Compositions and methods for recombinant adeno-associated virus production

Abstract

The present invention relates to methods and compositions for the production of recombinant Adeno-Associated Viruses (rAAV). In particular, the invention discloses nucleic acid constructs and packaging cells having improved properties for rAAV production, as well as novel methods of titration and characterization of rAAV preparations. The invention also describes novel sequences which promote or increase the packaging of nucleic acids in rAAV, and their use for producing rAAV with high efficiency. The invention can be used for producing or testing high quality rAAV preparations, for biological, preclinical, clinical or pharmaceutical uses.

1 . An isolated Replication Encapsidation Sequence (“RES”), which provides or promotes the packaging of a nucleic acid operably linked thereto into an Adeno-Associated Virus (AAV) particle, and which consists of the fragment from nucleotide position 190 to nucleotide position 540 of an AAV genome of SEQ ID No: 10.  
     
     
         2 . An isolated Replication Encapsidation Sequence (“RES”), which is a fragment of the RES of claim I and which provides or promotes the packaging of a nucleic acid operably linked thereto into an Adeno-Associated Virus (AAV) particle.  
     
     
         3 . The isolated Replication Encapsidation Sequence (“RES”) of  claim 2 , which consists of the fragment from nucleotide position 190 to nucleotide position 350 of an Adeno-Associated Virus (AAV) genome of SEQ ID No: 10.  
     
     
         4 . The isolated Replication Encapsidation Sequence (“RES”) of  claim 2 , which consists of the fragment from nucleotide position 230 to nucleotide position 350 of an Adeno-Associated Virus (AAV) genome of SEQ ID No: 10.  
     
     
         5 . The isolated Replication Encapsidation Sequence (“RES”) of  claim 2 , which consists of the fragment from nucleotide position 230 to nucleotide position 300 of an Adeno-Associated Virus (AAV) genome of SEQ ID No: 10.  
     
     
         6 . The isolated Replication Encapsidation Sequence (“RES”) of  claim 2 , which consists of the fragment from nucleotide position 250 to nucleotide position 300 of an Adeno-Associated Virus (AAV) genome of SEQ ID No: 10.  
     
     
         7 . An isolated Replication Encapsidation Sequence (“RES”), which is a functional variant of the RES of  claim 1 , and which differs from the fragment of the Adeno-Associated Virus (AAV) genome by 1, 2 or 3 mutation(s) or deletion(s).  
     
     
         8 . An isolated Replication Encapsidation Sequence (“RES”), which is a functional variant of the RES of  claim 2 , and which differs from the fragment of the Adeno-Associated Virus (AAV) genome by 1, 2 or 3 mutation(s) or deletion(s).  
     
     
         9 . The Replication Encapsidation Sequence (“RES”) according to any of  claims 1  to  8 , wherein said RES comprises a Rep Binding Element and a terminal resolution site.  
     
     
         10 . The Replication Encapsidation Sequence (“RES”) of  claim 9 , wherein said RES further comprises a palindromic region.  
     
     
         11 . The Replication Encapsidation Sequence (“RES”) according to any of  claims 1  to  8 , which comprises SEQ ID NO: 1 or a functional variant thereof, wherein said functional variant binds to Rep78 and/or Rep68.  
     
     
         12 . The Replication Encapsidation Sequence (“RES”) according to any of  claims 1  to  8 , which comprises SEQ ID NO: 3 or a functional variant thereof, wherein said functional variant binds to Rep78 and/or Rep68.  
     
     
         13 . The Replication Encapsidation Sequence (“RES”) according to any of  claims 1  to  8 , which comprises SEQ ID NO: 2 or a functional variant thereof, wherein said functional variant forms a hairpin structure.  
     
     
         14 . The isolated Replication Encapsidation Sequence (“RES”) of any one of  claims 1  to  8 , which consists of less than 400 bp.  
     
     
         15 . A nucleic acid consisting of one or several Replication Encapsidation Sequence(s) (“RES”) according to any one of  claims 1  to  8 , operably linked to a heterologous polynucleotide lacking a functional ITR sequence.  
     
     
         16 . A recombinant Adeno-Associated Virus (rAAV) vector plasmid, comprising a recombinant AAV genome and one or several Replication Encapsidation Sequence(s) (“RES”) according to any one of  claims 1  to  8 .  
     
     
         17 . The recombinant Adeno-Associated Virus (rAAV) vector plasmid of  claim 16 , comprising a Replication Encapsidation Sequence (“RES”) in antisense orientation.  
     
     
         18 . A composition comprising a recombinant Adeno-Associated Virus (rAAV) particle comprising a recombinant nucleic acid, wherein said recombinant nucleic acid comprises one or several Replication Encapsidation Sequence(s) (“RES”) according to any one of  claims 1  to  8 .  
     
     
         19 . The composition of  claim 18 , wherein said recombinant nucleic acid is a recombinant Adeno-Associated Virus genome comprising a Replication Encapsidation Sequence (“RES”) in antisense orientation.  
     
     
         20 . A method of producing recombinant Adeno-Associated Viruses (rAAVs) comprising: 
 (i) co-transfecting a cell culture with: 
 a rAAV vector plasmid,  
 a rep-cap plasmid devoid of ITR, containing a rep-cap unit consisting of residues 190-4484 of the AAV genome or fragments thereof encoding functional Rep and Cap proteins, and  
 an adenovirus plasmid containing the entire adenoviral genome or a genome lacking the left and right ITRs and the packaging region, and  
   (ii) recovering the rAAV produced,    wherein the rAAV vector plasmid comprises a Replication Encapsidation Sequence (“RES”) according to any one of  claims 1  to  8 , in antisense orientation.    
     
     
         21 . The method of  claim 20 , wherein said adenovirus plasmid lacks the E1 region.  
     
     
         22 . A method of producing recombinant Adeno-Associated Viruses (rAAVs) comprising: 
 (i) co-transfecting a culture of cells which contain, in their genome, nucleic acid construct(s) encoding the Rep and/or Cap functions, with: 
 a rAAV vector plasmid, and  
 a helper adenovirus or an adenovirus plasmid containing the entire adenoviral genome or a genome lacking the left and right ITRs and the packaging region, and  
   (ii) recovering the rAAV produced,    wherein the rAAV vector plasmid comprises a Replication Encapsidation Sequence (“RES”) according to any one of  claims 1  to  8 , in antisense orientation.    
     
     
         23 . The method of  claim 22 , wherein said helper adenovirus or adenovirus plasmid lacks the E1 region.