US5658776A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Generation of high titers of recombinant AAV vectors
Assignee: Johns Hopkins University
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Filing: 1995-06-07
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Granted: 1997-08-19
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Expires: 2015-06-07
Abstract
Abstract Adeno-associated virus (AAV) vectors may have utility for gene therapy but heretofore a significant obstacle has been the inability to generate sufficient quantifies of such recombinant vectors in amounts that would be clinically useful for human gene therapy application. Stable, helper-free AAV packaging cell lines have been elusive, mainly due to the activities of Rep protein, which down-regulates its own expression and reverses cellular immortalization. This invention provides packaging systems and processes for packaging AAV vectors that efficiently circumvent these problems by replacing the AAV p5 promoter with a heterologous promoter and that allow for substantially increased packaging efficiency.
Inventors
- 👤 Terence R. Flotte
- 👤 Barrie J. Carter
- 👤 William B. Guggino
- 👤 Rikki Solow
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 21:03
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; A; A61; A61P; A61P1/00; A61P1/18; A61P11/00; A61P3/00; A61P3/12; A61P43/00; A61P7/00; A61P7/06; C12N2750/00; C12N2750/00011; C12N2750/14011; C12N2750/14111; C12N2750/14141; C12N2750/14143
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
147 words
A process for the generation of high titers of AAV recombinant vectors comprising: (a) providing cells containing at least one intact copy of a stably integrated recombinant AAV vector, wherein the AAV vector comprises AAV inverted terminal repeat (ITR) regions and a transcription promoter operably linked to a target polynucleotide, and wherein the expression of the rep gene is limiting in said cells; (b) providing an AAV packaging plasmid that allows expression of the product of the rep gene, wherein in the plasmid the rep gene is operably linked to a heterologous promoter, and wherein the packaging plasmid lacks overlapping homology with AAV sequences in the vector in the cell provided in (a); (c) inserting the AAV packaging plasmid into the cell provided in and incubating the cell under conditions that allow replication and packaging of AAV; and (d) isolating recombinant AAV vectors produced in step (c).
Claim 2
Dependent Claim
147 words
A process for the generation of high titers of AAV recombinant vectors comprising: (a) providing cells containing at least one intact copy of a stably integrated recombinant AAV vector, wherein the AAV vector comprises AAV inverted terminal repeat (ITR) regions and a transcription promoter operably linked to a target polynucleotide, and wherein the expression of the rep gene is limiting in said cells; (b) providing an AAV packaging plasmid that allows expression of the product of the rep gene, wherein in the plasmid the rep gene is operably linked to a heterologous promoter, and wherein the packaging plasmid lacks overlapping homology with AAV sequences in the vector in the cell provided in (a); (c) inserting the AAV packaging plasmid into the cell provided in and incubating the cell under conditions that allow replication and packaging of AAV; and (d) isolating recombinant AAV vectors produced in step (c).
Claim 3
Dependent Claim
14 words
The process of claim 1 wherein the heterologous promoter of step (b) is HIV-LTR.
Claim 4
Dependent Claim
11 words
The process of claim 1 wherein the packaging plasmid is pRS5.
Claim 5
Dependent Claim
18 words
The process of claim 1 wherein the target polynucleotide encodes a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide.
Claim 6
Dependent Claim
18 words
The process of claim 2 wherein the target polynucleotide encodes a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide.
Claim 7
Dependent Claim
18 words
The process of claim 3 wherein the target polynucleotide encodes a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide.
Claim 8
Dependent Claim
113 words
A packaging system for the generation of high titers of AAV recombinant vectors comprising: (a) cells containing at least one intact copy of a stably integrated recombinant AAV vector, wherein the AAV vector comprises AAV inverted terminal repeat (ITR) regions and a transcription promoter operably linked to a target polynucleotide, and wherein the expression of the rep gene is limiting in said cells; and (b) an AAV packaging plasmid that allows expression of the product of the rep gene, wherein in the plasmid the rep gene is operably linked to a heterologous promoter, and wherein the packaging plasmid lacks overlapping homology with AAV sequences in the vector in the cell provided in (a).
Claim 9
Dependent Claim
113 words
A packaging system for the generation of high titers of AAV recombinant vectors comprising: (a) cells containing at least one intact copy of a stably integrated recombinant AAV vector, wherein the AAV vector comprises AAV inverted terminal repeat (ITR) regions and a transcription promoter operably linked to a target polynucleotide, and wherein the expression of the rep gene is limiting in said cells; and (b) an AAV packaging plasmid that allows expression of the product of the rep gene, wherein in the plasmid the rep gene is operably linked to a heterologous promoter, and wherein the packaging plasmid lacks overlapping homology with AAV sequences in the vector in the cell provided in (a).
Claim 10
Dependent Claim
15 words
The packaging system of claim 7 wherein the heterologous promoter in step (b) is HIV-LTR.
Claim 11
Dependent Claim
12 words
The packaging system of claim 7 wherein the packaging plasmid is pRS5.
Claim 12
Dependent Claim
19 words
The packaging system of claim 7 wherein the target polynucleotide encodes a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide.
Claim 13
Dependent Claim
19 words
The packaging system of claim 8 wherein the target polynucleotide encodes a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide.
Claim 14
Dependent Claim
19 words
The packaging system of claim 9 wherein the target polynucleotide encodes a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide.
Claim 15
Dependent Claim
41 words
A packaging plasmid for the production of high titers of AAV recombinant vectors wherein said packaging plasmid allows expression of the product of the rep gene, and further wherein the plasmid comprises the rep gene operably linked to a heterologous promoter.
Claim 16
Dependent Claim
41 words
A packaging plasmid for the production of high titers of AAV recombinant vectors wherein said packaging plasmid allows expression of the product of the rep gene, and further wherein the plasmid comprises the rep gene operably linked to a heterologous promoter.
Claim 17
Dependent Claim
21 words
The packaging plasmid of claim 13 wherein the promoter in the packaging plasmid to which rep is operably linked is HIV-LTR.
Claim 18
Dependent Claim
12 words
A packaging plasmid according to claim 13, wherein the plasmid is pRS5.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2000-08-18 | 2001-02-17 | 2001-08-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-08-18 | 2005-02-17 | 2005-08-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-08-18 | 2009-02-17 | 2009-08-19 | $7700.00 | Window Closed / Lapsed |