Patent US5910434A successfully refreshed from Google Patents.
US5910434A
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Method for obtaining retroviral packaging cell lines producing high transducing efficiency retroviral supernatant
Assignee: Systemix Inc
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Filing: 1995-12-15
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Granted: 1999-06-08
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Expires: 2015-12-15
Abstract
Abstract This invention provides a method for obtaining a recombinant retroviral packaging cell capable of producing retroviral vectors and the recombinant packaging cell obtained by the method. Also provided is a method of producing recombinant retroviral particles obtained by introducing into the packaging cells obtained according to the methods disclosed herein, a recombinant retroviral vector and propagating the resulting producer cells under conditions favorable for the production and secretion of retroviral vector supernatant. The retroviral supernatants produced by these methods also is claimed herein. This invention further provides a method for screening retroviral vector supernatant for high transduction efficiency and methods for producing retroviral vector supernatant for transducing cells with high efficiency in gene therapy applications.
Inventors
- 👤 Richard J. Rigg
- 👤 Jingyi Chen
- 👤 Jonathan S. Dando
- 👤 Ivan Plavec
- 👤 Sean P. Forestell
- 👤 Ernst Bohnlein
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 15:20
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; A; A61; A61K; A61K48/00; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13041; C12N2740/13043; C12N2740/13051; C12N2740/13052; C12N2840/00; C12N2840/20; C12N2840/203
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
227 words
A method for obtaining a recombinant retroviral packaging cell capable of producing retroviral vectors comprising: a. selecting a retrovirus; b. obtaining a eukaryotic cell free of endogenous retroviral nucleic acid of the same type as the retrovirus of step (a); c. preparing a minimal gag-pol open reading frame (ORF) insert from the retrovirus wherein the ORF contains no flanking sequences of the gag-pol gene; d. inserting the minimal gag-pol ORF prepared from step (c) into an appropriate expression plasmid, wherein the gag-pol ORF is operatively linked to a heterologous promoter having no overlap with the retroviral vector; e. preparing a minimal env open reading frame (ORF) insert from the retrovirus wherein the ORF contains no flanking sequences of the env gene; f. inserting the minimal env ORF prepared from step (e) into an appropriate expression plasmid, wherein the env ORF is operatively linked to a heterologous promoter having no overlap with the retroviral vector; g. inserting the expression plasmids of steps (d) and (f) into the cell of step (b); h. propagating the cell obtained from step (g) under conditions favorable for expression of the minimal retroviral gag-pol and env ORF; and i. screening a cell for retroviral Gag, Pol and Env production by the cell of step (h); thereby obtaining the retroviral packaging cell capable of packaging recombinant retroviral vector sequences to produce recombinant, transducing retrovirus.
Claim 2
Independent Claim
227 words
A method for obtaining a recombinant retroviral packaging cell capable of producing retroviral vectors comprising: a. selecting a retrovirus; b. obtaining a eukaryotic cell free of endogenous retroviral nucleic acid of the same type as the retrovirus of step (a); c. preparing a minimal gag-pol open reading frame (ORF) insert from the retrovirus wherein the ORF contains no flanking sequences of the gag-pol gene; d. inserting the minimal gag-pol ORF prepared from step (c) into an appropriate expression plasmid, wherein the gag-pol ORF is operatively linked to a heterologous promoter having no overlap with the retroviral vector; e. preparing a minimal env open reading frame (ORF) insert from the retrovirus wherein the ORF contains no flanking sequences of the env gene; f. inserting the minimal env ORF prepared from step (e) into an appropriate expression plasmid, wherein the env ORF is operatively linked to a heterologous promoter having no overlap with the retroviral vector; g. inserting the expression plasmids of steps (d) and (f) into the cell of step (b); h. propagating the cell obtained from step (g) under conditions favorable for expression of the minimal retroviral gag-pol and env ORF; and i. screening a cell for retroviral Gag, Pol and Env production by the cell of step (h); thereby obtaining the retroviral packaging cell capable of packaging recombinant retroviral vector sequences to produce recombinant, transducing retrovirus.
Claim 3
Dependent Claim
14 words
The method of claim 1, wherein the retrovirus is a Moloney murine leukemia virus.
Claim 4
Dependent Claim
12 words
The method of claim 1, wherein the cell is a non-murine cell.
Claim 5
Dependent Claim
13 words
The method of claim 3, wherein the non-murine cell is a primate cell.
Claim 6
Dependent Claim
13 words
The method of claim 4, wherein the primate cell is a human cell.
Claim 7
Dependent Claim
28 words
The method of claim 3, wherein the non-murine cell is selected from the group consisting of Vero, HT-1080, D17 MRC-5, FS-4, TEG71, human embryonic kidney (293), and HeLa.
Claim 8
Dependent Claim
17 words
The method of claim 6, wherein the human embryonic kidney cells are 293 cells (ATCC CRL 1573).
Claim 9
Dependent Claim
17 words
The method of claim 1, wherein the gag-pol ORF is a moloney murine leukemia virus gag-pol gene.
Claim 10
Dependent Claim
19 words
The method of claim 8, wherein the gag-pol gene is expressed from the CMV-IE promoter or the RSV-LTR promoter.
Claim 11
Dependent Claim
17 words
The method of claim 1, wherein the env ORF is a moloney murine leukemia virus env gene.
Claim 12
Dependent Claim
20 words
The method of claim 1, wherein the plasmid of steps (d) or (f) comprises a selectable or detectable marker gene.
Claim 13
Dependent Claim
53 words
The method of claim 1, wherein the gag-pol ORF and the env ORF expression plasmids of step (g) are amplified in bacterial host cells prior to inserting into the eukaryotic cell of step (b), wherein the bacterial host cells are propagated at a temperature range from about 28° C. to about 32° C.
Claim 14
Dependent Claim
16 words
The method of claim 12, wherein the bacterial host cells are propagated at about 30° C.
Claim 15
Dependent Claim
21 words
The method of claim 1, wherein in step (i), Gag, Pol and Env production is screened by a sandwich ELISA assay.
Claim 16
Dependent Claim
48 words
The method of claim 14, wherein Env is detected using a primary antibody from hybridoma 83A25 followed by antiserum 79S-834, enzyme-conjugated antispecies antibody and enzyme substrate; and Gag is detected separately using a primary antibody from hybridoma R187 followed by antiserum 77S-227, enzyme-conjugated antispecies antibody and enzyme substrate.
Claim 17
Dependent Claim
12 words
The recombinant retroviral packaging cell obtained by the method of claim 1.
Claim 18
Dependent Claim
15 words
The recombinant retroviral packaging cell of claim 16, wherein the cell produces an amphotropic env.
Claim 19
Dependent Claim
16 words
The recombinant retroviral retroviral packaging cell of claim 16, wherein the cell produces a xenotropic env.
Claim 20
Dependent Claim
13 words
A retroviral packaging cell line designated ProPak-A.6 having ATCC Accession No. CRL 12006.
Claim 21
Dependent Claim
13 words
A retroviral packaging cell line designated ProPak-A.6 having ATCC Accession No. CRL 12006.
Claim 22
Dependent Claim
37 words
A method of producing a retroviral producer cell which comprises transducing the cells of claim 16 with a retroviral-based vector and subsequently propagating the cells under conditions favorable for the production and secretion of retroviral vector supernatant.
Claim 23
Dependent Claim
54 words
The method of claim 20, further comprising screening the producer cell for the ability to produce a vector supernatant having high transduction efficiency, comprising measuring the ability of the vector supernatant to transduce a target cell population with a transduction efficiency greater than that achieved with a vector supernatant produced from murine PA317-based cells.
Claim 24
Dependent Claim
14 words
The method of claim 21, wherein the target cell population is human 293 cells.
Claim 25
Dependent Claim
11 words
The retroviral producer cell produced by the method of claim 20.
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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) | 2002-06-07 | 2002-12-07 | 2003-06-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2006-06-07 | 2006-12-07 | 2007-06-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2010-06-07 | 2010-12-07 | 2011-06-08 | $7700.00 | Window Closed / Lapsed |