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US 11,306,291 B2Granted
AAV vectors produced by insect cells comprising REP52 and REP78 coding sequences with differential codon biases
AssigneeuniQure IP B.V.
InventorsAndrew Christian Bakker, Wilhelmus Theodorus Johannes Maria Christiaan Hermens
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Abstract
The present invention relates to production of proteins in insect cells whereby repeated coding sequences are used in baculoviral vectors. In particular the invention relates to the production of parvoviral vectors that may be used in gene therapy and to improvements in expression of the viral rep proteins that increase the productivity of parvoviral vectors.
Claims19
A first baculovirus vector comprising:
- (i) a first nucleotide sequence encoding a first amino acid sequence of an AAV Rep52 protein selected from the group consisting of:
- (a) a sequence of at least 85% identity with SEQ ID NO:
- 10;
- (b) a nucleotide sequence complementary to the full length sequence of (a);
- and
- (c) a nucleotide sequence that differs from the sequence of (a) due to degeneracy of the genetic code;
- (ii) a second nucleotide sequence encoding a second amino acid sequence of an AAV Rep78 protein;
- and
- a further one or more baculoviral vectors comprising:
- (iii) a third nucleotide sequence comprising two AAV inverted terminal repeat (ITR) sequences and a nucleotide sequence encoding a gene product of interest located between the two AAV ITR sequences;
- and
- (iv) a fourth nucleotide sequence comprising AAV capsid protein-coding sequences operably linked to expression control sequences for expression in an insect cell;
- wherein the first and the second amino acid sequences share at least 90% sequence identity in a region from the second amino acid residue to the C-terminal residue of the AAV Rep52 protein, and
- wherein a portion of the first nucleotide sequence and a portion of the second nucleotide sequence that encode the region from the second amino acid residue to the C-terminal residue of the AAV Rep52 protein each comprise one or more contiguous stretches of at least 300 nucleotides that are less than 90% identical.
11 more elements
A first baculovirus vector comprising:
- (i) a first nucleotide sequence encoding a first amino acid sequence of an AAV Rep52 protein comprising SEQ ID NO:
- 10;
- (ii) a second nucleotide sequence encoding a second amino acid sequence of an AAV Rep78 protein;
- and
- a further one or more baculoviral vectors comprising:
- (iii) a third nucleotide sequence comprising two AAV inverted terminal repeat (ITR) sequences and a nucleotide sequence encoding a gene product of interest located between the two AAV ITR sequences;
- and
- (iv) a fourth nucleotide sequence comprising AAV capsid protein-coding sequences operably linked to expression control sequences for expression in an insect cell;
- wherein the Rep proteins encoded by the first and second nucleotide sequences are of the same AAV serotype;
- and
- wherein the translation initiation codon before the second nucleotide sequence encoding the AAV Rep78 protein is a suboptimal initiation codon selected from the group consisting of ACG, CTG, GTG and TTG.
8 more elements
Dependencies are inferred from the claim text.