Granted Patent B2

US 12,410,443 · App. 17/427,551 · Granted Sep 9, 2025

Inventors: Ryan Cawood (Oxford, GB); Tom Payne (Oxford, GB); Alissa Bray (Oxford, GB)

Assignee: Oxford Genetics

C12N15/86C12N2750/14143C12N2750/14152C12N2830/002C12N2840/203

Abstract

Claims (104)

1. A nucleic acid molecule comprising:

(i) a minimal promoter comprising 1, 2, 3, 4, 5 or 6 cumate operator (CuO) sites,

(ii) a cap gene, and

(iii) a rep gene,

in the above 5′-3′ order, wherein the cap gene and the rep gene are both operably-associated with the promoter.

2. The nucleic acid molecule as claimed in claim 1 , wherein the promoter is a minimal pol II promoter.

3. The nucleic acid molecule as claimed in claim 1 , wherein the promoter comprises 4, 5 or 6 CuO sites.

4. The nucleic acid molecule as claimed in claim 1 , wherein the CuO site(s) in the promoter are placed upstream of both the TATA box and the +1 site.

5. The nucleic acid molecule as claimed in claim 1 , wherein the rep gene encodes Rep78 and Rep68, but not Rep 52 or Rep 40.

6. The nucleic acid molecule as claimed in claim 1 , wherein the rep gene is operably-associated with an Internal Ribosome Entry Site (IRES) which is placed between the cap and rep genes.

7. The nucleic acid molecule as claimed in claim 1 , wherein the rep gene encodes Rep78 and Rep68.

8. The nucleic acid molecule as claimed in claim 1 , wherein the promoter comprises 6 CuO sites.

9. A vector or plasmid comprising the nucleic acid molecule of claim 1 .

10. A kit comprising:

(A) a nucleic acid molecule as claimed in claim 1 ;

and optionally one or more of the following:

(B) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter or a minimal promoter comprising one or more cumate operator sites, operably-associated with

(ii) a gene encoding a rc-CymR-TAD polypeptide;

(C) a nucleic acid molecule, vector or plasmid comprising

(i) a promoter or a constitutive promoter, operably-associated with

(ii) a gene encoding CymR;

(D) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter or a constitutive promoter, operably-associated with

(ii) a gene encoding a CymR-TAD polypeptide;

(E) a nucleic acid molecule, vector or plasmid comprising:

(i) one or more promoters, which may or may not comprise one or more cumate operator sites, operably-associated with

(ii) one or more adenoviral genes which are competent to support AAV production;

(F) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter, operably-associated with

(ii) a transgene,

wherein the promoter and transgene are flanked by ITRs.

11. A cell line, comprising:

(A) a nucleic acid molecule as claimed in claim 1 ;

and optionally one or more of the following:

(B) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter or a minimal promoter comprising one or more cumate operator sites, operably-associated with

(ii) a gene encoding a rc-CymR-TAD polypeptide;

(C) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter or a constitutive promoter, operably-associated with

(ii) a gene encoding CymR;

(D) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter or a constitutive promoter, operably-associated with

(ii) a gene encoding a CymR-TAD polypeptide;

(E) a nucleic acid molecule, vector or plasmid comprising:

(i) one or more promoters, which may or may not comprise one or more cumate operator sites, operably-associated with

(ii) one or more adenoviral genes which are competent to support AAV production;

(F) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter, operably-associated with

(ii) a transgene,

wherein the promoter and transgene are flanked by ITRs.

12. The cell line as claimed in claim 11 , wherein the cell line is a packaging cell line or a producer cell line.

13. The cell line as claimed in claim 11 , wherein the each of (A), (B), (C), (E), and (F), when present, are integrated into the genome of the cell line, and wherein the specified genes are expressed or expressible therefrom.

14. The cell line as claimed in claim 11 , wherein the cells of the cell line are HEK293, HEK293T, HEK293A, PerC6 or 911 cells.

15. A process for producing an AAV cell line, the process comprising the steps of integrating into the genome of the cells of the cell line:

(A) a nucleic acid molecule as claimed in claim 1 ,

thereby producing a cell line that inducibly expresses viral cap and rep genes;

and optionally one or more of the following:

(B) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter or a minimal promoter comprising one or more cumate operator sites, operably-associated with

(ii) a gene encoding a rc-CymR-TAD polypeptide;

(C) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter or a constitutive promoter, operably-associated with

(ii) a gene encoding a CymR;

(D) a nucleic acid molecule, vector or plasmid comprising:

(i) one or more promoters, which may or may not comprise one or more cumate operator sites, operably-associated with

(ii) one or more adenoviral genes which are competent to support AAV production.

16. A method of inducing transcription of cap and rep genes in a cell, the method comprising:

inducing the transcription of cap and rep genes in a cell which comprises in its genome:

(A) a nucleic acid molecule as claimed in claim 1 ,

wherein the inducing is by the presence, in the cell, of a CymR-TAD polypeptide or by the expression or transient expression, in the cell, of a nucleic acid molecule which encodes a CymR-TAD polypeptide.

17. The method as claimed in claim 16 , wherein the method additionally comprises the step of introducing into the cell:

(B) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter or a constitutive promoter, operably-associated with

(ii) a nucleotide sequence encoding a CymR-TAD;

and/or

(C) a nucleic acid molecule, vector or plasmid comprising:

(i) one or more promoters, which may or may not comprise one or more cumate operator sites, operably-associated with

(ii) one or more adenoviral genes which are competent to support AAV production;

and/or

(D) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter, operably-associated with

(ii) a transgene,

wherein the promoter and transgene are flanked by ITRs.

18. A method of inducing transcription of cap and rep genes in a cell, the method comprising:

inducing the transcription of cap and rep genes in a cell which comprises:

(A) a nucleic acid molecule as claimed in claim 1 ;

(B) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter comprising one or more cumate operator sites, operably-associated with

(ii) a gene encoding a rc-CymR-TAD polypeptide;

and

(C) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter, operably-associated with

(ii) a gene encoding a CymR;

and optionally

(D) a nucleic acid molecule, vector or plasmid comprising:

(i) one or more promoters, which may or may not comprise one or more cumate operator sites, operably-associated with

(ii) one or more adenoviral genes which are competent to support AAV production;

and/or

(E) a nucleic acid molecule, vector or plasmid comprising:

(i) a promoter, operably-associated with

(ii) a transgene, wherein the promoter and transgene are flanked by ITRs,

wherein the inducing is by the presence of or introduction into the cell of cumate.

19. The method of inducing transcription of cap and rep genes in a cell as claimed in claim 18 , wherein (A), (B), (C), (D) and (E), when present, are all integrated into the genome of the cell.

Assignments (1)

Priority Claims (1)

Continuity (1)

References Cited (98)