VECTORS WITH MODIFIED INITIATION CODON FOR THE TRANSLATION OF AAV-REP78 USEFUL FOR PRODUCTION OF AAV

• US 20200131535A1
• Filed: 01/10/2020
• Published: 04/30/2020
• Est. Priority Date: 06/21/2006
• Status: Active Grant

• Current Assignee: Uniqure Ip B.V. (uniQure N.V.)
• Original Assignee: Uniqure Ip B.V. (uniQure N.V.)
• Inventors: Hermens, Wilhelmus Theodorus Johannes Maria Christiaan, Haast, Saskia Jacoba Petronella, Biesmans, Dennis Johan, Bakker, Andrew Christian
• Granted Patent: US 10,865,423 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/14043 viral genome or elements thereof as genetic vectore
• CPC Class Codes: C12N 2710/14044 Chimeric viral vector comprising heterologous viral elements for production of another viral vector
• CPC Class Codes: C12N 2710/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/14144 Chimeric viral vector comprising heterologous viral elements for production of another viral vector
• CPC Class Codes: C12N 2710/14152 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 2750/14121 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2750/14122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14151 Methods of production or purification of viral material
• CPC Class Codes: C12N 2750/14152 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 2800/105 for insects
• CPC Class Codes: C12N 2800/50 Vectors for producing vectors
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

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.

Claims

1. A method of producing a recombinant parvoviral virion in an insect cell comprising, transfecting an insect cell with a nucleotide sequence encoding parvoviral Rep proteins Rep78 and Rep52, the nucleotide sequence comprising a Rep78 coding sequence and a Rep52 coding sequence, the Rep78 coding sequence and the Rep52 coding sequence being operably linked to expression control sequences for expression of both a Rep78 protein and a Rep52 protein in an insect cell;

wherein the Rep52 coding sequence is comprised within the Rep78 coding sequence, and wherein the nucleotide sequence is stably maintained within the insect cell over at least 5 passages.

2. The method of claim 1, wherein the Rep78 and Rep52 are expressed over at least 5 passages of the insect cell.

3. The method of claim 1, wherein Rep78 and Rep52 are produced in a molar ratio in the range of 1:

10 to 10;

1.

4. The method of claim 1, wherein the nucleotide sequence is comprised within a nucleic acid construct.

5. The method of claim 4, wherein the nucleic acid construct is comprised within a baculovirus vector.

6. The method of claim 1, wherein the insect cell is selected from the group consisting of Se301, Seizd2109, Seucr1, sf9, SP900+, Sf21, Bti-Tn-5b1-4, Mg-1, Tn368, Hzaml, Ha2302, Hz2e5, High five and Express+. The method of claim 6, wherein the insect cell is sf9.

8. The method of claim 1, wherein the nucleotide sequence is stably maintained within the insect cell over at least 8 passages.

9. A method of producing a recombinant parvoviral virion in an insect cell comprising, transfecting an insect cell with a nucleic acid construct encoding parvoviral Rep proteins Rep78 and Rep52, the nucleic acid construct comprising:

(a) a bicistronic nucleic acid encoding a parvovirus Rep78 protein and a parvovirus Rep52 protein, wherein the nucleic acid sequence encoding the Rep78 protein overlaps the nucleic acid sequence encoding the Rep52 protein, such that the nucleic acid sequence encoding the parvoviral Rep52 protein is not repeated in the bicistronic nucleic acid;

(b) a first expression control sequence that comprises an initiation codon and is active in an insect cell operably linked to the sequence encoding Rep78 protein;

(c) a second expression control sequence that is active in an insect cell operably linked to the sequence encoding Rep52 protein;

wherein both the parvoviral Rep78 protein and the parvoviral Rep52 protein are produced over at least 5 passages of the insect cell when the nucleic acid construct is expressed by the insect cell.

10. The method of claim 9, wherein the nucleic acid construct is stably maintained within the insect cell over at least 5 passages.

11. The method of claim 9, wherein the parvoviral Rep78 protein and the parvoviral Rep52 protein are produced in a molar ratio in the range of 1:

10 to 10;

1.

12. The method of claim 9, wherein the nucleic acid construct is comprised within a baculovirus vector.

13. The method of claim 9, wherein the insect cell is selected from the group consisting of Se301, Seizd2109, Seucr1, sf9, Sf900+, Sf21, Bti-Tn-5b1-4, Mg-1, Tn368, Hzam 1, Ha2302, Hz2e5, High five and Express+.

14. The method of claim 9, wherein the insect cell is sf9.

15. The method of claim 9, wherein the nucleotide acid construct is stably maintained within the insect cell over at least 8 passages.