US6017761A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for obtaining retroviral packaging cell lines producing high transducing efficiency retroviral supernatant
Assignee: Systemix Inc
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Filing: 1996-12-13
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Granted: 2000-01-25
•
Expires: 2016-12-13
Abstract
Abstract This invention provides a method for obtaining a recombinant retroviral packaging cell capable of producing retroviral vectors as well as 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 supernatant 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 13:41
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: 49
Claim 1
Independent Claim
162 words
A method for obtaining a recombinant retroviral packaging cell comprising: a. isolating a retroviral nucleic acid sequence encoding a minimal gag-pol open reading frame (ORF), said nucleic acid sequence having no flanking sequences of the gag-pol ORF, and inserting said nucleic acid sequence into a first expression plasmid; b. isolating a retroviral nucleic acid sequence encoding a minimal env ORF, said nucleic acid sequence having no flanking sequences of the env ORF, and inserting said nucleic acid sequence into a second expression plasmid, c. obtaining a eukaryotic cell free of an endogenous nucleic acid sequence which encodes the gag-pol ORF or the env ORF and which is derived from the retrovirus from which the minimal gag-pol ORF or env ORF is isolated; and d. introducing the first and second expression plasmids into the eukaryotic cell and expressing the nucleic acids encoding the minimal gag-pol ORF and env ORF to produce Gag, Pol and Env proteins, thereby producing the recombinant retroviral packaging cell.
Claim 2
Independent Claim
162 words
A method for obtaining a recombinant retroviral packaging cell comprising: a. isolating a retroviral nucleic acid sequence encoding a minimal gag-pol open reading frame (ORF), said nucleic acid sequence having no flanking sequences of the gag-pol ORF, and inserting said nucleic acid sequence into a first expression plasmid; b. isolating a retroviral nucleic acid sequence encoding a minimal env ORF, said nucleic acid sequence having no flanking sequences of the env ORF, and inserting said nucleic acid sequence into a second expression plasmid, c. obtaining a eukaryotic cell free of an endogenous nucleic acid sequence which encodes the gag-pol ORF or the env ORF and which is derived from the retrovirus from which the minimal gag-pol ORF or env ORF is isolated; and d. introducing the first and second expression plasmids into the eukaryotic cell and expressing the nucleic acids encoding the minimal gag-pol ORF and env ORF to produce Gag, Pol and Env proteins, thereby producing the recombinant retroviral packaging cell.
Claim 3
Dependent Claim
13 words
The method of claim 1, wherein the retrovirus is a 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
27 words
The method of claim 3, wherein the non-murine cell is selected from the group consisting of Vero, HT-1080, D17 MRC-5, TE671, human embryonic kidney, and HeLa cells.
Claim 8
Dependent Claim
18 words
The method of claim 6, wherein the human embryonic kidney cells are human 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
16 words
The method of claim 1, wherein the env ORF is a murine leukemia virus env gene.
Claim 11
Dependent Claim
21 words
The method of claim 1, wherein at least one of the expression plasmids further comprises a selectable or detectable marker gene.
Claim 12
Dependent Claim
12 words
The method of claim 1, wherein the screening is done by ELISA.
Claim 13
Dependent Claim
51 words
The method of claim 11, wherein in the ELISA, 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 14
Dependent Claim
15 words
The method of claim 8 wherein the gag-pol gene is expressed from the MMLV-LTR promoter.
Claim 15
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 16
Dependent Claim
18 words
The method of claim 1 wherein the gag-pol ORF and env ORF are isolated from the same retrovirus.
Claim 17
Dependent Claim
17 words
The method of claim 1, wherein the gag-pol ORF and env ORF are isolated from different retroviruses.
Claim 18
Dependent Claim
23 words
The method of claim 1, wherein the first and second expression plasmids are introduced into the eukaryotic cell in separate and sequential steps.
Claim 19
Dependent Claim
20 words
The method of claim 1, further comprising screening the cell of step (d) for retroviral Gag, Pol and Env production.
Claim 20
Dependent Claim
12 words
The recombinant retroviral packaging cell obtained by the method of claim 1.
Claim 21
Dependent Claim
14 words
The retroviral packaging cell of claim 19, wherein the cell produces an amphotropic Env.
Claim 22
Dependent Claim
14 words
The retroviral packaging cell of claim 19, wherein the cell produces a xenotropic Env.
Claim 23
Dependent Claim
15 words
The retroviral packaging cell of claim 19, wherein the cell produces a chimeric amphotropic/xenotropic Env.
Claim 24
Dependent Claim
16 words
The recombinant retroviral packaging cell of claim 19 wherein the packaging cell is a non-murine cell.
Claim 25
Dependent Claim
23 words
The recombinant retroviral packaging cell of claim 19 wherein the packaging cell is derived from human 293 having ATCC Accession No. CRL 1573.
Claim 26
Dependent Claim
16 words
The recombinant retroviral packaging cell of claim 19 wherein the packaging cell is a primate cell.
Claim 27
Dependent Claim
40 words
A method of producing a retroviral vector producer cell which comprises transducing the cells of claim 19 or 23 with a retroviral-based vector and subsequently propagating the cell under conditions favorable for the production and secretion of retroviral vector supernatant.
Claim 28
Dependent Claim
54 words
The method of claim 26, 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 29
Dependent Claim
17 words
The method of claim 27, wherein the target cell population is human 293 cells (ATCC CRL 1573).
Claim 30
Dependent Claim
19 words
The method of claim 26, wherein the retroviral-based vector used to transduce the cells was produced in human cells.
Claim 31
Dependent Claim
12 words
The retroviral vector producer cell produced by the method of claim 26.
Claim 32
Dependent Claim
53 words
A method of increasing the gene transduction efficiency of a cell, comprising transducing the cell with a retroviral vector supernatant produced from the culture of at least one retroviral vector producer cell of claim 30, wherein the transduction efficiency is increased over that achieved with a vector supernatant produced from murine PA317-based cells.
Claim 33
Dependent Claim
40 words
The method of claim 31, wherein the retroviral vector producer cell is derived from a packaging cell selected from group consisting of ProPak-A.6 (ATCC Accession No. CRL 12006), ProPak-A.52 (ATCC Accession No. CRL-12479) or ProPak-X.36 (ATCC Accession No. CRL 12007).
Claim 34
Dependent Claim
31 words
The method of claim 31 wherein the retroviral vector supernatant is produced from the co-culture of a first and a second complementary retroviral vector producer cell without replication competent retrovirus generation.
Claim 35
Dependent Claim
32 words
The method of claim 30 wherein the first retroviral vector producer cell is derived from an amphotropic packaging cell and the second vector producer cell is derived from a xenotropic packaging cell.
Claim 36
Dependent Claim
35 words
The method of claim 34 wherein the amphotropic packaging cell is ProPak-A.6 (ATCC Accesssion No. CRL 12006) or ProPak-A.52 (ATCC Accession No. CRL-12479), and the xenotropic packaging cell is ProPak-X.36 (ATCC Accesssion No. CRL 12007).
Claim 37
Dependent Claim
22 words
The method of claim 34 wherein both the amphotropic and xenotropic packaging cells are produced from human 293 cells (ATCC CRL 1573).
Claim 38
Dependent Claim
20 words
The method of claim 31 wherein the retroviral vector supernatant is produced from a stable retroviral vector producer cell culture.
Claim 39
Dependent Claim
15 words
The method of claim 31 wherein the transduced cell is a primary human hematopoietic cell.
Claim 40
Dependent Claim
15 words
The method of claim 31 wherein the transduced cell is a human hematopoietic stem cell.
Claim 41
Dependent Claim
22 words
The method of claim 38 wherein the hematopoietic cell is a CD34+ Thyl+ cell from mobilized peripheral blood or a CD4+ PBL.
Claim 42
Dependent Claim
63 words
The retroviral packaging cell of claim 19, capable of packaging retroviral vector sequences to form a retroviral vector producer cell that does not generate RCR after continuous culture for up to at least 12 weeks and that produces a recombinant, transducing retroviral vector particle, the retroviral vector particle characterized by: a. being resistant to human complement; and b. having a high transduction efficiency.
Claim 43
Dependent Claim
22 words
The retroviral packaging cell line of claim 41 wherein the cell line is designated ProPak-A.6 and has ATCC Accesssion No. CRL 12006.
Claim 44
Dependent Claim
21 words
The retroviral packaging cell line of claim 41 wherein the cell line is designated ProPak A.52 having ATCC Accession No. CRL-12479.
Claim 45
Dependent Claim
21 words
The retroviral packaging cell line of claim 41 wherein the cell line is designated ProPak-X.36 and has ATCC Accession No.CRL 12007.
Claim 46
Dependent Claim
29 words
An expression plasmid for expressing gag-pol, comprising a gag-pol open reading frame from the start codon to the stop codon with no flanking sequences of the open reading frame.
Claim 47
Dependent Claim
29 words
An expression plasmid for expressing gag-pol, comprising a gag-pol open reading frame from the start codon to the stop codon with no flanking sequences of the open reading frame.
Claim 48
Dependent Claim
29 words
An expression plasmid for expressing env, comprising an env open reading frame from the start codon to the stop codon with no flanking sequences of the open reading frame.
Claim 49
Dependent Claim
29 words
An expression plasmid for expressing env, comprising an env open reading frame from the start codon to the stop codon with no flanking sequences of the open reading frame.
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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) | 2003-01-24 | 2003-07-26 | 2004-01-25 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-01-24 | 2007-07-26 | 2008-01-25 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-01-24 | 2011-07-26 | 2012-01-25 | $7700.00 | Window Closed / Lapsed |