US5830725A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Rapid, stable high-titre production of recombing retrovirus
Assignee: Leland Stanford Junior University
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Filing: 1995-04-28
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Granted: 1998-11-03
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Expires: 2015-04-28
Abstract
Abstract High titre helper-free recombinant retrovirus are produced by (a) growing a transfected host cell, produced by transfecting a eukaryotic host cell with a recombinant vector capable of stable episomal maintenance in the host cell, in a medium under conditions whereby the recombinant vector is stably maintained as an episome in the transfected host cell and transcripts of said vector form, with retroviral gag, pol and env gene products, an infectious retrovirus; and (b) isolating from the medium helper-free infectious retrovirus formed in the transfected host cell. The recombinant vectors comprise (i) a retroviral construct comprising an exogenous gene; (ii) a eukaryotic origin of replication sequence providing a substrate for replicase activity capable of replicating the vector in the host cell; and, (iii) a copy control sequence providing a substrate for a copy control activity capable of maintaining the vector at a stable copy number in the host cell. The transfected host cell provides retroviral pol reverse transcriptase and integrase activity, the replicase activity and copy control activity, as well as retroviral gag and env gene products capable of forming, with a transcript of the retroviral construct, an infectious retrovirus.
Inventors
- 👤 Garry P. Nolan
- 👤 Todd Kinsella
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 20:01
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13041; C12N2740/13043
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
186 words
A method for making high titre helper-free retrovirus comprising steps: (a) growing a transfected host cell produced by transfecting a eukaryotic host cell with a recombinant vector, wherein said recombinant vector is stably maintained as an episome in said transfected host cell and transcripts of said vector form, with retroviral gag, pol and env gene products, an infectious retrovirus, said vector comprising: (i) a retroviral construct comprising an exogenous gene; (ii) a eukaryotic origin of replication sequence providing a substrate for replicase activity which replicates said vector in said host cell; and, (iii) a copy control sequence providing a substrate for a copy control activity which maintains said vector at a stable copy number in said host cell; said transfected host cell providing retroviral pol reverse transcriptase and integrase activity, said replicase activity and said copy control activity, as well as said retroviral gag, pol and env gene products which activities and gene products form, with a transcript of said retroviral construct, an infectious retrovirus; and, (b) isolating from said medium helper-free infectious retrovirus comprising said exogenous gene, said retrovirus formed in said transfected host cell.
Claim 2
Independent Claim
186 words
A method for making high titre helper-free retrovirus comprising steps: (a) growing a transfected host cell produced by transfecting a eukaryotic host cell with a recombinant vector, wherein said recombinant vector is stably maintained as an episome in said transfected host cell and transcripts of said vector form, with retroviral gag, pol and env gene products, an infectious retrovirus, said vector comprising: (i) a retroviral construct comprising an exogenous gene; (ii) a eukaryotic origin of replication sequence providing a substrate for replicase activity which replicates said vector in said host cell; and, (iii) a copy control sequence providing a substrate for a copy control activity which maintains said vector at a stable copy number in said host cell; said transfected host cell providing retroviral pol reverse transcriptase and integrase activity, said replicase activity and said copy control activity, as well as said retroviral gag, pol and env gene products which activities and gene products form, with a transcript of said retroviral construct, an infectious retrovirus; and, (b) isolating from said medium helper-free infectious retrovirus comprising said exogenous gene, said retrovirus formed in said transfected host cell.
Claim 3
Dependent Claim
16 words
A method according to claim 1, wherein said copy control sequence is a Herpes virus sequence.
Claim 4
Dependent Claim
32 words
A method according to claim 1, wherein said copy control sequence comprises at least a portion of the Epstein-Barr virus Ori sequence sufficient to provide a substrate for said copy control activity.
Claim 5
Dependent Claim
18 words
A method according to claim 1, wherein said copy control activity is provided by a Herpes virus protein.
Claim 6
Dependent Claim
18 words
A method according to claim 1, wherein said copy control activity is provided by Epstein-Barr Virus Nuclear Antigen.
Claim 7
Dependent Claim
26 words
A method according to claim 1, wherein said vector encodes said copy control activity and said copy control activity is provided by a Herpes virus protein.
Claim 8
Dependent Claim
44 words
A method according to claim 1 wherein said recombinant vector further comprises a eukaryotic selectable marker operably linked to a transcription regulatory element and said medium comprises a selective agent, which agent permits the selective growth of eukaryotic cells expressing said eukaryotic selectable marker.
Claim 9
Dependent Claim
45 words
A method according to claim 1 wherein said recombinant vector further comprises a prokaryotic selectable marker operably linked to a transcription regulatory element and a prokaryotic origin of replication sequence providing a substrate for replicase activity capable of replicating said vector in a prokaryotic cell.
Claim 10
Dependent Claim
31 words
A method according to claim 1 wherein both said eukaryotic origin of replication and said copy control sequence of said recombinant vector are provided by a single continuous viral ori sequence.
Claim 11
Dependent Claim
32 words
A method according to claim 1, wherein said host cell is stably transfected with nucleic acid encoding said retroviral gag and pol genes, said genes operably linked to a transcription regulatory element.
Claim 12
Dependent Claim
36 words
A method according to claim 1, wherein said host cell is stably transfected with nucleic acid encoding a viral gene, the transcription product of said viral gene providing both said replicase and said copy control activity.
Claim 13
Dependent Claim
24 words
A method according to claim 1, wherein said isolating step comprises isolating at least 10 7 helper-free infectious retrovirus per milliliter of said medium.
Claim 14
Dependent Claim
24 words
A method according to claim 1, wherein said isolating step comprises isolating at least 10 8 helper-free infectious retrovirus per milliliter of said medium.
Claim 15
Dependent Claim
161 words
A method according to claim 1 wherein said recombinant vector further comprises a eukaryotic selectable marker operably linked to a transcription regulatory element, a prokaryotic selectable marker operably linked to a transcription regulatory element, a prokaryotic origin of replication sequence providing a substrate for replicase activity capable of replicating said vector in a prokaryotic cell, a transcription regulatory element operably linked to said exogenous gene; said medium comprises a selective agent permit the selective growth of eukaryotic cells expressing said eukaryotic selectable marker; said copy control sequence comprises at least a portion of the Epstein-Barr virus Ori sequence and said copy control activity is provided by Epstein-Barr Virus Nuclear Antigen; said host cell is stably transfected with nucleic acid comprising gag and pol genes encoding said gag and pol gene products, said gag and pol genes operably linked to a transcription regulatory element; and said isolating step comprises isolating at least 10 7 helper-free infectious retrovirus per milliliter of said medium.
Claim 16
Dependent Claim
73 words
A recombinant vector capable of stable episomal maintenance in a eukaryotic host cell, said vector comprising: (a) a retroviral construct comprising an exogenous gene; (b) a eukaryotic origin of replication sequence providing a substrate for a replicase activity which replicates said vector in said host cell; and, (c) a copy control sequence providing a substrate for a copy control activity which maintains said vector at a stable copy number in said host cell.
Claim 17
Dependent Claim
73 words
A recombinant vector capable of stable episomal maintenance in a eukaryotic host cell, said vector comprising: (a) a retroviral construct comprising an exogenous gene; (b) a eukaryotic origin of replication sequence providing a substrate for a replicase activity which replicates said vector in said host cell; and, (c) a copy control sequence providing a substrate for a copy control activity which maintains said vector at a stable copy number in said host cell.
Claim 18
Dependent Claim
17 words
A recombinant vector according to claim 15, wherein said copy control sequence is a Herpes virus sequence.
Claim 19
Dependent Claim
33 words
A recombinant vector according to claim 15, wherein said copy control sequence comprises at least a portion of the Epstein-Barr virus Ori sequence sufficient to provide a substrate for said copy control activity.
Claim 20
Dependent Claim
27 words
A recombinant vector according to claim 15, wherein said vector encodes said copy control activity and said copy control activity is provided by a Herpes virus protein.
Claim 21
Dependent Claim
27 words
A recombinant vector according to claim 15, wherein said vector encodes said copy control activity and said copy control activity is provided by Epstein-Barr Virus Nuclear Antigen.
Claim 22
Dependent Claim
20 words
A recombinant vector according to claim 15 further comprising a eukaryotic selectable marker operably linked to a transcription regulatory element.
Claim 23
Dependent Claim
42 words
A recombinant vector according to claim 15 further comprising a prokaryotic selectable marker operably linked to a transcription regulatory element and a prokaryotic origin of replication sequence providing a substrate for replicase activity capable of replicating said vector in a prokaryotic cell.
Claim 24
Dependent Claim
101 words
A recombinant vector according to claim 15 further comprising a eukaryotic selectable marker operably linked to a transcription regulatory element, a prokaryotic selectable marker operably linked to a transcription regulatory element, a prokaryotic origin of replication sequence providing a substrate for replicase activity which replicates said vector in a prokaryotic cell; said copy control and said eukaryotic origin of replication sequences comprising at least a portion of the Epstein-Barr virus Ori sequence sufficient to provide a substrate for said copy control activity; said vector encodes said copy control activity and said copy control activity is provided by Epstein-Barr Virus Nuclear Antigen.
Claim 25
Dependent Claim
52 words
A host cell comprising a recombinant vector according to claim 15, said retroviral pol reverse transcriptase and integrase activity, said replicase activity and said copy control activity, as well as retroviral gag and env gene products which activities and gene products form, with a transcript of said retroviral construct, an infectious retrovirus.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-11-02 | 2002-05-04 | 2002-11-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-11-02 | 2006-05-04 | 2006-11-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-11-02 | 2010-05-04 | 2010-11-03 | $7700.00 | Window Closed / Lapsed |