Method for producing a recombinant adeno-associated virus, suitable means therefor and use for producing a medicament

Abstract

The present invention relates to a method for producing a recombinant adeno-associated virus (rAAV), in which at different times a helper construct and also a vector construct are introduced into a suitable cell, and preferably the helper construct does not comprise, in particular with the exception of the AAV promoters, any nucleic acid sequences to which at least a Rep protein can essentially specifically bind and preferably the vector construct comprises ITR sequences in flop orientation. The recombinant adeno-associated viruses produced according to the method of the invention are suitable in particular for producing a tumor cell into which additionally nucleic acids coding for GM-CSF and B7.2 has been introduced, which in turn can be used in the form of a medicament for the treatment of cancers.

1 - 28 . (Cancelled)  
     
     
         29 . A method for producing a recombinant adeno-associated virus (rAAV), in which one or more helper construct(s) comprising nucleic acids coding for at least one Rep protein and/or for the Cap proteins (first construct) and also a vector construct (second construct) comprising one or more nucleic acids which are heterologous to AAV and are flanked by ITR sequences are introduced into a suitable cell, wherein the helper construct(s) and also the vector construct are introduced into the suitable cell at different times.  
     
     
         30 . The method of  claim 29 , wherein the helper construct(s) is introduced first into the suitable cell.  
     
     
         31 . The method of  claim 29 , wherein, after introducing said first construct(s) and before introducing said second construct, the cell is purified.  
     
     
         32 . The method of  claim 29 , wherein in another step a helper virus is added at a different time.  
     
     
         33 . The method of  claim 29  or  32 , wherein the time differences between the individual steps independently of one another are at least approximately 1 hour.  
     
     
         34 . The method of  claim 29  or  32 , wherein the time differences between the individual steps independently of one another are at least approximately 12 hours.  
     
     
         35 . The method of  claim 29  or  32 , wherein the time differences between the individual steps independently of one another are at least approximately 1 day.  
     
     
         36 . The method of  claim 29  or  32 , wherein the time differences between the individual steps independently of one another are at least approximately 1-2 days.  
     
     
         37 . The method of  claim 29 , wherein the genetically modified cells are cultured in a serum-free medium.  
     
     
         38 . The method of  claim 29 , wherein the helper construct does not comprise any nucleic acid sequences to which at least one Rep protein can essentially specifically bind with the exception of the AAV promoters.  
     
     
         39 . The method of  claim 38 , wherein the helper construct does not comprise SEQ ID NO:1 in the vector backbone.  
     
     
         40 . The method of  claim 38 , wherein the helper construct includes nucleic acid sequences coding Rep 68, Rep 52 and Rep 40 and wherein the helper construct comprises nucleic acid sequences coding for Cap proteins VP 1, VP 2 and VP 3.  
     
     
         41 . The method of  claim 38 , wherein the helper construct includes nucleic acid sequences coding Rep 68 and Rep 52 and wherein the helper construct comprises nucleic acid sequences coding for Cap proteins VP 1, VP 2 and VP 3.  
     
     
         42 . The method of  claim 38 , wherein an additional nucleic acid sequence which impairs packaging of the helper construct into an AAV capsid is incorporated between the nucleic acid sequence coding for Rep and a nucleic acid sequence within the helper construct that encodes AAV Cap protein.  
     
     
         43 . The method of  claim 42 , wherein the additional nucleic acid sequence is at least approximately 300 nucleotides in length.  
     
     
         44 . The method of  claim 42 , wherein the additional nucleic acid sequence is at least approximately 500 nucleotides in length.  
     
     
         45 . The method of  claim 42 , wherein the additional nucleic acid sequence is at least approximately 600 to approximately 700 nucleotides in length.  
     
     
         46 . The method of  claim 38 , wherein the helper construct does not comprise any nucleic acid sequences which are homologous to the vector construct and allow homologous recombination.  
     
     
         47 . The method of  claim 38 , wherein the AAV is AAV-2 and bases 4461-4497 of AAV-2 have been deleted in the helper construct.  
     
     
         48 . The method of  claim 38 , wherein in the helper construct expression of the Rep proteins and/or Cap proteins is controlled by the AAV promoters or by a heterologous promoter and/or enhancer.  
     
     
         49 . The method of  claim 38 , wherein the helper construct is replication-deficient.  
     
     
         50 . The method of  claim 38 , wherein the helper construct is self-replicating.  
     
     
         51 . The method of  claim 50 , wherein the helper construct comprises both SV40ori nucleic acid sequences and nucleic acid sequences coding for the SV40 T antigen  
     
     
         52 . The method of  claim 50 , wherein the helper construct comprises both EBV oriP nucleic acid sequences and EBV EBNA sequences.  
     
     
         53 . The method of  claim 29 , wherein in the helper construct coding for at least one Rep protein the rep gene(s) is/are controlled by their natural promoters p5, p19 and p40.  
     
     
         54 . The method of  claim 53 , wherein the Rep proteins are Rep 68 and Rep 52.  
     
     
         55 . The method of  claim 53 , wherein the Rep proteins are Rep 68, Rep 52 and Rep 40.  
     
     
         56 . The method of  claim 29 , wherein the ITR sequences in the vector construct are present in flop orientation.  
     
     
         57 . The method of  claim 56 , wherein in the vector construct expression of the heterologous nucleic acid(s) is controlled by a promoter and/or enhancer heterologous to AAV.  
     
     
         58 . The method of  claim 57 , wherein in the vector construct the heterologous promoter and/or enhancer is orientated toward the 3′ located ITR sequence.  
     
     
         59 . The method of  claim 56 , wherein the construct is replication-deficient.  
     
     
         60 . The method of  claim 56 , wherein the construct is self-replicating.  
     
     
         61 . The method of  claim 60 , wherein the construct comprises both SV40ori nucleic acid sequences and nucleic acid sequences coding for the SV40 T antigen.  
     
     
         62 . The method of  claim 60 , wherein the construct comprises both EBV oriP nucleic acid sequences and EBV EBNA sequences.  
     
     
         63 . The method of  claim 29 , wherein one or more heterologous nucleic acid sequence(s) is/are coding for GM-CSF and a B7 molecule.  
     
     
         64 . The method of  claim 63 , wherein the B7 molecule is B7.2.  
     
     
         65 . A method for transducing a tumor cell, the method comprising using a rAAV produced according to the method as claimed in  claim 29 .  
     
     
         66 . The method of  claim 65 , wherein the tumor cell is treated with radiation.