US5834256A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells
Assignee: GBP IP LLC
•
Filing: 1993-06-11
•
Granted: 1998-11-10
•
Expires: 2013-06-11
Abstract
Abstract The invention provides a novel retroviral packaging system, in which retroviral packaging constructs and packagable vector transcripts are produced from high expression plasmids by transfection in human cells. High titers of recombinant retrovirus are produced in infected cells. The methods of the invention include the use of the novel retroviral constructs to transduce primary human cells, including T cells and bone marrow stem cells, with foreign genes by cocultivation at high efficiencies. The invention is useful for the rapid production of high viral supernatants, and to transduce with high efficiency cells that are refractory to transduction by conventional means.
Inventors
- 👤 Mitchell H. Finer
- 👤 Margo R. Roberts
- 👤 Thomas J. Dull
- 👤 Krisztina M. Zsebo
- 👤 Lu Qin
- 👤 Deborah A. Farson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 04:59
CPC Classifications
C; C12; C12N; C12N7/00; C07; C07K; C07K14/00; C07K14/005; C07K14/435; C07K14/705; C07K14/70503; C07K14/7051; C07K14/70514; C07K14/72; C07K14/721; C07K16/00; C07K16/08; C07K16/10; C12N15/00; C12N15/09; C12N15/10; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C12N15/87; A; A61; A61K; A61K48/00; C07K2319/00; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13022; C12N2740/13041; C12N2740/13043; C12N2740/13051; C12N2740/13052; C12N2740/13061; C12N2740/13062
Claims Analysis
Total Claims: 116
Claim 1
Independent Claim
204 words
A method for transducing, mammalian target cells with foreign genes, said method comprising: A) transient cotransfection of a first population of mammalian cells that can produce virus with: (i) at least one retroviral packaging plasmid comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer without the production of replication competent helper virus said retroviral helper DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a SV40 polyadenylation site; and (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells; and B) cocultivation of said first population of mammalian cells producing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, to transduce said second population of target cells with said foreign gene, whereby target cells transduced with said foreign gene are obtained.
Claim 2
Independent Claim
204 words
A method for transducing, mammalian target cells with foreign genes, said method comprising: A) transient cotransfection of a first population of mammalian cells that can produce virus with: (i) at least one retroviral packaging plasmid comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer without the production of replication competent helper virus said retroviral helper DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a SV40 polyadenylation site; and (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells; and B) cocultivation of said first population of mammalian cells producing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, to transduce said second population of target cells with said foreign gene, whereby target cells transduced with said foreign gene are obtained.
Claim 3
Dependent Claim
48 words
The method of claim 1, wherein said target cells are selected from the group consisting of lymphocytes, human hematopoietic stem cells, fibroblasts, epithelial cells, endothelial cells, myoblasts, retinal epithelial cells, islets of Langerhans, adrenal medulla cells, osteoblasts, osteoclasts, neurons, glial cells, ganglion cells, embryonic stem cells, and hepatocytes.
Claim 4
Dependent Claim
15 words
The method of claim 1, wherein said population of mammalian target cells are human cells.
Claim 5
Dependent Claim
17 words
The method of claim 1, wherein said population of mammalian target cells are human hematopoietic stem cells.
Claim 6
Dependent Claim
17 words
The method of claim 1, wherein said first population of mammalian cells are human embryonic kidney cells.
Claim 7
Dependent Claim
35 words
The method of claim 1, wherein said retroviral genome is a leukemia viral genome selected from the group consisting of Moloney murine leukemia virus (MMLV), Human immunodeficiency virus (HIV) and Gibbon ape leukemia virus (GALV).
Claim 8
Dependent Claim
16 words
The method of claim 1, wherein said retroviral packaging plasmid comprises two retroviral helper DNA sequences.
Claim 9
Dependent Claim
25 words
The method of claim 1, wherein said foreign gene is selected from the group consisting of gene coding growth factors, lymphokines, hormones and coagulation factors.
Claim 10
Dependent Claim
15 words
The method of claim 1, wherein said foreign gene encodes a chimeric T cell receptor.
Claim 11
Dependent Claim
12 words
The method of claim 3 wherein said human target cells are lymphocytes.
Claim 12
Dependent Claim
11 words
The method of claim 10, wherein said lymphocytes are T cells.
Claim 13
Dependent Claim
27 words
The method of claim 10, wherein said lymphocytes are selected from the group consisting of CD8 positive cytotoxic T cells, CD4 positive T cells and tumor-infiltrating lymphocytes.
Claim 14
Dependent Claim
13 words
The method of claim 11, wherein said T cells are cytotoxic T cells.
Claim 15
Dependent Claim
14 words
The method of claim 5, wherein said human embryonic kidney cells are 293 cells.
Claim 16
Dependent Claim
12 words
The method of claim 14 wherein said 293 cells are tsa201 cells.
Claim 17
Dependent Claim
25 words
The method of claim 6, wherein said foreign enhancer is the human cytomegalovirus (CMV) immediate early enhancer and said promoter is the native MMLV promoter.
Claim 18
Dependent Claim
20 words
The method of claim 6, wherein said foreign enhancer and promoter is the human CMV immediate early enhancer and promoter.
Claim 19
Dependent Claim
21 words
The method of claim 6, wherein said foreign enhancer and promoter is the Moloney murine sarcoma virus (MMSV) enhancer and promoter.
Claim 20
Dependent Claim
76 words
The method of claim 7, wherein a first helper sequence codes for ecotropic MMLV gag and pol proteins and a second helper sequence codes for env proteins, or combinations thereof, selected from virus of the group consisting of xenotropic murine leukemia virus, amphotropic murine leukemia virus, ecotropic murine leukemia virus, polytropic murine leukemia virus, 10A1 murine leukemia virus, GALV, HIV, Vesicular Stomatitis Virus (VSV), human T cell leukemia virus (HTLV) type I and HTLV type II.
Claim 21
Dependent Claim
80 words
The method of claim 7 wherein a first helper sequence codes for HIV gag and pol proteins or GALV gag and pol proteins and a second helper sequence codes for env proteins, or combinations thereof, selected from virus of the group consisting of xenotropic murine leukemia virus, amphotropic murine leukemia virus, ecotropic murine leukemia virus, polytropic murine leukemia virus, 10A1 murine leukemia virus, GALV, HIV, Vesicular Stomatitis Virus, human T cell leukemia virus (HTLV) type I and HTLV type II.
Claim 22
Dependent Claim
70 words
The method of claim 9, wherein said chimeric T cell receptor is a receptor encoded by a DNA sequence comprising in reading frame: a sequence encoding a signal sequence; a sequence encoding a non-MHC restricted extracellular surface membrane protein domain binding specifically to at least one ligand; a sequence encoding a transmembrane domain; and a sequence encoding a cytoplasmic signal-transducing domain of a protein that activates an intracellular messenger system.
Claim 23
Dependent Claim
38 words
The method of claim 21, wherein said cytoplasmic domain is selected from the group consisting of gene coding the CD3 zeta chain, the eta chain, the CD3 gamma chain, the CD3 delta chain and the CD3 epsilon chain.
Claim 24
Dependent Claim
17 words
The method of claim 21, wherein said cytoplasmic domain is the gamma chain of the FcεR1 receptor.
Claim 25
Dependent Claim
17 words
The method of claim 21, wherein said extracellular domain is a single-chain antibody, or functional portion thereof.
Claim 26
Dependent Claim
25 words
The method of claim 21, wherein said extracellular domain is a single-chain antibody specific for the HIV env glycoprotein and said cytoplasmic domain is zeta.
Claim 27
Dependent Claim
15 words
The method of claim 21, wherein said chimeric T cell receptor is a CD4/zeta receptor.
Claim 28
Dependent Claim
13 words
The method of claim 23, wherein said extracellular domain is a CD antigen.
Claim 29
Dependent Claim
13 words
The method of claim 27, wherein said extracellular domain is CD4 or CD8.
Claim 30
Dependent Claim
16 words
The method of claim 24, wherein said single-chain antibody is specific for the HIV env glycoprotein.
Claim 31
Independent Claim
189 words
A method for transducing mammalian target cells with foreign genes, said method comprising: A) transient cotransfection of 293 cells with (i) at least one retroviral packaging plasmid comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and promoter functional in a selected mammalian cell and a SV40 polyadenylation site; and (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said 293 cells; and B) cocultivation of said 293 cells producing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, to transduce said population of target cells with said foreign gene, whereby target cells efficiently transduced with said foreign gene are obtained.
Claim 32
Independent Claim
189 words
A method for transducing mammalian target cells with foreign genes, said method comprising: A) transient cotransfection of 293 cells with (i) at least one retroviral packaging plasmid comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and promoter functional in a selected mammalian cell and a SV40 polyadenylation site; and (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said 293 cells; and B) cocultivation of said 293 cells producing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, to transduce said population of target cells with said foreign gene, whereby target cells efficiently transduced with said foreign gene are obtained.
Claim 33
Dependent Claim
13 words
The method of claim 30, wherein said target cells are human target cells.
Claim 34
Dependent Claim
12 words
The method of claim 31, wherein said human target cells are lymphocytes.
Claim 35
Dependent Claim
14 words
The method of claim 31, wherein said human target cells are hematopoietic stem cells.
Claim 36
Independent Claim
51 words
A method for transducing mammalian target cells with foreign genes, said method comprising cocultivation of transfected 293 cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene with mammalian target cells, to transduce said target cells with said foreign gene, whereby target cells transduced with said foreign gene are obtained.
Claim 37
Independent Claim
51 words
A method for transducing mammalian target cells with foreign genes, said method comprising cocultivation of transfected 293 cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene with mammalian target cells, to transduce said target cells with said foreign gene, whereby target cells transduced with said foreign gene are obtained.
Claim 38
Dependent Claim
12 words
The method of claim 34, wherein said 293 cells are transiently cotransfected.
Claim 39
Dependent Claim
12 words
The method of claim 34, wherein said 293 cells are stably transfected.
Claim 40
Dependent Claim
13 words
The method of claim 34, wherein said mammalian target cells are human cells.
Claim 41
Dependent Claim
136 words
The method of claim 34, wherein said 293 cells are transiently cotransfected with: (a) at least one retroviral packaging plasmid comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and promoter functional in a selected mammalian cell and a SV40 polyadenylation site; and (b) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said 293 cells.
Claim 42
Dependent Claim
11 words
The method of claim 37, wherein said human cells are lymphocytes.
Claim 43
Dependent Claim
13 words
The method of claim 37, wherein said human cells are hematopoietic stem cells.
Claim 44
Dependent Claim
109 words
A retroviral packaging plasmid for the production of high titers of recombinant retrovirus in human cells comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and promoter functional in a selected mammalian cell and a SV40 polyadenylation site.
Claim 45
Dependent Claim
109 words
A retroviral packaging plasmid for the production of high titers of recombinant retrovirus in human cells comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and promoter functional in a selected mammalian cell and a SV40 polyadenylation site.
Claim 46
Dependent Claim
14 words
The retroviral packaging plasmid of claim 41, wherein said retrovirus is a leukemia retrovirus.
Claim 47
Dependent Claim
72 words
The retroviral packaging plasmid according to claim 41 wherein said helper DNA sequence codes for ecotropic MMLV gag and pol, and an envelope protein, or combination thereof, selected from virus of the group consisting of xenotropic murine leukemia virus, amphotropic murine leukemia virus, ecotropic murine leukemia virus, polytropic murine leukemia virus, 10A1 murine leukemia virus, GALV, HIV, vesicular stomatitis virus, human T cell leukemia virus (HTLV) type I and HTLV type II.
Claim 48
Dependent Claim
59 words
A method for transiently producing replication-defective recombinant retrovirus in mammalian cells at high titer comprising introducing into mammalian cells that can produce virus at least one retroviral packaging plasmid according to claim 41 and a retroviral vector encoding a foreign gene, whereby mammalian cells containing said retroviral packaging plasmid and retroviral vector produce high titers of retrovirus for infection.
Claim 49
Dependent Claim
31 words
The retroviral packaging plasmid of claim 42, wherein said leukemia retrovirus is selected from the group consisting of Moloney murine leukemia viruses (MMLV), Gibbon ape leukemia viruses (GALV), and HIV viruses.
Claim 50
Dependent Claim
26 words
The retroviral packaging plasmid of claim 45, wherein said foreign enhancer is the human CMV immediate early enhancer and said promoter is the native MMLV promoter.
Claim 51
Dependent Claim
22 words
The retroviral packaging plasmid of claim 45, wherein said foreign enhancer and promoter is the human CMV immediate early enhancer and promoter.
Claim 52
Dependent Claim
19 words
The retroviral packaging plasmid of claim 45, wherein said foreign enhancer and promoter is the MMSV enhancer and promoter.
Claim 53
Dependent Claim
16 words
The retroviral packaging plasmid of claim 45, wherein said plasmid comprises two retroviral helper DNA sequences.
Claim 54
Dependent Claim
78 words
The retroviral packaging plasmid of claim 49, wherein a first helper sequence codes for ecotropic MMLV gag and pol proteins and a second helper sequence codes for env proteins, or combinations thereof, selected from virus of the group consisting of xenotropic murine leukemia virus, amphotropic murine leukemia virus, ecotropic murine leukemia virus, polytropic murine leukemia virus, 10A1 murine leukemia virus, GALV, HIV, Vesicular Stomatitis Virus (VSV), human T cell leukemia virus (HTLV) type I and HTLV type II.
Claim 55
Dependent Claim
12 words
The method of claim 44 wherein said mammalian cells are human cells.
Claim 56
Dependent Claim
19 words
A transfected cell producing replication-defective recombinant retroviruses at high titer, said cell prepared by the method of claim 44.
Claim 57
Dependent Claim
14 words
The method of claim 51, wherein said human cells are human embryonic kidney cells.
Claim 58
Dependent Claim
14 words
The method of claim 53, wherein said human embryonic kidney cells are 293 cells.
Claim 59
Dependent Claim
12 words
The method of claim 54 wherein said 293 cells are tsa201 cells.
Claim 60
Dependent Claim
13 words
The transfected cell of claim 52, wherein said cell is a human cell.
Claim 61
Dependent Claim
16 words
The transfected cell of claim 56, wherein said human cell is a human embryonic kidney cell.
Claim 62
Dependent Claim
15 words
The transfected cell of claim 57, wherein said embryonic kidney cell is a 293 cell.
Claim 63
Dependent Claim
13 words
The transfected cells of claim 58 wherein said 293 cells are tsa201 cells.
Claim 64
Dependent Claim
54 words
A replication-defective retroviral vector comprising in the 5' to 3' direction, a modified 5' MMLV LTR region wherein the U3 region of the 5' LTR is replaced with the U3 region of MMSV, viral gag sequences up to the Nar I site of MMLV, a retroviral splice acceptor and a 3' MMLV LTR region.
Claim 65
Dependent Claim
54 words
A replication-defective retroviral vector comprising in the 5' to 3' direction, a modified 5' MMLV LTR region wherein the U3 region of the 5' LTR is replaced with the U3 region of MMSV, viral gag sequences up to the Nar I site of MMLV, a retroviral splice acceptor and a 3' MMLV LTR region.
Claim 66
Dependent Claim
81 words
A replication-defective retroviral vector comprising in the 5' to 3' direction, a modified 5' MMLV LTR region wherein the 5' LTR is replaced with the human CMV immediate early enhancer/promoter fused to the MMLV R region by an oligonucleotide encoding nucleotides 19 (Sac I) to +1 of the human CMV promoter linked to nucleotides +1 to +32 (KpnI) of MMLV, viral gag sequences up to the Nar I site of MMLV, a retroviral splice acceptor and a MMLV 3' LTR region.
Claim 67
Dependent Claim
81 words
A replication-defective retroviral vector comprising in the 5' to 3' direction, a modified 5' MMLV LTR region wherein the 5' LTR is replaced with the human CMV immediate early enhancer/promoter fused to the MMLV R region by an oligonucleotide encoding nucleotides 19 (Sac I) to +1 of the human CMV promoter linked to nucleotides +1 to +32 (KpnI) of MMLV, viral gag sequences up to the Nar I site of MMLV, a retroviral splice acceptor and a MMLV 3' LTR region.
Claim 68
Dependent Claim
40 words
A replication-defective retroviral vector comprising a modification of the vector of claim 61 wherein the Sac I to Bst EII fragment of the vector of claim 64 is replaced with the Sac I to Bst EII fragment of vector LXSN.
Claim 69
Dependent Claim
55 words
A replication-defective retroviral vector comprising a modification of pIK1.1 which contains the SV40 T antigen polyadenylation site and the SV40 origin of replication, wherein said modification consists of an insertion of the DNA sequence between the 5' LTR and 3' LTR of the vector of claim 61 between the SacI and EcoRI sites of pIK1.1.
Claim 70
Dependent Claim
79 words
A replication-defective retroviral vector comprising a modification of the pIK1.1 vector containing the SV40 T antigen polyadenylation site and the SV40 origin of replication, wherein the DNA, defined at its 5' end by the Sac I site in the human CMV promoter and defined at its 3' end by an Eco RI site located approximately 750 bp downstream of the 3' LTR of the vector of claim 62 is inserted, between the SacI and Eco RI sites of pIK1.1.
Claim 71
Dependent Claim
36 words
The retroviral vector of claim 64, wherein the splice acceptor is replaced with a transcriptional control element internal to the vector selected from the group consisting of a promoter, enhancer, enhancer/promoter and a dominant control region.
Claim 72
Dependent Claim
24 words
The retroviral vector of claim 60, 61, 62, 63 or 64 further comprising DNA encoding a foreign gene inserted downstream of said splice acceptor.
Claim 73
Dependent Claim
71 words
A replication-defective retroviral vector comprising a modification of pIK1.1 in which the sequences of pIK1.1 downstream of the human CMV immediate early enhancer/promoter and upstream of the SV40 origin of replication and SV40 polyadenylation site are replaced with a fragment of a first retroviral vector consisting of the 5' R region of the first retroviral vector up to a restriction site downstream of the 3' LTR of said first retroviral vector.
Claim 74
Dependent Claim
71 words
A replication-defective retroviral vector comprising a modification of pIK1.1 in which the sequences of pIK1.1 downstream of the human CMV immediate early enhancer/promoter and upstream of the SV40 origin of replication and SV40 polyadenylation site are replaced with a fragment of a first retroviral vector consisting of the 5' R region of the first retroviral vector up to a restriction site downstream of the 3' LTR of said first retroviral vector.
Claim 75
Dependent Claim
16 words
The replication-defective retroviral vector of claim 67, wherein said first retroviral vector is an MMLV vector.
Claim 76
Dependent Claim
16 words
The retroviral vector of claim 66 wherein said foreign gene encodes a chimeric T cell receptor.
Claim 77
Dependent Claim
19 words
The retroviral vector of claim 69 wherein said receptor is a CD4/zeta or single-chain antibody chain/zeta T cell receptor.
Claim 78
Dependent Claim
45 words
A method of using the replication-defective retroviral vector of claim 66 to express high levels of packagable genomic retroviral transcipts in mammalian cells comprising transiently cotransfecting a first population of mammalian cells with a packaging plasmid and said retroviral vector whereby said transcripts are produced.
Claim 79
Dependent Claim
13 words
A mammalian cell which produces recombinant retrovirus by the method of claim 71.
Claim 80
Dependent Claim
30 words
The method of claim 71, further comprising cocultivating said first population of mammalian cells with a second population of target cells to transduce said target cells with the foreign gene.
Claim 81
Dependent Claim
15 words
The mammalian cell according to claim 72, wherein said mammalian cell is a human cell.
Claim 82
Dependent Claim
15 words
The mammalian cell according to claim 74, wherein said human cell is a 293 cell.
Claim 83
Dependent Claim
11 words
The method of claim 73 wherein said target cells are lymphocytes.
Claim 84
Independent Claim
211 words
A method for transducing mammalian target cells with foreign genes, said method comprising: A) transient cotransfection of a first population of mammalian cells that can produce virus with: (i) at least one retroviral packaging plasmid comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a SV40 polyadenylation site; and (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells; B) separation of said first population of mammalian cells from cell supernatant; and C) incubating of said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, to transduce said second population of target cells with said foreign gene, whereby target cells transduced with said foreign gene are obtained.
Claim 85
Independent Claim
211 words
A method for transducing mammalian target cells with foreign genes, said method comprising: A) transient cotransfection of a first population of mammalian cells that can produce virus with: (i) at least one retroviral packaging plasmid comprising at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral helper DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5' LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3' LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a SV40 polyadenylation site; and (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells; B) separation of said first population of mammalian cells from cell supernatant; and C) incubating of said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, to transduce said second population of target cells with said foreign gene, whereby target cells transduced with said foreign gene are obtained.
Claim 86
Dependent Claim
17 words
The method of claim 77 wherein said first population of mammalian cells are human embryonic kidney cells.
Claim 87
Dependent Claim
16 words
The method of claim 77 wherein said retroviral packaging plasmid comprises two retroviral helper DNA sequences.
Claim 88
Dependent Claim
14 words
The method of claim 78 wherein said human embryonic kidney cells are 293 cells.
Claim 89
Dependent Claim
75 words
The method of claim 79 wherein a first helper sequence codes for ecotropic MMLV gag and pol proteins and a second helper sequence codes for env proteins, or combinations thereof, selected from virus of the group consisting of xenotropic murine leukemia virus, amphotropic murine leukemia virus, ecotropic murine leukemia virus, polytropic murine leukemia virus, 10A1 murine leukemia virus, GALV, HIV, Vesicular Stomatitis Virus, human T cell leukemia virus (HTLV) type I and HTLV type II.
Claim 90
Dependent Claim
80 words
The method of claim 79 wherein a first helper sequence codes for HIV gag and pol proteins or GALV gag and pol proteins and a second helper sequence codes for env proteins, or combinations thereof, selected from virus of the group consisting of xenotropic murine leukemia virus, amphotropic murine leukemia virus, ecotropic murine leukemia virus, polytropic murine leukemia virus, 10A1 murine leukemia virus, GALV, HIV, Vesicular Stomatitis Virus, human T cell leukemia virus (HTLV) type I and HTLV type II.
Claim 91
Dependent Claim
20 words
The retroviral vector of claim 65, further comprising DNA encoding a foreign gene inserted downstream of said transcriptional control element.
Claim 92
Dependent Claim
16 words
The retroviral vector of claim 83, wherein said foreign gene is a chimeric T cell receptor.
Claim 93
Dependent Claim
47 words
A method of using the retroviral vector of claim 83 to express high levels of packagable genomic retroviral transcripts in mammalian cells which produce virus comprising transiently cotransfecting a first population of mammalian cells with a packaging plasmid and said retroviral vector whereby said transcripts are produced.
Claim 94
Dependent Claim
18 words
The retroviral vector of claim 84, wherein said receptor is a CD4/zeta or single-chain antibody/zeta T cell receptor.
Claim 95
Dependent Claim
30 words
The method of claim 85, further comprising cocultivating said first population of mammalian cells with a second population of target cells to transduce said target cells with the foreign gene.
Claim 96
Dependent Claim
13 words
A mammalian cell producing recombinant retroviruses produced by the method of claim 85.
Claim 97
Dependent Claim
11 words
The method of claim 87, wherein said target cells are lymphocytes.
Claim 98
Dependent Claim
11 words
Retroviral packaging plasmid pIK6.1MMSVampac, having the structure shown in FIG. 1.
Claim 99
Dependent Claim
11 words
Retroviral packaging plasmid pIK6.1MMSVampac, having the structure shown in FIG. 1.
Claim 100
Dependent Claim
11 words
Retroviral packaging plasmid pIK6.1MCVampac, having the structure shown in FIG. 1.
Claim 101
Dependent Claim
11 words
Retroviral packaging plasmid pIK6.1MCVampac, having the structure shown in FIG. 1.
Claim 102
Dependent Claim
11 words
Retroviral packaging plasmid pIK6.1gagpolATG, having the structure shown in FIG. 1.
Claim 103
Dependent Claim
11 words
Retroviral packaging plasmid pIK6.1gagpolATG, having the structure shown in FIG. 1.
Claim 104
Dependent Claim
11 words
Retroviral packaging plasmid pIK6.1amenvATG, having the structure shown in FIG. 1.
Claim 105
Dependent Claim
11 words
Retroviral packaging plasmid pIK6.1amenvATG, having the structure shown in FIG. 1.
Claim 106
Dependent Claim
22 words
The method of claim 1, wherein said retroviral packaging plasmid is the retroviral packaging plasmid of claim 90, 91, 92 or 93.
Claim 107
Dependent Claim
5 words
A retroviral vector designated pRTD4.2.
Claim 108
Dependent Claim
5 words
A retroviral vector designated pRTD4.2.
Claim 109
Dependent Claim
5 words
A retroviral vector designated pRTD2.2.
Claim 110
Dependent Claim
5 words
A retroviral vector designated pRTD2.2.
Claim 111
Dependent Claim
5 words
A retroviral vector designated pRTD2.2SVG.
Claim 112
Dependent Claim
5 words
A retroviral vector designated pRTD2.2SVG.
Claim 113
Dependent Claim
5 words
A retroviral vector designated pIKT2.2.
Claim 114
Dependent Claim
5 words
A retroviral vector designated pIKT2.2.
Claim 115
Dependent Claim
5 words
A retroviral vector designated pIKT2.2SVG.
Claim 116
Dependent Claim
5 words
A retroviral vector designated pIKT2.2SVG.
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) | 2001-11-09 | 2002-05-11 | 2002-11-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-11-09 | 2006-05-11 | 2006-11-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-11-09 | 2010-05-11 | 2010-11-10 | $7700.00 | Window Closed / Lapsed |