US5681731A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for producing recombinant adeno-associated virus vectors
Assignee: Gencell SAS
•
Filing: 1995-06-02
•
Granted: 1997-10-28
•
Expires: 2015-06-02
Abstract
Abstract Simplified methods to produce recombinant adeno-associated virus (rAAV) vectors are described. The methods involve the use of chimeric plasmids which incorporate the Epstein Barr nuclear antigen (EBNA) gene, the latent origin of replication of Epstein Barr virus (oriP), and a rAAV genome. The chimeric plasmids themselves are also a part of the present invention. These plasmids are maintained as multicopy extra-chromosomal elements in cells, such as human 293 cells. Permanent cell lines carrying these EBV/AAV plasmids are induced to produce large amounts of rAAV upon addition of wild-type, adeno-associated virus helper functions. Vectors produced in this manner are capable of transducing exogenous genes into other human cell lines and exhibit the attributes of viral elements produced by conventional methods.
Inventors
- 👤 Jane S. Lebkowski
- 👤 Maureen A. McNally
- 👤 Thomas B. Okarma
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 09:54
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C07; C07K; C07K14/00; C07K14/005; C12N7/00; C12N2710/00; C12N2710/00011; C12N2710/16011; C12N2710/16211; C12N2710/16222; C12N2710/16241; C12N2710/16244; C12N2710/16261; C12N2710/16262; C12N2750/00; C12N2750/00011; C12N2750/14011; C12N2750/14111; C12N2750/14122; C12N2750/14141; C12N2750/14143
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
35 words
An adeno-associated virus transducing vector having the cellular tropism of a parvovirus different from that of adeno-associated virus comprising: (A) a gene encoding a capsid protein of said parvovirus; and (B) other exogenous genetic material.
Claim 2
Dependent Claim
35 words
An adeno-associated virus transducing vector having the cellular tropism of a parvovirus different from that of adeno-associated virus comprising: (A) a gene encoding a capsid protein of said parvovirus; and (B) other exogenous genetic material.
Claim 3
Dependent Claim
32 words
An adeno-associated virus transducing vector having the cellular tropism of a parvovirus different from that of adeno-associated virus comprising: (A) a capsid protein of said parvovirus; and (B) other exogenous genetic material.
Claim 4
Dependent Claim
32 words
An adeno-associated virus transducing vector having the cellular tropism of a parvovirus different from that of adeno-associated virus comprising: (A) a capsid protein of said parvovirus; and (B) other exogenous genetic material.
Claim 5
Dependent Claim
54 words
An AAV/EBV plasmid vector having the cellular tropism of a parvovirus different from that of adeno-associated virus, wherein said vector comprises: (A) an Epstein Barr virus nuclear antigen gene; (B) an Epstein Barr virus latent origin of replication; (C) a gene encoding a capsid protein of said parvovirus; and (D) other exogenous genetic material.
Claim 6
Dependent Claim
54 words
An AAV/EBV plasmid vector having the cellular tropism of a parvovirus different from that of adeno-associated virus, wherein said vector comprises: (A) an Epstein Barr virus nuclear antigen gene; (B) an Epstein Barr virus latent origin of replication; (C) a gene encoding a capsid protein of said parvovirus; and (D) other exogenous genetic material.
Claim 7
Dependent Claim
16 words
The plasmid vector according to claim 3 lacking one or more functioning adeno-associated virus cap genes.
Claim 8
Dependent Claim
14 words
The plasmid vector according to claim 3 having no functioning adeno-associated virus cap genes.
Claim 9
Dependent Claim
12 words
The plasmid vector according to claim 3 wherein said parvovirus is MVM(p).
Claim 10
Dependent Claim
12 words
The plasmid vector according to claim 3 wherein said parvovirus is B19.
Claim 11
Dependent Claim
10 words
A cell comprising the plasmid vector according to claim 3.
Claim 12
Dependent Claim
77 words
A method for producing a recombinant adeno-associated virus comprising the steps of: (a) introducing into a cell the plasmid vector according to claim 3, thereby producing a recombinant adeno-associated virus producer cell; (b) introducing into said producer cell an adenovirus vector comprising an adeno-associated virus rep gene; (c) growing said producer cell in a cell growth medium; (d) introducing adenovirus to the producer cell, thereby producing a recombinant adeno-associated virus; and (e) isolating said recombinant adeno-associated virus.
Claim 13
Dependent Claim
22 words
The method according to claim 9 further comprising the step of infecting said producer cell with wild type adenovirus or herpes virus.
Claim 14
Dependent Claim
28 words
The method according to claim 9 further comprising introducing into said producer cell a vector comprising adenovirus or herpes virus helper functions sufficient to permit adeno-associated virus replication.
Claim 15
Independent Claim
134 words
A method for producing a recombinant adeno-associated virus comprising the steps of: (a) introducing one or more DNA sequences into the genome of a cell, said sequences comprising a parvovirus rep gene from other than adeno-associated virus and a parvovirus cap gene from other than adeno-associated virus, thereby producing a recombinant adeno-associated virus producer cell; (b) growing said producer cell in a cell growth medium; (c) introducing into said producer cell an AAV/EBV plasmid vector comprising: an Epstein Barr virus nuclear antigen gene; an Epstein Barr virus latent origin of replication; and an adeno-associated virus transducing vector comprising exogenous genetic material and lacking functional adeno-associated virus rep and cap genes, thereby producing a transfected cell; (d) introducing adenovirus to the transfected cell, thereby producing recombinant adeno-associated virus; and (e) isolating said recombinant adeno-associated virus.
Claim 16
Independent Claim
134 words
A method for producing a recombinant adeno-associated virus comprising the steps of: (a) introducing one or more DNA sequences into the genome of a cell, said sequences comprising a parvovirus rep gene from other than adeno-associated virus and a parvovirus cap gene from other than adeno-associated virus, thereby producing a recombinant adeno-associated virus producer cell; (b) growing said producer cell in a cell growth medium; (c) introducing into said producer cell an AAV/EBV plasmid vector comprising: an Epstein Barr virus nuclear antigen gene; an Epstein Barr virus latent origin of replication; and an adeno-associated virus transducing vector comprising exogenous genetic material and lacking functional adeno-associated virus rep and cap genes, thereby producing a transfected cell; (d) introducing adenovirus to the transfected cell, thereby producing recombinant adeno-associated virus; and (e) isolating said recombinant adeno-associated virus.
Claim 17
Dependent Claim
11 words
The method according to claim 12 wherein said parvovirus is MVM(p).
Claim 18
Dependent Claim
11 words
The method according to claim 12 wherein the parvovirus is β19.
Claim 19
Independent Claim
134 words
A method for producing recombinant adeno-associated virus comprising the steps of: (a) introducing into a cell an AAV/EBV plasmid vector comprising: an Epstein Barr virus nuclear antigen gene; an Epstein Barr virus latent origin of replication; and an adeno-associated virus transducing vector comprising exogenous genetic material and lacking functional adeno-associated virus rep and cap genes, thereby producing a recombinant adeno-associated virus producer cell; (b) growing said producer cell in a cell growth medium; (c) introducing into said producer cell a rep/cap vector comprising: genes encoding helper functions from adenovirus or herpes virus the expression of which is sufficient to permit adeno-associated virus replication; a parvovirus cap gene from other than adeno-associated virus; and a parvovirus rep gene from other than adeno-associated virus; thereby producing recombinant adeno-associated virus; and (d) isolating said recombinant adeno-associated virus.
Claim 20
Independent Claim
134 words
A method for producing recombinant adeno-associated virus comprising the steps of: (a) introducing into a cell an AAV/EBV plasmid vector comprising: an Epstein Barr virus nuclear antigen gene; an Epstein Barr virus latent origin of replication; and an adeno-associated virus transducing vector comprising exogenous genetic material and lacking functional adeno-associated virus rep and cap genes, thereby producing a recombinant adeno-associated virus producer cell; (b) growing said producer cell in a cell growth medium; (c) introducing into said producer cell a rep/cap vector comprising: genes encoding helper functions from adenovirus or herpes virus the expression of which is sufficient to permit adeno-associated virus replication; a parvovirus cap gene from other than adeno-associated virus; and a parvovirus rep gene from other than adeno-associated virus; thereby producing recombinant adeno-associated virus; and (d) isolating said recombinant adeno-associated virus.
Claim 21
Dependent Claim
11 words
The method according to claim 15 wherein said parvovirus is MVM(p).
Claim 22
Dependent Claim
11 words
The method according to claim 15 wherein said parvovirus is β19.
Claim 23
Independent Claim
53 words
A method for producing recombinant adeno-associated virus comprising introducing into a cell: (A) a DNA sequence encoding a cap gene of a parvovirus other than adeno-associated virus; (B) exogenous genetic material; (C) an adeno-associated virus rep gene; and (D) adenovirus genes which encode helper functions or herpes virus genes which encode helper functions.
Claim 24
Independent Claim
53 words
A method for producing recombinant adeno-associated virus comprising introducing into a cell: (A) a DNA sequence encoding a cap gene of a parvovirus other than adeno-associated virus; (B) exogenous genetic material; (C) an adeno-associated virus rep gene; and (D) adenovirus genes which encode helper functions or herpes virus genes which encode helper functions.
Claim 25
Dependent Claim
123 words
The method of claim 18 for producing a recombinant adeno-associated virus further, said method comprising the steps of: (a) introducing into a cell an AAV/EBV plasmid vector, said AAV/EBV plasmid vector comprising an Epstein Barr nuclear antigen gene, an Epstein Barr virus latent origin of replication and an adeno-associated virus transducing vector comprising exogenous genetic material and lacking functional adeno-associated virus rep and cap genes; thereby producing a recombinant adeno-associated virus producer cell; (b) growing said producer cell in a cell growth medium; (c) introducing into said producer cell an adenovirus vector comprising a parvovirus cap gene from a parvovirus other than adeno-associated virus and an adeno-associated virus rep gene, thereby producing a recombinant adeno-associated virus; and, (d) isolating said recombinant adeno-associated virus.
Claim 26
Dependent Claim
136 words
The method of claim 18 for producing a recombinant adeno-associated virus further, said method comprising the steps of: (a) introducing one or more DNA sequences into the genome of a cell, said DNA sequences comprising an adeno-associated virus rep gene and a parvovirus cap gene from a parvovirus other than adeno-associated virus, thereby producing a permanent packaging cell; (b) introducing into said permanent packaging cell an adeno-associated virus transducing vector which comprises an Epstein Barr nuclear antigen gene; an Epstein Barr virus latent origin of replication; and exogenous genetic material; and which lacks functional adeno-associated virus rep and cap genes, thereby producing a permanent producer cell; (c) growing said permanent producer cell in a cell growth medium; (d) introducing adenovirus to the producer cell, thereby producing recombinant adeno-associated virus; and (e) isolating said recombinant adeno-associated virus.
Claim 27
Dependent Claim
141 words
The method of claim 18 for producing recombinant adeno-associated virus further, said method comprising the steps of: (a) introducing into a cell an adeno-associated virus transducing vector, said vector comprising an Epstein Barr nuclear antigen gene, an Epstein Barr virus latent origin of replication, and exogenous genetic material, said vector lacking a functional adeno-associated rep gene and lacking a functional parvovirus cap gene; thereby producing a recombinant adeno-associated virus producer cell; (b) growing said producer cell in a cell growth medium; (c) introducing into said producer cell a rep/cap vector comprising genes encoding helper functions from adenovirus or herpes virus the expression of which is sufficient to permit adeno-associated virus replication, an adeno-associated virus cap gene from a parvovirus other than adeno-associated virus, and an adeno-associated virus rep gene, thereby producing a transfected cell; and (d) isolating said recombinant adeno-associated virus.
Claim 28
Dependent Claim
17 words
The method according to claim 18 wherein the genes which encode helper functions are said adenovirus genes.
Claim 29
Independent Claim
49 words
A method for making a producer cell which expresses a cap gene of a parvovirus other than adeno-associated virus comprising introducing into a cell a DNA sequence encoding a cap gene of a parvovirus other than adeno-associated virus and introducing into said producer cell an adeno-associated virus rep gene.
Claim 30
Independent Claim
49 words
A method for making a producer cell which expresses a cap gene of a parvovirus other than adeno-associated virus comprising introducing into a cell a DNA sequence encoding a cap gene of a parvovirus other than adeno-associated virus and introducing into said producer cell an adeno-associated virus rep gene.
Claim 31
Dependent Claim
16 words
The method according to claim 23 wherein said DNA sequence encodes a cap gene from MVM(p).
Claim 32
Dependent Claim
16 words
The method according to claim 23 wherein said DNA sequence encodes a cap gene from β19.
References Cited (Backward Citations)
US4686186A
Cited
US4797368A
Cited
US5585254A
Cited
WO1991018088A1
Cited
US5173414A
Cited
US5354678A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2000-10-27 | 2001-04-28 | 2001-10-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-10-27 | 2005-04-28 | 2005-10-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-10-27 | 2009-04-28 | 2009-10-28 | $7700.00 | Window Closed / Lapsed |