US2006166318A1PendingUtilityA1

Methods, compositions, and cells for encapsidating recombinant vectors in AAV particles

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Assignee: TARGETED GENETICS CORPPriority: Apr 28, 1999Filed: Jan 21, 2005Published: Jul 27, 2006
Est. expiryApr 28, 2019(expired)· nominal 20-yr term from priority
Inventors:Dara LockertCarmel LynchHaim BursteinAnthony Stepan
C12N 7/00A61K 48/00C07K 14/005C12N 15/86C12N 2750/14122C12N 2750/14143C12N 2750/14152
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Claims

Abstract

Isolated recombinant polynucleotides comprising elements which promote encapsidation into AAV particles, packaging cells comprising the recombinant polynucleotides, and methods for their use are provided in the present invention. These isolated recombinant polynucleotides comprise a non-AAV ITR encapsidation element (such as the P1 sequence located within the AAV S1 integration site of human chromosome 19) operably linked to one or more heterologous genes to be encapsidated. The constructs may be either integrated into a mammalian cell genome, maintained episomally, or provided transiently.

Claims

exact text as granted — not AI-modified
1 . A method for producing a recombinant polynucleotide encapsidated in an adeno-associated virus (AAV) particle, comprising providing a mammalian cell which produces AAV rep and cap gene products, wherein said mammalian cell contains the recombinant polynucleotide which comprises a heterologous gene operably linked to an encapsidation element which promotes encapsidation of said heterologous gene into an AAV particle, and wherein said encapsidation element is other than an AAV inverted terminal repeat (ITR) or its D sequence.  
     
     
         2 . The method of  claim 1 , wherein said encapsidation element has a nucleotide sequence which has at least about 30% nucleotide sequence identity with SEQ ID NO:1.  
     
     
         3 . The method of  claim 1 , wherein said encapsidation element has a nucleotide sequence which has at least about 47% nucleotide sequence identity with SEQ ID NO:1.  
     
     
         4 . The method of  claim 1 , wherein said encapsidation element comprises at least about 35 contiguous nucleotides of the nucleotide sequence depicted in SEQ ID NO:1.  
     
     
         5 . The method of  claim 1 , wherein said encapsidation element comprises a terminal resolution site.  
     
     
         6 . The method of  claim 1 , wherein said encapsidation element comprises a binding site for AAV Rep68 and Rep78 proteins.  
     
     
         7 . The method of  claim 1 , wherein said encapsidation element comprises a terminal resolution site and a binding site for AAV Rep68 and Rep78 proteins.  
     
     
         8 . The method of  claim 1 , wherein said encapsidation element comprises the nucleotide sequence GGTTGG(X)nGCXCGCTCGCTCGCTX, wherein X is any nucleotide and n is an integer from 1 to about 20.  
     
     
         9 . The method of  claim 1 , wherein said encapsidation element comprises a nucleotide sequence having the sequence of nucleotides 19 to 48 of SEQ ID NO:1.  
     
     
         10 . The method of  claim 1 , wherein said encapsidation element has the nucleotide sequence depicted in SEQ ID NO:1.  
     
     
         11 . The method of  claim 1 , wherein the encapsidation activity of said encapsidation element is activated by helper function.  
     
     
         12 . The method of  claim 11 , wherein said helper function is provided by an adenovirus.  
     
     
         13 . The method of  claim 1 , wherein said AAV rep and cap gene products produced by said mammalian cell are encoded by AAV rep and cap genes which are stably integrated into the genome of said cell.  
     
     
         14 . The method of  claim 1 , wherein said AAV rep and cap gene products are encoded by an extrachromosomal polynucleotide.  
     
     
         15 . An isolated recombinant polynucleotide sequence comprising a heterologous gene flanked by encapsidation elements other than adeno-associated virus (AAV) inverted terminal repeats (ITR) or AAV ITR D sequences, wherein said encapsidation elements promote encapsidation of said heterologous gene in the presence of AAV rep and cap gene products and helper virus function.  
     
     
         16 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element has a nucleotide sequence which has at least about 30% nucleotide sequence identity with SEQ ID NO:1.  
     
     
         17 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element has a nucleotide sequence which has at least about 47% nucleotide sequence identity with SEQ ID NO:1.  
     
     
         18 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element comprises at least about 35 contiguous nucleotides of the nucleotide sequence depicted in SEQ ID NO:1.  
     
     
         19 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element comprises a terminal resolution site.  
     
     
         20 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element comprises a binding site for AAV Rep68 and Rep78 proteins.  
     
     
         21 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element comprises a terminal resolution site and a binding site for AAV Rep68 and Rep78 proteins.  
     
     
         22 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element comprises the nucleotide sequence GGTTGG(X)nGCXCGCTCGCTCGCTX, wherein X is any nucleotide and n is an integer from 1 to about 20.  
     
     
         23 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element comprises a nucleotide sequence having the sequence of nucleotides 19 to 48 of SEQ ID NO:1.  
     
     
         24 . The isolated recombinant polynucleotide of  claim 15 , wherein said encapsidation element has the nucleotide sequence depicted in SEQ ID NO:1.  
     
     
         25 . The isolated recombinant polynucleotide of  claim 15 , wherein the encapsidation activity of said encapsidation element is activated by helper function.  
     
     
         26 . The isolated recombinant polynucleotide of  claim 15 , wherein said helper function is provided by an adenovirus.  
     
     
         27 . The isolated recombinant polynucleotide of  claim 15 , wherein said AAV rep and cap gene products produced by said mammalian cell are encoded by AAV rep and cap genes which are stably integrated into the genome of said cell.  
     
     
         28 . The isolated recombinant polynucleotide of  claim 15 , wherein said AAV rep and cap gene products are encoded by an extrachromosomal polynucleotide.  
     
     
         29 . The isolated recombinant polynucleotide of  claim 15 , wherein said heterologous gene encodes a functional CFTR polypeptide.  
     
     
         30 . A method for generating a packaging cell capable of producing stocks of a recombinant polynucleotide comprising a heterologous gene encapsidated in an adeno-associated virus (AAV) particle, comprising transfecting mammalian cells which produce AAV rep and cap gene products with the recombinant polynucleotide, wherein said recombinant polynucleotide comprises a heterologous gene operably linked to an encapsidation element other than an AAV inverted terminal repeat (ITR) or an AAV ITR D sequence, wherein said encapsidation element promotes encapsidation of said heterologous gene into an AAV particle.  
     
     
         31 . The method of  claim 30 , wherein said AAV rep and cap gene products produced by said mammalian cell are encoded by AAV rep and cap genes stably integrated into the genome of the cell.  
     
     
         32 . The method of  claim 30 , wherein said AAV rep and cap gene products are encoded by an extrachromosomal polynucleotide.  
     
     
         33 . The method of  claim 30 , wherein said recombinant polynucleotide further comprises a selectable marker.  
     
     
         34 . The method of  claim 30 , wherein said recombinant polynucleotide integrates into the genome of the cell.  
     
     
         35 . A packaging cell produced by the method of  claim 18 .  
     
     
         36 . A mammalian packaging cell for producing stocks of a recombinant polynucleotide encapsidated in an adeno-associated virus (AAV) particle, wherein said packaging cell comprises the recombinant polynucleotide, wherein said cell produces adeno-associated virus (AAV) rep and cap gene products, and wherein said recombinant polynucleotide comprises a heterologous gene operably linked to an encapsidation element other than an AAV inverted terminal repeat (ITR) or an AAV ITR D sequence, wherein said encapsidation element promotes encapsidation of said heterologous gene.  
     
     
         37 . The mammalian packaging cell of  claim 36 , wherein said rep and said cap gene products are encoded by genes stably integrated into the genome of the cell.  
     
     
         38 . The mammalian packaging cell of  claim 36 , wherein said rep and said cap gene products are encoded by extrachromosomal genes.  
     
     
         39 . The mammalian packaging cell of  claim 36 , wherein said recombinant vector is maintained episomally.

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