Vectors with modified initiation codon for the translation of aav-rep78 useful for production of aav

Abstract

The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second nucleotide sequence comprising at least one parvoviral (AA V) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.

1 . A nucleic acid construct comprising a first nucleotide sequence that comprises a single open reading frame (ORF) encoding parvoviral Rep proteins Rep78 and Rep52, wherein the ORF comprises a non-ATG initiation codon selected from the group consisting of ACG, TTG, CTG and GTG. 
     
     
         2 . The nucleic acid construct of  claim 1 , wherein the non-ATG initiation codon is ACG or CTG. 
     
     
         3 . The nucleic acid construct of  claim 1 , wherein the parvoviral Rep proteins are AAV Rep proteins. 
     
     
         4 . The nucleic acid construct of  claim 1 , wherein one or more false translation initiation sites between the Rep78 initiation site and the Rep52 initiation site have been eliminated. 
     
     
         5 . The nucleic acid construct of  claim 4 , wherein all false translation initiation sites between the Rep78 initiation site and the Rep52 initiation site have been eliminated. 
     
     
         6 . The nucleic acid construct of  claim 1 , wherein the single ORF encoding the parvoviral Rep proteins is operably linked to an expression control sequence for expression in insect cells. 
     
     
         7 . The nucleic acid construct of  claim 6 , wherein the expression control sequence comprises a polyhedron promoter. 
     
     
         8 . The nucleic acid construct according to  claim 1 , wherein the nucleic acid construct is a recombinant viral vector. 
     
     
         9 . The nucleic acid construct according to  claim 8 , wherein the vector is a baculoviral vector. 
     
     
         10 . An insect cell comprising a first nucleotide sequence that comprises a single open reading frame (ORF) encoding parvoviral Rep proteins Rep78 and Rep52, wherein the ORF comprises a non-ATG initiation codon selected from the group consisting of ACG, TTG, CTG and GTG. 
     
     
         11 . The insect cell of  claim 10 , wherein the non-ATG initiation codon is ACG or CTG. 
     
     
         12 . The insect cell of  claim 10 , wherein the parvoviral Rep proteins are AAV Rep proteins. 
     
     
         13 . The insect cell of  claim 10 , wherein one or more false translation initiation sites between the Rep78 initiation site and the Rep52 initiation site have been eliminated. 
     
     
         14 . The insect cell of  claim 13 , wherein all false translation initiation sites between the Rep78 initiation site and the Rep52 initiation site have been eliminated. 
     
     
         15 . The insect cell of  claim 10 , wherein the single ORF encoding the parvoviral Rep proteins is operably linked to an expression control sequence for expression in insect cells. 
     
     
         16 . The insect cell of  claim 15 , wherein the expression control sequence comprises a polyhedron promoter. 
     
     
         17 . The insect cell of  claim 15 , wherein the single ORF encoding the parvoviral Rep proteins is operably linked to an expression control sequence for expression in insect cells is part of a first nucleic acid construct. 
     
     
         18 . The insect cell of  claim 17 , wherein the first nucleic acid construct is a baculoviral vector. 
     
     
         19 . The insect cell of  claim 17 , further comprising:
 (i) a second nucleotide sequence comprising at least one parvoviral inverted terminal repeat (ITR) sequence; and,   (ii) a third nucleotide sequence comprising parvoviral capsid protein-coding sequences operably linked to an expression control sequence for expression in the insect cell, wherein the capsid protein-coding sequences encodes parvoviral VP1, VP2, and VP3 capsid proteins, and wherein the initiation codon for translation of the VP1 capsid protein is ACG, TTG, CTG, or GTG.   
     
     
         20 . The insect cell of  claim 19 , wherein the first recombinant viral vector further comprises the third nucleotide sequence. 
     
     
         21 . The insect cell of  claim 19 , wherein the expression control sequence operable linked to the parvoviral capsid protein-coding sequences comprises a p10 promoter. 
     
     
         22 . The insect cell of  claim 19 , wherein the second nucleotide sequence comprising at least one parvoviral inverted terminal repeat (ITR) sequence is part of a second nucleic acid construct. 
     
     
         23 . The insect cell of  claim 19 , wherein the second nucleotide sequence further comprises a nucleotide sequence encoding a gene product of interest wherein the nucleotide sequence encoding the gene product of interest becomes incorporated into the genome of the parvoviral vector produced in the cell. 
     
     
         24 . The insect cell of  claim 23 , wherein the second nucleotide sequence comprises two parvoviral ITR sequences which flank the nucleotide sequence encoding the gene product of interest. 
     
     
         25 . The insect cell of  claim 22 , wherein the first and second nucleic acid constructs are insect cell-compatible vectors. 
     
     
         26 . The insect cell of  claim 25 , wherein the insect cell-compatible vectors are baculoviral vectors. 
     
     
         27 . A recombinant AAV virion produced by:
 (i) culturing the insect cell of  claim 10  under conditions that permit production of the recombinant AAV virion; and   (ii) recovering the recombinant AAV virion.   
     
     
         28 . A recombinant AAV virion produced by:
 (i) culturing the insect cell of  claim 19  under conditions that permit production of the recombinant AAV virion; and   (ii) recovering the recombinant AAV virion.   
     
     
         29 . A recombinant AAV virion produced by:
 (i) culturing the insect cell of  claim 20  under conditions that permit production of the recombinant AAV virion; and   (ii) recovering the recombinant AAV virion.   
     
     
         30 . A method for producing a recombinant parvoviral virion in an insect cell, comprising:
 (a) culturing an insect cell comprising a first nucleotide sequence comprising a single open reading frame (ORF) encoding parvoviral Rep proteins Rep78 and Rep52 with a non-ATG initiation codon selected from the group consisting of ACG, TTG, CTG and GTG, under conditions such that the recombinant parvoviral virion is produced; and   (b) recovering the recombinant parvoviral virion.   
     
     
         31 . The method of  claim 30 , wherein the non-ATG initiation codon is ACG or CTG. 
     
     
         32 . The method of  claim 30 , wherein the insect cell does not comprise a nucleotide sequence encoding parvoviral Rep proteins other than the first nucleotide sequence. 
     
     
         33 . The method of  claim 30 , wherein the parvoviral Rep proteins are adeno-associated virus (AAV) Rep proteins. 
     
     
         34 . The method of  claim 30 , wherein the single ORF encoding the parvoviral Rep proteins is operably linked to an expression control sequence for expression in the insect cells. 
     
     
         35 . The method of  claim 34 , wherein the expression control sequence comprises a polyhedron promoter. 
     
     
         36 . The method of  claim 30 , wherein the first nucleotide sequence comprising the single ORF encoding the parvoviral Rep proteins is part of a first nucleic acid construct. 
     
     
         37 . The method of  claim 36 , wherein the insect cell further comprises:
 (i) a second nucleotide sequence comprising at least one parvoviral inverted terminal repeat (ITR) sequence; and,   (ii) a third nucleotide sequence comprising parvoviral capsid protein-coding sequences operably linked to an expression control sequence for expression in the insect cell, wherein the capsid protein-coding sequences encodes parvoviral VP1, VP2, and VP3 capsid proteins, and wherein the initiation codon for translation of the VP1 capsid protein is ACG, TTG, CTG, or GTG.   
     
     
         38 . The method of  claim 37 , wherein the first nucleic acid construct further comprises the third nucleotide sequence. 
     
     
         39 . The method of  claim 38 , wherein the expression control sequence operable linked to the parvoviral capsid protein-coding sequences comprises a p10 promoter. 
     
     
         40 . The method of  claim 37 , wherein the second nucleotide sequence comprising at least one parvoviral inverted terminal repeat (ITR) sequence is part of a second nucleic acid construct. 
     
     
         41 . The method of  claim 37 , wherein the second nucleotide sequence further comprises a nucleotide sequence encoding a gene product of interest wherein the nucleotide sequence encoding the gene product of interest becomes incorporated into the genome of the parvoviral vector produced in the cell. 
     
     
         42 . The method of  claim 41 , wherein the second nucleotide sequence comprises two parvoviral ITR sequences which flank the nucleotide sequence encoding the gene product of interest. 
     
     
         43 . The method of  claim 40 , wherein the first and second nucleic acid constructs are insect cell-compatible vectors. 
     
     
         44 . The method of  claim 43 , wherein the insect cell-compatible vectors are baculoviral vectors.