucleotide in a host cell, comprising: contacting a host cell comprising a recombinant AAV vector corresponding to the vector of  claim 5 , with a composition comprising a further recombinant AAV vector comprising a polynucleotide segment which encodes a polypeptide, in an amount which enhances expression of the polynucleotide. 
     
     
         16 . A method to enhance the expression of a polynucleotide in a host cell, comprising: contacting a host cell with a recombinant AAV vector corresponding to the vector of  claim 5  and a further recombinant AAV vector comprising a polynucleotide segment which encodes a polypeptide, in an amount which enhances expression of the polynucleotide in the cell. 
     
     
         17 . The method of  claim 14  or  15  wherein the composition further comprises a delivery vehicle. 
     
     
         18 . The method of  claim 17  wherein the delivery vehicle is a pharmaceutically acceptable carrier. 
     
     
         19 . The method of  claim 11 ,  12 ,  14 ,  15  or  16  wherein expression of the gene product in the host cell does not rely on splicing. 
     
     
         20 . A first rAAV comprising a first recombinant DNA molecule comprising linked:
 a first DNA segment comprising a 5′-inverted terminal repeat of AAV; a second DNA segment which comprises a heterologous enhancer and a heterologous promoter; and a third DNA segment comprising a 3′-inverted terminal repeat of AAV; and   a second rAAV comprising a second recombinant DNA molecule comprising linked: a first DNA segment comprising a 5′-inverted terminal repeat of AAV; a second DNA segment which comprises an entire open reading frame for a therapeutic gene product; and a third DNA segment comprising a 3′-inverted terminal repeat of AAV,   
       wherein the heterologous promoter in the first rAAV initiates transcriptional expression of the gene product encoded by the open reading frame in the second rAAV in a host cell contacted with the first and second rAAVs and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame. 
     
     
         21 . The rAAVs of  claim 20  wherein the first recombinant DNA molecule does not encode a protein.