Proviral plasmids and production of recombinant adeno-associated virus

Patent metadata

Field	Value
Publication number	US-9896665-B2
Application number	US-201514979786-A
Country	US
Kind code	B2
Filing date	Dec 28, 2015
Priority date	May 16, 2011
Publication date	Feb 20, 2018
Grant date	Feb 20, 2018

Abstract

Proviral plasmids contain a modular gene expression cassette with one or a combination of (i) a wildtype 5′ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of the ITR; (ii) a promoter flanked by unique restriction sites that permit ready removal or replacement of the entire promoter sequence; (iii) a polylinker sequence that permits insertion of a gene coding sequence without modification thereof, wherein the gene is operatively linked to, and under the regulatory control of, the aforementioned promoter; (iv) a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that permit ready removal or replacement of the polyA sequence; and (v) a wildtype 3′ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of the 3′ ITR. These plasmids enable rapid manipulation of the components of the cassette, such as rapid mutation and/or replacement of any component, and thereby increase the efficiency of recombinant viral vector, such as rAAV, production.

First claim

The invention claimed is:

1. A proviral plasmid comprising a modular recombinant AAV genome comprising:
(a) a 5′ AAV ITR sequence, the ITR flanked upstream by restriction site 1 and downstream by restriction site 2;
(b) a promoter flanked upstream by restriction site 2 and downstream by restriction site 3;
(c) a polylinker sequence comprising restriction site 3, restriction site 4 and restriction site 5, that permits insertion of a heterologous nucleic acid sequence between any two of the restriction sites 3, 4 and 5, without modification thereof, wherein the gene is operatively linked to, and under the regulatory control of, said promoter;
(d) a polyadenylation sequence flanked upstream by restriction site 4 or 5 and downstream by restriction site 6; and
(e) a 3′ AAV ITR sequence flanked upstream by restriction site 6 and downstream by restriction site 7;
wherein each said restriction site 1 through 7 occurs only once in the plasmid and is cleaved by a different enzyme that cannot cleave another restriction site in the plasmid, and wherein said restriction sites 1 through 7 are positioned to permit ready removal, replacement or substitution of one or more of element (a), (b), (c), (d) and (e) or the entire AAV genome (a) through (e) from the plasmid.

2. The plasmid according to claim 1 , wherein the polyadenylation sequence is a bovine growth hormone polyadenylation sequence.

3. The plasmid according to claim 1 , further comprising a plasmid backbone comprising the elements necessary for replication in bacterial cells, and further comprising a kanamycin resistance gene (Kan R ).

4. The plasmid according to claim 3 , further comprising 5′ and 3′ transcriptional terminator/insulator sequences that isolate transcription in the backbone from transcription in the gene cassette.

5. The plasmid according to claim 3 , wherein the plasmid backbone further comprises a non-coding stuffer sequence that increases the backbone length and prevents reverse packaging of non-functional AAV genomes.

6. A proviral plasmid comprising:
(a) a modular recombinant AAV genome comprising in operative association:
(i) a 5′ AAV ITR sequence flanked upstream by restriction site 1 and downstream by restriction site 2 that permit ready removal or replacement of said ITR;
(ii) a hybrid promoter comprising a 49 nucleic acid cytomegalovirus sequence upstream of a cytomegalovirus (CMV)-chicken beta actin sequence, the hybrid promoter flanked upstream by restriction site 2 and downstream by restriction site 4 that permit ready removal or replacement of the entire promoter sequence, and the upstream sequence flanked by restriction site 2 and downstream by restriction site 3 that permit ready removal or replacement of only the upstream CMV sequence, from the hybrid promoter sequence;
(iii) a multi-cloning polylinker sequence comprising restriction site 4 and restriction site 5 that permits insertion of a gene coding sequence without modification thereof, wherein the gene is operatively linked to, and under the regulatory control of, promoter (ii);
(iv) a bovine growth hormone polyadenylation sequence flanked upstream by restriction site 4 and downstream by restriction site 6 that permit ready removal or replacement of said polyA sequence; and
(v) a wildtype 3′ AAV2 ITR sequence flanked upstream by restriction site 6 and downstream by restriction site 7 that permit ready removal or replacement of the 3′ ITR; and

(b) a plasmid backbone comprising the elements necessary for replication in bacterial cells, and further comprising a kanamycin resistance gene, said plasmid backbone flanked by transcriptional terminator/insulator sequences that isolate transcription in the backbone from transcription in the gene cassette.

7. The plasmid according to claim 6 , wherein said plasmid backbone (b) further comprises a non-coding lambda phage 5.1 kb stuffer sequence to increase backbone length and prevent reverse packaging of non-functional AAV genomes.

8. The plasmid according to claim 1 , further comprising in the recombinant AAV genome a gene encoding sequence of up to 2.6 kb in length.

9. The plasmid according to claim 1 , further comprising as the gene encoding sequence a nucleotide sequence encoding an open reading frame of human rod derived cone viability factor exon 1.

10. The plasmid according to claim 1 , wherein the gene encoding sequence encodes a detectable reporter gene.

11. The plasmid according to claim 10 , wherein the detectable reporter gene is selected from the group consisting of green fluorescent protein, red fluorescent protein, and beta-galactosidase.

12. A method of generating a rAAV virus comprising:
(a) obtaining a plasmid of claim 1 ;
(b) modifying the plasmid by removing or replacing at least one of an ITR sequence, the promoter or the upstream CMV sequence of the promoter, the BGH polyA sequence, or inserting a gene of interest into the polylinker, of said plasmid using the appropriate restriction enzymes;
(c) culturing a packaging cell carrying the modified in the presence of sufficient viral sequences to permit packaging of the gene expression cassette viral genome into an infectious AAV envelope or capsid.

13. A recombinant AAV produced according to the method of claim 12 .

14. A proviral plasmid comprising a modular recombinant AAV genome comprising:
(a) a 5′AAV ITR sequence, the ITR flanked upstream by restriction site 1 and downstream by restriction site 2;
(b) a promoter flanked upstream by restriction site 2 and downstream by restriction site 3 wherein the promoter is a CMV promoter sequence;
(c) a polylinker sequence comprising restriction site 3, restriction site 4 and restriction site 5, that permits insertion of a heterologous nucleic acid sequence between any two of the restriction sites 3, 4 and 5, without modification thereof, wherein the gene is operatively linked to, and under the regulatory control of, said promoter;
(d) a polyadenylation sequence flanked upstream by restriction site 4 or 5 and downstream by restriction site 6; and
(e) a 3′ AAV ITR sequence flanked upstream by restriction site 6 and downstream by restriction site 7;
wherein each said restriction site 1 through 7 occurs only once in the plasmid and is cleaved by a different enzyme that cannot cleave another restriction site in the plasmid, and wherein said restriction sites 1 through 7 are positioned to permit ready removal, replacement or substitution of one or more of element (a), (b), (c), (d) and (e) or the entire AAV genome (a) through (e) from the plasmid.

15. The plasmid according to claim 14 , wherein the promoter is a hybrid promoter comprising a CMV promoter sequence and a chicken beta actin (CBA) promoter sequence.

Assignees

• Univ Pennsylvania

Inventors

• Bennett Jean
• Bennicelli Jeannette L

Classifications

• C12N2800/107

  for mammalian · CPC title

• C12N2830/60

  from viruses · CPC title

• C12N2750/14143

  viral genome or elements thereof as genetic vector · CPC title

• C12N7/00 Primary

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

• C12N15/86 Primary

  Viral vectors · CPC title