AAV vectors produced by insect cells comprising Rep52 and Rep78 coding sequences with differential codon biases

Patent metadata

Field	Value
Publication number	US-12486496-B2
Application number	US-202217722275-A
Country	US
Kind code	B2
Filing date	Apr 15, 2022
Priority date	Jul 26, 2007
Publication date	Dec 2, 2025
Grant date	Dec 2, 2025

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.

First claim

What is claimed is:

1 . A recombinant adeno-associated virus (rAAV) virion obtainable by culturing an insect cell under conditions such that rAAV virions are produced and recovering the rAAV virions, wherein the insect cell comprises a baculoviral 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; and
(ii) a second nucleotide sequence encoding a second amino acid sequence of an AAV Rep78 protein;
(iii) a third nucleotide sequence comprising two AAV inverted terminal repeat (ITR) sequences and a nucleotide sequence encoding a gene product located between the two AAV ITR sequences, wherein the gene product, when expressed in a target cell, provides ablation of an undesired activity or complementation of a genetic defect; 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.

2 . The rAAV virion according to claim 1 wherein the first nucleotide sequence is selected from the group consisting of
(a) a sequence of at least 85% identity with SEQ ID NO:10; and
(b) nucleotide sequence complementary to the full length sequence of (a).

3 . The rAAV virion according to claim 1 , wherein:
(a) the first nucleotide sequence is operably linked to a polyhedron (polH) promoter and the second nucleotide sequence is operably linked to a p10 or deltaIE-1 promoter; or (b) the first nucleotide sequence is operably linked to a p10 promoter and the second nucleotide sequence is operably linked to a polyhedron (polH) or deltaIE-1 promoter.

4 . The rAAV virion according to claim 1 , wherein the first nucleotide sequence has the sequence SEQ ID NO:10.

5 . The rAAV virion according to claim 4 , wherein the second nucleotide sequence is operably linked to a deltaIE-1 promoter.

6 . The rAAV virion according to claim 1 , 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.

7 . The rAAV virion cell according to claim 6 , wherein all ATG codons that occur between the translation start codons of the AAV Rep78 protein and the translation start codon of the AAV Rep52 protein in the second nucleotide sequence are mutated.

8 . The rAAV virion according to claim 7 , wherein the first nucleotide sequence has the sequence of SEQ ID NO:10.

9 . The rAAV virion according to claim 7 , wherein the second nucleotide sequence is operably linked to a p10 or to a polH promoter.

10 . The rAAV virion according to claim 9 , wherein the second nucleotide sequence is operably linked to a p10 promoter.

11 . The rAAV virion according to claim 10 , wherein the Rep proteins encoded by the first and second nucleotide sequences are of the same AAV serotype.

12 . The rAAV virion according to claim 9 , wherein the Rep proteins encoded by the first and second nucleotide sequences are of the same AAV serotype.

13 . The rAAV virion according to claim 1 , wherein the gene product complements a genetic defect causing a deficiency in an enzymatic activity.

14 . The rAAV virion according to claim 13 , wherein the enzymatic activity is Factor IX activity.

15 . The rAAV virion according to claim 1 , wherein recovering the rAAV virions comprises affinity-purifying the virion using an anti-AAV antibody.

16 . The rAAV virion according to claim 15 , wherein the anti-AAV antibody is immobilized.

17 . The rAAV virion according to claim 15 , wherein the anti-AAV antibody is a monoclonal antibody.

18 . The rAAV virion according to claim 17 , wherein the monoclonal anti-AAV antibody is single chain camelid antibody or a fragment thereof.

19 . The rAAV virion according to claim 17 , wherein the single chain camelid antibody or fragment is a camel or llama antibody.

Assignees

• Uniqure Ip Bv

Inventors

• Bakker Andrew Christian
• Hermens Wilhelmus Theodorus Johannes Maria Christiaan

Classifications

• C12N2750/14121

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N2710/14044

  Chimeric viral vector comprising heterologous viral elements for production of another viral vector · CPC title

• C12N15/86

  Viral vectors · CPC title

• C12N2800/50

  Vectors for producing vectors · CPC title

• C12N2800/22

  Vectors comprising a coding region that has been codon optimised for expression in a respective host · CPC title