Further improved AAV vectors produced in insect cells

Patent metadata

Field	Value
Publication number	US-12448628-B2
Application number	US-202017090807-A
Country	US
Kind code	B2
Filing date	Nov 5, 2020
Priority date	Mar 10, 2014
Publication date	Oct 21, 2025
Grant date	Oct 21, 2025

Abstract

The present invention relates to the production of adeno-associated viral vectors in insect cells. The insect cells therefore comprise a first nucleotide sequence encoding the adeno-associated virus (AAV) capsid proteins, whereby the initiation codon for translation of the AAV VP1 capsid protein is a non-ATG, suboptimal initiation codon and wherein the coding sequence for one or more amino acid residues have been inserted between the suboptimal translation initiation codon and the codon encoding the amino acid residue that corresponds to the amino acid residue at position 2 of the wild type capsid amino acid sequence of which the first amino acid residue is alanine, glycine, valine, aspartic acid or glutamic acid. The insect cell further comprises a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence; a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell; and, a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell. The invention further relates to adeno-associated viral vectors with an altered ratio of the viral capsid proteins.

First claim

The invention claimed is:

1. A method for producing an AAV in an insect cell, comprising:
(a) culturing an insect cell comprising a nucleic acid construct comprising a nucleic acid molecule having a nucleotide sequence comprising an open reading frame, wherein the reading frame in 5′ to 3′ order consists of:
(i) a first codon, which is a suboptimal translation initiation codon selected from the group consisting of CTG and ACG;
(ii) a second codon encoding an amino acid residue selected from the group consisting of alanine, glycine, valine, aspartic acid and glutamic acid; and
(iii) a sequence encoding wild-type adeno-associated virus (AAV) serotype 5 capsid proteins, wherein the sequence lacks only the wild-type VP1 translation initiation codon,

(b) recovering of the AAV.

2. The method according to claim 1 , wherein the wildtype AAV serotype 5 capsid proteins consist of amino acid sequence of SEQ ID NO: 22.

3. The method according to claim 1 , wherein the second codon encodes alanine.

4. The method according to claim 1 , wherein the second codon is selected from the group consisting of GCT, GCC, GCA, GCG and GGU.

5. The method according to claim 3 , wherein the second codon is GCT.

6. The method according to claim 1 , wherein the nucleotide sequence of the reading frame is operably linked to a promoter selected from the group consisting of polyhedrin promoter, plO promoter, 4xHsp27 EcRE+minimal Hsp70 promoter, deltaE1 promoter, and E1 promoter.

7. The method according to according to claim 6 , wherein the polyhedrin promoter has SEQ ID No: 54.

8. The method according to claim 1 , wherein the construct is an insect-compatible vector.

9. The method according to claim 8 , wherein the insect-compatible vector is a baculoviral vector.

10. The method according to claim 1 , wherein the nucleic acid molecule comprises an open reading frame selected from the group consisting of: SEQ ID NO: 51, 69, 42, 47, 48 and 50.

11. The method according to claim 10 , wherein the open reading frame comprises SEQ ID NO 51.

12. The method according to claim 1 , wherein the insect cell further comprises:
(a) a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence.

13. The method according to claim 12 , wherein the nucleic acid construct comprises a third nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell and further comprises a fourth nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell and
wherein the insect cell further comprises a second nucleic acid construct comprising a nucleotide sequence comprising at least one AAV ITR nucleotide sequence.

14. The method according to claim 13 , wherein the second nucleic acid construct is an insect cell-compatible vector.

15. The method according to claim 14 , wherein the insect cell-compatible vector is a baculoviral vector.

16. The method according to claim 12 , wherein the second nucleotide sequence further comprises at least one nucleotide sequence encoding a gene product of interest for expression in a mammalian cell and wherein the at least one nucleotide sequence encoding a gene product of interest becomes incorporated into the genome of an AAV serotype 5 produced in the insect cell.

17. The method according to claim 16 , wherein the second nucleotide sequence comprises two AAV ITR nucleotide sequences and wherein the at least one nucleotide sequence encoding a gene product of interest is located between the two AAV ITR nucleotide sequences.

18. The method according to claim 12 , wherein the insect cell comprises the first nucleotide sequence, the second nucleotide sequence, and further comprises a third nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell, and a fourth nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell.

19. The method according to claim 12 , wherein the insect cell further comprises a third nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell.

20. The method according to claim 18 , wherein the first thru fourth nucleotide sequences are stably integrated in the genome of the insect cell.

Assignees

• Uniqure Ip Bv

Inventors

• Lubelski Jacek
• Bosma Sebastiaan
• Petry Harald Peter Albert
• Hermens Wilhelmus Theodorus Johannes Maria Christ

Classifications

• C12N2830/008

  cell type or tissue specific enhancer/promoter combination · CPC title

• C12N2750/14122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N2750/14121

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

• C12N2710/14043

  viral genome or elements thereof as genetic vectore · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title