US6451595B1
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High efficiency retroviral vectors that contain none of viral coding sequences
Assignee: Helixmith Co Ltd
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Filing: 1999-07-24
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Granted: 2002-09-17
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Expires: 2019-07-24
Abstract
Abstract The present invention relates to improved retroviral vectors for gene therapy. In this invention, retroviral vectors with higher safety and efficiency are constructed from MLV-based starting vectors, MON and MIN. The improved vectors have the following features: 1) sequences corresponding to MLV-derived pol gene are completely deleted in the vectors, avoiding homologous recombination which has been a baffling problem in conventional retroviral vectors, 2) a heterologous intron, splicing acceptor and/or non-coding sequence are/is inserted into the upstream position of a cloning site, maximizing the expression of a foreign gene through efficient splicing, 3) the vectors contain either the full-length U3 sequence of 5' LTR or a strong heterologous promoter instead, permitting the abundant production of RNA, 4) either IRES (Internal Ribosomal Entry Site) or internal SV40 minimal promoter is inserted into the downstream position of cloning site, enabling the simultaneous expression of two or more foreign genes. Since the improved retroviral vectors of this invention turn out to be safe and to express the foreign gene efficiently, they are useful for gene therapy and the like.
Inventors
- 👤 Sunyoung Kim
- 👤 Seung Shin Yu
- 👤 Jong-mook Kim
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 15:56
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; A; A61; A61P; A61P43/00; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13041; C12N2740/13043; C12N2830/00; C12N2830/42; C12N2830/50; C12N2840/00; C12N2840/20; C12N2840/203; C12N2840/44
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
47 words
A murine leukemia virus (MLV)-based retroviral vector comprising a MLV 5′ LTR (long terminal repeat), a MLV packaging signal, a cloning site for a foreign gene, and A MLV 3′ LTR in order, wherein the gag, env and pol coding regions of MLV have been completely deleted.
Claim 2
Dependent Claim
47 words
A murine leukemia virus (MLV)-based retroviral vector comprising a MLV 5′ LTR (long terminal repeat), a MLV packaging signal, a cloning site for a foreign gene, and A MLV 3′ LTR in order, wherein the gag, env and pol coding regions of MLV have been completely deleted.
Claim 3
Dependent Claim
36 words
The MLV-based retroviral vector of claim 1 , which comprises an intron, a splicing acceptor, or both inserted upstream of the cloning site for the foreign gene, said intron and splicing acceptor being heterologous to MLV.
Claim 4
Dependent Claim
48 words
The MLV-based retroviral vector of claim 2 , wherein the intron is selected from the group consisting of the introns of a human cytomegalovirus (HCMV) IE1 (U123) gene, an elongation factor 1α (EF1α) gene, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, a β-actin gene and a mouse immunoglobulin gene.
Claim 5
Dependent Claim
30 words
The MLV-based retroviral vector of claim 1 , which comprises a non-coding sequence inserted upstream of the cloning site for the foreign gene, said non-coding sequence being heterologous to MLV.
Claim 6
Dependent Claim
39 words
The MLV-based retroviral vector of claim 4 , wherein the non-coding sequence is selected from the group consisting of the non-coding sequences of a HCMV IE1 (UL123) gene, an EF1 α gene, a GAPDH gene and a β-actin gene.
Claim 7
Dependent Claim
19 words
The MLV-based retroviral vector of claim 4 , wherein the non-coding sequence comprises the HCMV major immediate early promoter.
Claim 8
Dependent Claim
28 words
The MLV-based retroviral vector of claim 1 , which comprises a promoter inserted downstream of the cloning site for the foreign gene, said promoter being heterologous to MLV.
Claim 9
Dependent Claim
16 words
The MLV-based retroviral vector of claim 7 , wherein the promoter is the SV40 minimal promoter.
Claim 10
Dependent Claim
22 words
The MLV-based retroviral vector of claim 4 , which is the DONSA1 vector having the genetic map depicted in FIG. 4 c.
Claim 11
Dependent Claim
22 words
The MLV-based retroviral vector of claim 2 , which is the DON2 vector having the genetic map depicted in FIG. 4 b.
Claim 12
Dependent Claim
22 words
The MLV-based retroviral vector of claim 2 , which is the MIN-EI vector having the genetic map depicted in FIG. 7 b.
Claim 13
Dependent Claim
22 words
The MLV-based retroviral vector of claim 2 , which is the MIN-2 vector having the genetic map depicted in FIG. 7 d.
Claim 14
Dependent Claim
17 words
The E. coli strain Top10-DONSA1 (Accession No.: KCCM-10127) transformed with the DONSA1 vector of claim 9 .
Claim 15
Dependent Claim
17 words
The E. coli strain Top10-DON2 (Accession No.: KCCM-10128) transformed with the DONSA1 vector of claim 10 .
Claim 16
Dependent Claim
32 words
The E. coli strain MIN-EICAT(Top10) (Accession No.: KCCM-10163) transformed with the MIN-EICAT vector which comprises a bacterial chloramphenicol acetyltransferase (CAT) gene in the cloning site of the vector of claim 11 .
Claim 17
Dependent Claim
31 words
E. coli strain MIN-2CAT(Top10) (Accession No.: KCCM-10164) transformed with the MIN-2CAT vector which comprises a bacterial chloramphenicol acetyltransferase (CAT) gene in the cloning site of the vector of claim 12 .
Claim 18
Dependent Claim
50 words
The MLV-based retroviral vector of claim 2 , wherein the splicing acceptor is selected from the group consisting of the splicing acceptors of a human cytomegalovirus (HCMV) IE1 (UL123) gene, an elongation factor 1α (EF1α) gene, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, a β-actin gene and a mouse immunoglobulin gene.
Claim 19
Dependent Claim
29 words
The MLV-based retroviral vector of claim 1 , wherein the full-length U3 sequence (−419 to −1 bp) of MLV 5′ LTR is replaced with a promoter heterologous to MLV.
Claim 20
Dependent Claim
18 words
The MLV-based retroviral vector of claim 18 , wherein the promoter is the HCMV major immediate early promoter.
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) | 2005-09-16 | 2006-03-18 | 2006-09-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2009-09-16 | 2010-03-18 | 2010-09-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2013-09-16 | 2014-03-18 | 2014-09-17 | $7700.00 | Window Closed / Lapsed |