US6165715A
Expired / Lapsed
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Expression systems
Assignee: Cancer Research Campaign Technology Ltd
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Filing: 1996-08-23
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Granted: 2000-12-26
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Expires: 2016-08-23
Abstract
Abstract PCT No. PCT/GB96/02061 Sec. 371 Date Jun. 22, 1998 Sec. 102(e) Date Jun. 22, 1998 PCT Filed Aug. 23, 1996 PCT Pub. No. WO97/08330 PCT Pub. Date Mar. 6, 1997The invention relates to new expression systems and in particular to an expression system in which a gene of interest is expressed at an optimal level. The invention provides a recombinant expression vector comprising a gene of interest and a selectable marker gene, wherein the selectable marker gene is arranged downstream of the gene of interest and a stop codon associated with the gene of interest is spaced from a start codon of said selectable marker gene at a distance which is sufficient to ensure that translation reinitiation is required before said selectable marker protein is expressed from the corresponding mRNA. Examples of such expression systems are vector viral packaging cell lines and a number of preferred cell lines have been identified.
Inventors
- 👤 Mary Katherine Levinge Collins
- 👤 Robin Anthony Weiss
- 👤 Yasuhiro Takeuchi
- 👤 Francois-Lois Cosset
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 11:28
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C12N15/67; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13041; C12N2740/13043
Claims Analysis
Total Claims: 39
Claim 1
Independent Claim
73 words
A recombinant expression vector comprising a gene of interest and a selectable marker gene, wherein the selectable marker gene is arranged downstream of the gene of interest and a stop codon associated with the gene of interest is spaced from a start codon of said selectable marker gene at a distance which is sufficient to ensure that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation.
Claim 2
Dependent Claim
73 words
A recombinant expression vector comprising a gene of interest and a selectable marker gene, wherein the selectable marker gene is arranged downstream of the gene of interest and a stop codon associated with the gene of interest is spaced from a start codon of said selectable marker gene at a distance which is sufficient to ensure that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation.
Claim 3
Dependent Claim
15 words
A recombinant expression vector according to claim 1 wherein the vector is a viral vector.
Claim 4
Dependent Claim
22 words
A recombinant expression vector according to claim 1 wherein the gene of interest is included as part of a viral packaging construct.
Claim 5
Dependent Claim
43 words
A recombinant expression vector according to claim 1, wherein the number of nucleotides in the space between the stop codon of the gene of interest and the start codon of the selectable marker is in the range of from 20 to 200 nucleotides.
Claim 6
Dependent Claim
13 words
A host cell transformed with a recombinant expression vector according to claim 1.
Claim 7
Dependent Claim
61 words
A retroviral packaging cell line according to claim 1, wherein a packaging-deficient construct comprising a viral env gene and second selectable marker is the FBdelPASAF (SEQ ID No. 5), the FBdelPMOSAF (SEQ ID No. 6), FbdelPGASAF (SEQ ID No. 7), the FbdelPRDSAF (SEQ ID No. 8), the FbdelPXSAF (FIG. 3), the FbdelP10A1SAF (FIG. 3), or the FbdelPVSVGSAF (FIG. 3) expression construct.
Claim 8
Dependent Claim
15 words
A recombinant expression vector according to claim 2 wherein the vector is a retroviral vector.
Claim 9
Dependent Claim
17 words
A recombinant expression vector according to claim 7, wherein the vector is a human complement-resistant retroviral vector.
Claim 10
Dependent Claim
43 words
A recombinant expression vector according to claim 4, wherein the number of nucleotides in the space between the stop codon of the gene of interest and the start codon of the selectable marker is in the range of from 60 to 80 nucleotides.
Claim 11
Dependent Claim
69 words
A nucleic acid construct comprising a gene of interest and a selectable marker gene, the selectable marker gene being operably linked 3' to the gene of interest, said gene of interest associated with a stop codon spaced from a start codon of said selectable marker gene at a distance sufficient to ensure that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation.
Claim 12
Dependent Claim
69 words
A nucleic acid construct comprising a gene of interest and a selectable marker gene, the selectable marker gene being operably linked 3' to the gene of interest, said gene of interest associated with a stop codon spaced from a start codon of said selectable marker gene at a distance sufficient to ensure that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation.
Claim 13
Dependent Claim
10 words
A vector comprising the nucleic acid construct of claim 10.
Claim 14
Dependent Claim
65 words
A process for producing a cell line in which a gene of interest is expressed, which process comprises: transforming host cells with a nucleic acid construct according to claim 10; selecting those cells where expression of the selectable marker gene may be detected, and growing said transformed cells in the presence of a selection agent, thereby producing a cell line expressing said gene of interest.
Claim 15
Dependent Claim
14 words
A process according to claim 12, wherein the host cell is a eukaryotic cell.
Claim 16
Dependent Claim
23 words
A vector as claimed in claim 11, said vector being selected from the group consisting of plasmids, recombinant retroviral vectors and viral vectors.
Claim 17
Dependent Claim
114 words
A retroviral packaging cell line comprising a host cell transformed with a first and a second recombinant expression vector, said first recombinant expression vector having a packaging-deficient construct comprising a viral gag-pol gene and a first selectable marker gene downstream thereof, and said second recombinant expression vector having a packaging-deficient construct comprising a viral env gene and a second selectable marker gene downstream thereof; wherein the start codon of the first and second selectable markers are spaced from the stop codons of the viral gag-pol gene and the viral env gene respectively by a distance which ensures that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation.
Claim 18
Dependent Claim
114 words
A retroviral packaging cell line comprising a host cell transformed with a first and a second recombinant expression vector, said first recombinant expression vector having a packaging-deficient construct comprising a viral gag-pol gene and a first selectable marker gene downstream thereof, and said second recombinant expression vector having a packaging-deficient construct comprising a viral env gene and a second selectable marker gene downstream thereof; wherein the start codon of the first and second selectable markers are spaced from the stop codons of the viral gag-pol gene and the viral env gene respectively by a distance which ensures that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation.
Claim 19
Dependent Claim
18 words
A retroviral packaging cell line according to claim 15, wherein said retroviral packaging cell line is human complement-resistant.
Claim 20
Dependent Claim
29 words
A retroviral packaging cell line according to claim 15, wherein the first selectable marker is a bsr selectable marker and the second selectable marker is a phleo selectable marker.
Claim 21
Dependent Claim
31 words
A retroviral packaging cell line according to claim 15, wherein the packaging-deficient construct comprising the viral gag-pol gene and first selectable marker is the CeB (SEQ ID No. 2) expression construct.
Claim 22
Dependent Claim
19 words
A retroviral packaging cell line according to claim 15, wherein recombinant expression vector is a packaging-deficient retroviral helper construct.
Claim 23
Dependent Claim
27 words
A retroviral packaging cell line according to claim 15, wherein the viral gag-pol gene and the selectable marker are expressed under the control of a non-retroviral promoter.
Claim 24
Dependent Claim
26 words
A retroviral packaging cell line according to claim 15, wherein the viral env gene and the selectable marker are under the control of a non-retroviral promoter.
Claim 25
Dependent Claim
30 words
A retroviral packaging cell line according to claim 15, wherein the cell line is the HT1080 line, the TE671 line, the 3T3 line, the 293 line or the MV-1-1U line.
Claim 26
Dependent Claim
24 words
A retroviral packaging cell line according to claim 15, wherein the retroviral packaging cell is a human HT 1080 cell and expresses RD114 envelopes.
Claim 27
Dependent Claim
21 words
A retroviral packaging cell line according to claim 15, wherein said second recombinant expression vector is a packaging-deficient retroviral helper construct.
Claim 28
Dependent Claim
22 words
A retroviral packaging cell line according to claim 17, wherein the retroviral packaging cells comprises human TE671 cells and express RD114 envelopes.
Claim 29
Dependent Claim
38 words
A retroviral packaging cell line according to claim 19, wherein overlapping sequences between genomes of a retroviral vector sequence and a packaging-deficient construct are reduced by minimizing the extent of non-coding retroviral sequences in a packaging deficient genome.
Claim 30
Dependent Claim
19 words
A retroviral packaging cell line according to claim 20, wherein the promoter is fused to rabbit beta-1 globin intron.
Claim 31
Dependent Claim
16 words
A retroviral packaging cell line according to claim 20, wherein the promoter is a hCMV promoter.
Claim 32
Dependent Claim
34 words
A retroviral packaging cell line according to claim 20, wherein the viral gag-pol gene and the selectable marker is a hCMV+intron (SEQ ID No. 3) or a hCMV+intronkaSD (SEQ ID No. 4) expression construct.
Claim 33
Dependent Claim
19 words
A retroviral packaging cell line according to claim 21, wherein the promoter is fused to rabbit beta-1 globin intron.
Claim 34
Dependent Claim
16 words
A retroviral packaging cell line according to claim 21, wherein the promoter is a hCMV promoter.
Claim 35
Dependent Claim
27 words
A retroviral packaging cell line according to claim 21, wherein the viral env gene and the selectable marker is a CMV10A1 (SEQ ID No. 9) expression construct.
Claim 36
Dependent Claim
185 words
A process for producing a retroviral packaging cell line in which at least one gene of interest is expressed, which process comprises: transforming host cells with a first and a second recombinant expression vector, said first recombinant expression vector having a packaging-deficient construct comprising a viral gag-pol gene, said gag-pol gene optionally being operably linked to a gene of interest and a first selectable marker gene downstream thereof, and said second recombinant expression vector having a packaging-deficient construct comprising a viral env gene, said env gene optionally being operably linked to a gene of interest and a second selectable maker gene downstream thereof; wherein the start codon of the first and second selectable markers are spaced from the stop codons of the viral gag-pol gene and the viral env gene respectively by a distance which ensures that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation; and selecting transformed cells which express at least one and optionally both first and second marker genes, thereby producing a retroviral packaging cells line expressing said at least one gene of interest.
Claim 37
Dependent Claim
185 words
A process for producing a retroviral packaging cell line in which at least one gene of interest is expressed, which process comprises: transforming host cells with a first and a second recombinant expression vector, said first recombinant expression vector having a packaging-deficient construct comprising a viral gag-pol gene, said gag-pol gene optionally being operably linked to a gene of interest and a first selectable marker gene downstream thereof, and said second recombinant expression vector having a packaging-deficient construct comprising a viral env gene, said env gene optionally being operably linked to a gene of interest and a second selectable maker gene downstream thereof; wherein the start codon of the first and second selectable markers are spaced from the stop codons of the viral gag-pol gene and the viral env gene respectively by a distance which ensures that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation; and selecting transformed cells which express at least one and optionally both first and second marker genes, thereby producing a retroviral packaging cells line expressing said at least one gene of interest.
Claim 38
Dependent Claim
64 words
A packaging deficient construct for use in a process according to claim 33, which expresses a viral gag-pol gene and a selectable marker wherein a start codon of the selectable marker is spaced from a stop codon of the viral gag-pol gene by a distance which ensures that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation.
Claim 39
Dependent Claim
65 words
A packaging deficient construct for use in a process according to claim 33, which expresses a viral env gene and a selectable marker gene; wherein a start codon of the selectable marker is spaced from a stop codon of the viral env gene by a distance which ensures that said selectable marker protein is expressed from the corresponding mRNA as a result of translation reinitiation.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2003-12-26 | 2004-06-26 | 2004-12-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-12-26 | 2008-06-26 | 2008-12-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-12-26 | 2012-06-26 | 2012-12-26 | $7700.00 | Window Closed / Lapsed |