Patent US3640450A successfully refreshed from Google Patents.
US5858740A
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Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells
Assignee: Miltenyi Biotec Technology Inc
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Filing: 1995-05-10
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Granted: 1999-01-12
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Expires: 2015-05-10
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 human hematopoietic 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
2026-11-02 15:40
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: 60
Claim 1
Independent Claim
79 words
A mammalian target cell transduced with retroviral vector pDRTD4.2 comprising in the 5' to 3' direction, a modified 5' Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) region comprising replacement of the U3 region of the 5' LTR with the U3 region of Moloney murine sarcoma virus (MMSV) viral gag sequences up to the Nar I site of MMLV, a retroviral splice acceptor, a 3' MMLV LTR region and a foreign gene inserted downstream of said splice acceptor.
Claim 2
Dependent Claim
79 words
A mammalian target cell transduced with retroviral vector pDRTD4.2 comprising in the 5' to 3' direction, a modified 5' Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) region comprising replacement of the U3 region of the 5' LTR with the U3 region of Moloney murine sarcoma virus (MMSV) viral gag sequences up to the Nar I site of MMLV, a retroviral splice acceptor, a 3' MMLV LTR region and a foreign gene inserted downstream of said splice acceptor.
Claim 3
Dependent Claim
22 words
The target cell of claim 1, wherein said foreign gene encodes a growth factor, a lymphokine, a hormone or a coagulation factor.
Claim 4
Dependent Claim
16 words
The target cell of claim 1, wherein said foreign gene encodes a chimeric T cell receptor.
Claim 5
Dependent Claim
68 words
The target cell of claim 3, wherein said chimeric T cell receptor is encoded by a DNA 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 6
Dependent Claim
38 words
The target cell of claim 4, wherein said cytoplasmic domain is selected from the group consisting of the CD3 zeta chain, the CD3 eta chain, the CD3 gamma chain, the CD3 delta chain and the CD3 epsilon chain.
Claim 7
Dependent Claim
18 words
The target cell of claim 4, wherein said cytoplasmic domain is the gamma chain of the FcεR1 receptor.
Claim 8
Dependent Claim
18 words
The target cell of claim 4, wherein said extracellular domain is a single-chain antibody, or antigen-binding portion thereof.
Claim 9
Dependent Claim
26 words
The target cell of claim 4, wherein said extracellular domain is a single-chain antibody specific for the HIV env glycoprotein and said cytoplasmic domain is zeta.
Claim 10
Dependent Claim
16 words
The target cell of claim 4, wherein said chimeric T cell receptor is a CD4/zeta receptor.
Claim 11
Dependent Claim
14 words
The target cell of claim 6, wherein said extracellular domain is a CD antigen.
Claim 12
Dependent Claim
14 words
The target cell of claim 10, wherein said extracellular domain is CD4 or CD8.
Claim 13
Dependent Claim
17 words
The target cell of claim 7, wherein said single-chain antibody is specific for the HIV env glycoprotein.
Claim 14
Independent Claim
61 words
A method for transducing with high efficiency mammalian target cells with foreign genes, said method comprising: A) culturing stably transfected 293 cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene; B) separating said transfected 293 cells from supernatant; and C) exposing said mammalian target cells to said supernatant, whereby target cells efficiently transduced with said foreign gene are obtained.
Claim 15
Independent Claim
61 words
A method for transducing with high efficiency mammalian target cells with foreign genes, said method comprising: A) culturing stably transfected 293 cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene; B) separating said transfected 293 cells from supernatant; and C) exposing said mammalian target cells to said supernatant, whereby target cells efficiently transduced with said foreign gene are obtained.
Claim 16
Dependent Claim
13 words
The method of claim 13, wherein said mammalian target cells are human cells.
Claim 17
Dependent Claim
11 words
The method of claim 14, wherein said human cells are lymphocytes.
Claim 18
Dependent Claim
13 words
The method of claim 14, wherein said human cells are hematopoietic stem cells.
Claim 19
Dependent Claim
97 words
A mammalian target cell transduced with replication-defective retroviral vector pRTD2.2 comprising in the 5' to 3' direction, a modified 5' MMLV LTR region comprising replacement of the 5' LTR with the human cytomegalovirus 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 acceptors, an MMLV 3' LTR region and a foreign gene inserted downstream of said splice acceptor.
Claim 20
Dependent Claim
97 words
A mammalian target cell transduced with replication-defective retroviral vector pRTD2.2 comprising in the 5' to 3' direction, a modified 5' MMLV LTR region comprising replacement of the 5' LTR with the human cytomegalovirus 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 acceptors, an MMLV 3' LTR region and a foreign gene inserted downstream of said splice acceptor.
Claim 21
Dependent Claim
45 words
A mammalian target cell transduced with replication-defective retroviral vector pRTD2.2SVG comprising a modification of the pRTD2.2 vector of claim 17 consisting of replacement of the Sac I to Bst EII fragment of vector pRTD2.2 with the Sac I to Bst EII fragment of vector LXSN.
Claim 22
Dependent Claim
62 words
A mammalian target cell transduced with replication-defective retroviral vector pIKT2.2 comprising a modification of the pIK1.1 vector containing the SV40 T antigen polyadenylation site and the SV40 origin of replication, said modification consisting of insertion of the DNA sequence between the 5' LTR and 3' LTR of vector pRTD2.2 of claim 17 between the Sac I and Eco RI sites of pIK1.1.
Claim 23
Dependent Claim
87 words
A mammalian target cell transduced with replication-defective retroviral vector pIKT2.2 comprising a modification of the pIK1.1 vector containing the SV40 T antigen polyadenylation site and the SV40 origin of replication, consisting of insertion of 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 vector pRTD2.2SVG of claim 18, between the Sac I and Eco RI sites of pIK1.1.
Claim 24
Dependent Claim
98 words
A mammalian target cell transduced with a replication-defective retroviral vector comprising (a) a modification of the pIK1.1 vector 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, wherein said vector comprises a splice acceptor, and (b) a foreign gene inserted downstream of said splice acceptor.
Claim 25
Dependent Claim
98 words
A mammalian target cell transduced with a replication-defective retroviral vector comprising (a) a modification of the pIK1.1 vector 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, wherein said vector comprises a splice acceptor, and (b) a foreign gene inserted downstream of said splice acceptor.
Claim 26
Dependent Claim
22 words
A mammalian target cell transduced with the replication-defective retroviral vector of claim 21, wherein said first retroviral vector is an MMLV vector.
Claim 27
Dependent Claim
20 words
The target cell of claim 1, 17, 18, 19, 20, 21 or 22, wherein said cell is a human cell.
Claim 28
Dependent Claim
56 words
The target cell of claim 1, 17, 18, 19, 20, 21 or 22, wherein said cell is selected from the group consisting of lymphocyte, human hematopoietic stem cell, fibroblast, epithelial cell, endothelial cell, myoblast, retinal epithelial cell, islet of Langerhans cell, adrenal medulla cell, osteoblast, osteoclast, neuron, glial cell, ganglion cell, embryonic stem cell and hepatocyte.
Claim 29
Dependent Claim
44 words
The mammalian target cell of claim 1, 17, 18, 19, 20, 21 or 22, wherein the splice receptor 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 30
Dependent Claim
23 words
The mammalian target cell of claim 17, 18, 19, 20, 21 or 22, wherein said foreign gene encodes a chimeric T cell receptor.
Claim 31
Independent Claim
45 words
A method for transducing with high efficiency mammalian target cells with foreign genes, said method comprising cocultivating transfected human cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene with mammalian target cells, wherein said vectors transduce said target cells with said foreign gene.
Claim 32
Independent Claim
45 words
A method for transducing with high efficiency mammalian target cells with foreign genes, said method comprising cocultivating transfected human cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene with mammalian target cells, wherein said vectors transduce said target cells with said foreign gene.
Claim 33
Dependent Claim
13 words
The method of claim 27, wherein said transfected human cells are transiently cotransfected.
Claim 34
Dependent Claim
13 words
The method of claim 27, wherein said transfected human cells are stably cotransfected.
Claim 35
Dependent Claim
13 words
The method of claim 27, wherein said mammalian target cells are human cells.
Claim 36
Dependent Claim
11 words
The method of claim 30, wherein said human cells are lymphocytes.
Claim 37
Dependent Claim
13 words
The method of claim 30, wherein said human cells are hematopoietic stem cells.
Claim 38
Independent Claim
60 words
A method for transducing with high efficiency mammalian target cells with foreign genes, said method comprising: A) culturing transfected human cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene; B) separating said transfected human cells from supernatant; and C) exposing said mammalian target cells to said supernatant, whereby target cells efficiently transduced with said foreign gene are obtained.
Claim 39
Independent Claim
60 words
A method for transducing with high efficiency mammalian target cells with foreign genes, said method comprising: A) culturing transfected human cells producing replication-defective recombinant retroviral vectors carrying a selected foreign gene; B) separating said transfected human cells from supernatant; and C) exposing said mammalian target cells to said supernatant, whereby target cells efficiently transduced with said foreign gene are obtained.
Claim 40
Dependent Claim
13 words
The method of claim 33, wherein said transfected human cells are transiently cotransfected.
Claim 41
Dependent Claim
13 words
The method of claim 33, wherein said transfected human cells are stably cotransfected.
Claim 42
Dependent Claim
13 words
The method of claim 33, wherein said mammalian target cells are human cells.
Claim 43
Dependent Claim
11 words
The method of claim 36, wherein said human cells are lymphocytes.
Claim 44
Dependent Claim
13 words
The method of claim 36, wherein said human cells are hematopoietic stem cells.
Claim 45
Dependent Claim
129 words
A cell stably transfected with 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 the gag and pol virion proteins for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said 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 containing an SV40 polyadenylation site.
Claim 46
Dependent Claim
129 words
A cell stably transfected with 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 the gag and pol virion proteins for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said 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 containing an SV40 polyadenylation site.
Claim 47
Dependent Claim
12 words
The cell of claim 39 wherein said cell is a human cell.
Claim 48
Dependent Claim
20 words
The mammalian target cell of claim 26, wherein said receptor is a CD4/zeta or single-chain antibody chain/zeta T cell receptor.
Claim 49
Independent Claim
204 words
A method for transducing mammalian target cells with foreign genes, said method comprising: A) transiently transfecting a first population of mammalian cells that produce virus, wherein the first population of mammalian cells are stably transfected with at least two retroviral packaging plasmids, which are transfected sequentially, comprising retroviral helper DNA sequences 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 including an SV40 polyadenylation site with 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) cocultivating said first population of mammalian cells producing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, wherein said recombinant retroviral vectors transduce said second population of target cells with said foreign gene.
Claim 50
Independent Claim
204 words
A method for transducing mammalian target cells with foreign genes, said method comprising: A) transiently transfecting a first population of mammalian cells that produce virus, wherein the first population of mammalian cells are stably transfected with at least two retroviral packaging plasmids, which are transfected sequentially, comprising retroviral helper DNA sequences 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 including an SV40 polyadenylation site with 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) cocultivating said first population of mammalian cells producing replication-defective recombinant retroviral vectors carrying said foreign gene with a second population of mammalian target cells, wherein said recombinant retroviral vectors transduce said second population of target cells with said foreign gene.
Claim 51
Independent Claim
206 words
A method for transducing mammalian target cells with foreign genes, said method comprising: A) transiently transfecting a first population of mammalian cells that produce virus, wherein the first population of mammalian cells are stably transfected with at least two retroviral packaging plasmids, which are transfected sequentially, comprising retroviral helper DNA sequences 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 including an SV40 polyadenylation site with 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) separating said first population of mammalian cells from supernatant containing recombinant retroviral vectors; and C) incubating said supernatant with a second population of mammalian target cells, wherein said recombinant retroviral vector transduces said second population of target cells with said foreign gene.
Claim 52
Independent Claim
206 words
A method for transducing mammalian target cells with foreign genes, said method comprising: A) transiently transfecting a first population of mammalian cells that produce virus, wherein the first population of mammalian cells are stably transfected with at least two retroviral packaging plasmids, which are transfected sequentially, comprising retroviral helper DNA sequences 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 including an SV40 polyadenylation site with 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) separating said first population of mammalian cells from supernatant containing recombinant retroviral vectors; and C) incubating said supernatant with a second population of mammalian target cells, wherein said recombinant retroviral vector transduces said second population of target cells with said foreign gene.
Claim 53
Dependent Claim
124 words
A human cell stably transfected with a retroviral packaging plasmid for producing high titers of recombinant retrovirus in human target 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 and for producing virion proteins which package said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral 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 the helper genome and the 3' LTR, and encoding a foreign enhancer and/or promoter functional in said human cell and a SV40 polyadenylation site.
Claim 54
Dependent Claim
124 words
A human cell stably transfected with a retroviral packaging plasmid for producing high titers of recombinant retrovirus in human target 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 and for producing virion proteins which package said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral 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 the helper genome and the 3' LTR, and encoding a foreign enhancer and/or promoter functional in said human cell and a SV40 polyadenylation site.
Claim 55
Dependent Claim
12 words
The human cell of claim 44 which is an embryonic kidney cell.
Claim 56
Dependent Claim
16 words
The human cell of claim 44, wherein said human cell comprises two retroviral helper DNA sequences.
Claim 57
Dependent Claim
15 words
The human cell of claim 45, wherein said embryonic kidney cell is a 293 cell.
Claim 58
Dependent Claim
14 words
The human cell of claim 47, wherein said 293 cell is a tsa54 cell.
Claim 59
Dependent Claim
85 words
The human cell of claim 46, wherein a first helper sequence codes for human immunodeficiency virus (HIV) or gibbon ape leukemia virus (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 (VSV), human T cell leukemia virus (HTLV) type I and HTLV type II.
Claim 60
Dependent Claim
18 words
The human cell of claim 49, wherein said first helper sequence codes for HIV gag and pol proteins.
References Cited (Backward Citations)
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