US5674722A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Genetic modification of endothelial cells
Assignee: The Brigham and Women's Hospital Inc
•
Filing: 1994-05-25
•
Granted: 1997-10-07
•
Expires: 2014-05-25
Abstract
Abstract Endothelial cells transduced with genetic material encoding a polypeptide or protein of interest and, optionally, a selectable marker, as well as methods for making and using the transduced endothelial cells are disclosed. Such endothelial cells are useful in improving the performance of vascular grafts and in delivering the encoded polypeptide or protein, such as an enzyme, a hormone, a receptor or a drug, to an individual.
Inventors
- 👤 Richard C. Mulligan
- 👤 Lawrence K. Cohen
- 👤 Lori F. Rafield
- 👤 Louis K. Birinyi
- 👤 Allan D. Callow
- 👤 James M. Wilson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-22 22:01
CPC Classifications
A; A61; A61L; A61L27/00; A61L27/36; A61L27/38; A61L27/3804; A61L27/3808; A61K; A61K38/00; A61K38/16; A61K38/17; A61K38/177; A61K38/22; A61K38/29; A61K38/36; A61K38/37; A61K38/43; A61K38/46; A61K38/47; A61K38/48; A61K38/49; A61L27/3895; C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C12N5/00; C12N5/06; C12N5/0602; C12N5/069; C12Y; C12Y302/00; C12Y302/01; C12Y302/01023; C12Y304/00; C12Y304/21; C12Y304/21068; A61K48/00; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13041; C12N2740/13043; Y; Y10; Y10S; Y10S623/00; Y10S623/92; Y10S623/921
Claims Analysis
Total Claims: 68
Claim 1
Independent Claim
19 words
Transduced endothelial cells which have been engineered to express stably maintained genetic material encoding a polypeptide or protein product.
Claim 2
Dependent Claim
19 words
Transduced endothelial cells which have been engineered to express stably maintained genetic material encoding a polypeptide or protein product.
Claim 3
Dependent Claim
77 words
Endothelial cells according to claim 1 wherein the genetic material is DNA, and wherein the genetic material is selected from the group consisting of: DNA that is present in and expressed by normal endothelial cells; DNA that does not normally occur in endothelial cells; or normally occurs in endothelial cells but is not expressed in them at levels which are biologically significant; and DNA that can be modified so that it can be expressed in endothelial cells.
Claim 4
Dependent Claim
46 words
Endothelial cells according to claim 1, wherein said genetic material encodes a protein or polypeptide selected from the group consisting: (a) a hormone; (b) an enzyme; (c) a selectable marker; (d) a protein or polypeptide which is secreted from the cells; and (e) a membrane receptor.
Claim 5
Dependent Claim
16 words
Endothelial cells of claim 1 wherein said stably maintained genetic material has been introduced by transduction.
Claim 6
Dependent Claim
62 words
Encothelial cells of claim 4 wherein said transduction is via a retrovirus having a recombinant genome comprised of; (a) genetic material encoding the product of interest, (b) the long terminal repeat sequences, tRNA binding site and Psi packaging site derived from a retrovirus; and (c) at least one promoter of eukaryotic origin which is capable of being modulated by an external cue.
Claim 7
Dependent Claim
14 words
Endothelial cells according to claim 1 wherein said genetic material encodes a thrombolytic protein.
Claim 8
Dependent Claim
41 words
Endothelial cells according to claim 1 wherein said endothelial cells produce a confluent layer of endothelial cells lining the inner surface of a prosthetic vessel, and wherein said genetic material encodes an endothelial cell mitogen or an endothelial cell growth factor.
Claim 9
Dependent Claim
45 words
Endothelial cells according to claim 1 wherein said genetic material encodes a membrane receptor which binds a ligand, and wherein said ligand may be attached to a prosthetic vessel so that binding of the endothelial cells to the surface of the prosthetic vessel is enhanced.
Claim 10
Dependent Claim
32 words
Endothelial cells according to claim 1 wherein said genetic material encodes a product which inhibits smooth muscle cell growth, for inhibiting the growth of smooth muscle cells in a synthetic vascular implant.
Claim 11
Dependent Claim
13 words
Transduced endothelial cells according to claim 1 wherein said expressing is in vitro.
Claim 12
Dependent Claim
75 words
Transduced endothelial cells according to claim 10 wherein said genetic material of interest is selected from the group consisting of: DNA which is present in and expressed by normal endothelial cells; DNA which does not normally occur in endothelial cells; DNA which normally occurs in endothelial cells but is not expressed in them at levels which are biologically significant; and any DNA which can be modified so that it can be expressed in endothelial cells.
Claim 13
Dependent Claim
17 words
Transduced endothelial cells according to claim 11, further comprising DNA which encodes at least one selectable marker.
Claim 14
Dependent Claim
33 words
Transduced endothelial cells according to claim 11, wherein said genetic material of interest encodes a protein or polypeptide selected from the group consisting of: a hormone, a receptor, an enzyme and a polypeptide.
Claim 15
Dependent Claim
39 words
Transduced endothelial cells according to claim 13, wherein said genetic material of interest encodes a polypeptide or protein product selected from the group consisting of human parathyroid hormone, tissue plasminogen activator, Factor VIII, low density lipoprotein receptor or beta-galactosidase.
Claim 16
Dependent Claim
22 words
Transduced human endothelial cells that have been engineered to express recombinant genetic material of interest encoding a polypeptide or protein in vivo.
Claim 17
Dependent Claim
22 words
Transduced human endothelial cells that have been engineered to express recombinant genetic material of interest encoding a polypeptide or protein in vivo.
Claim 18
Dependent Claim
80 words
Transduced human endothelial cells according to claim 15, wherein said genetic material of interest is selected from the group consisting of: genetic material which is present in and expressed by normal endothelial cells; genetic material which does not normally occur in endothelial cells; genetic material which normally occurs in endothelial cells but is not expressed in them at levels which are biologically significant; and any genetic material which can be modified so that it can be expressed in endothelial cells.
Claim 19
Dependent Claim
18 words
Transduced human endothelial cells of claim 16, further comprising DNA which encodes at least one dominant selectable marker.
Claim 20
Dependent Claim
35 words
Transduced human endothelial cells according to claim 16 in which said genetic material of interest encodes a protein or polypeptide selected from the group consisting of: a hormone, a receptor, an enzyme and a polypeptide.
Claim 21
Dependent Claim
29 words
Transduced human endothelial cells according to claim 15 wherein said genetic material of interest encodes human parathyroid hormone, tissue plasminogen activator, Factor VIII, low density lipoprotein receptor, or beta-galactosidase.
Claim 22
Dependent Claim
29 words
Cultured endothelial cells containing DNA that is integrated into said cultured endothelial cells, wherein said DNA encodes a protein not made by naturally-occurring endothelial cells and a selectable marker.
Claim 23
Dependent Claim
29 words
Cultured endothelial cells containing DNA that is integrated into said cultured endothelial cells, wherein said DNA encodes a protein not made by naturally-occurring endothelial cells and a selectable marker.
Claim 24
Dependent Claim
51 words
Transduced endothelial cells according to claim 5 having genetic material of interest integrated therein, wherein said genetic material of interest was reverse transcribed from RNA which was provided as part of a retroviral vector, and wherein said transduced endothelial cells have been engineered to express said incorporated genetic material of interest.
Claim 25
Dependent Claim
13 words
Transduced endothelial cells according to claim 21, wherein said cells are human cells.
Claim 26
Independent Claim
78 words
A method of making transduced endothelial cells which express integrated genetic material of interest encoding at least one polypeptide of interest, comprising the steps of: a) contacting endothelial cells with media containing an infectious recombinant retrovirus having a recombinant genome comprising the genetic material of interest; and b) maintaining the endothelial cells and the media containing the infectious recombinant retrovirus under conditions appropriate for infection of the endothelial cells by said recombinant retrovirus, thereby producing transduced endothelial cells.
Claim 27
Independent Claim
78 words
A method of making transduced endothelial cells which express integrated genetic material of interest encoding at least one polypeptide of interest, comprising the steps of: a) contacting endothelial cells with media containing an infectious recombinant retrovirus having a recombinant genome comprising the genetic material of interest; and b) maintaining the endothelial cells and the media containing the infectious recombinant retrovirus under conditions appropriate for infection of the endothelial cells by said recombinant retrovirus, thereby producing transduced endothelial cells.
Claim 28
Dependent Claim
16 words
A method according to claim 23 wherein said infection of the endothelial cells occurs in vitro.
Claim 29
Independent Claim
46 words
A method of producing a confluent layer of endothelial cells lining the inner surface of a synthetic prosthetic vessel, comprising applying to the inner surface of the vessel endothelial cells comprising incorporated genetic material encoding an endothelial cell mitogen, under conditions appropriate for endothelial cell proliferation.
Claim 30
Independent Claim
46 words
A method of producing a confluent layer of endothelial cells lining the inner surface of a synthetic prosthetic vessel, comprising applying to the inner surface of the vessel endothelial cells comprising incorporated genetic material encoding an endothelial cell mitogen, under conditions appropriate for endothelial cell proliferation.
Claim 31
Dependent Claim
15 words
A method according to claim 25, wherein the genetic material encodes endothelial cell growth factor.
Claim 32
Independent Claim
76 words
A method of enhancing binding of endothelial cells to a surface of a synthetic vessel, comprising the steps of: a) applying to the surface of said vessel a ligand, to produce a ligand-bearing surface; b) seeding onto said ligand-bearing surface, said endothelial cells wherein said endothelial cells contain integrated genetic material encoding a membrane receptor which binds the ligand; and c) maintaining the vessel under conditions appropriate for binding of said ligand and said membrane receptor.
Claim 33
Independent Claim
76 words
A method of enhancing binding of endothelial cells to a surface of a synthetic vessel, comprising the steps of: a) applying to the surface of said vessel a ligand, to produce a ligand-bearing surface; b) seeding onto said ligand-bearing surface, said endothelial cells wherein said endothelial cells contain integrated genetic material encoding a membrane receptor which binds the ligand; and c) maintaining the vessel under conditions appropriate for binding of said ligand and said membrane receptor.
Claim 34
Dependent Claim
42 words
A method of producing a prosthetic vessel having endothelial cells which express incorporated genetic material of interest on the inner surface of the vessel, comprising lining the inner surface of the vessels with endothelial cells produced by the method of claim 24.
Claim 35
Independent Claim
97 words
A method of producing a prosthetic vessel lined with endothelial cells which express incorporated genetic material of interest, comprising the steps of: a) contacting cultured endothelial cells with media containing an infectious recombinant retrovirus having a recombinant genome comprised of the genetic material of interest; b) maintaining the cultured endothelial cells with media containing an infectious recombinant retrovirus, under conditions appropriate for infection of the endothelial cells by recombinant retrovirus; and c) lining the inner surface of a prosthetic vessel with the endothelial cells infected in step (b) under conditions appropriate for maintenance of the endothelial cells.
Claim 36
Independent Claim
97 words
A method of producing a prosthetic vessel lined with endothelial cells which express incorporated genetic material of interest, comprising the steps of: a) contacting cultured endothelial cells with media containing an infectious recombinant retrovirus having a recombinant genome comprised of the genetic material of interest; b) maintaining the cultured endothelial cells with media containing an infectious recombinant retrovirus, under conditions appropriate for infection of the endothelial cells by recombinant retrovirus; and c) lining the inner surface of a prosthetic vessel with the endothelial cells infected in step (b) under conditions appropriate for maintenance of the endothelial cells.
Claim 37
Dependent Claim
83 words
Transduced endothelial cells containing stably maintained genetic material of interest, said transduced endothelial cells having been engineered to express the polypeptide or protein product encoded by the genetic material of interest in vivo, wherein said genetic material comprises a retroviral vector, wherein said retroviral vector comprises in operable combination: 5' LTR sequence and 3' LTR sequence derived from a retrovirus of interest, an insertion site for a gene of interest, and said vector does not contain a complete gag, env, or pol gene.
Claim 38
Dependent Claim
83 words
Transduced endothelial cells containing stably maintained genetic material of interest, said transduced endothelial cells having been engineered to express the polypeptide or protein product encoded by the genetic material of interest in vivo, wherein said genetic material comprises a retroviral vector, wherein said retroviral vector comprises in operable combination: 5' LTR sequence and 3' LTR sequence derived from a retrovirus of interest, an insertion site for a gene of interest, and said vector does not contain a complete gag, env, or pol gene.
Claim 39
Dependent Claim
75 words
Transduced endothelial cells of claim 30 in which said genetic material of interest is selected from the group consisting of: DNA which is present in and expressed by normal endothelial cells; DNA which does not normally occur in endothelial cells; DNA which normally occurs in endothelial cells but is not expressed in them at levels which are biologically significant; and any DNA which can be modified so that it can be expressed in endothelial cells.
Claim 40
Dependent Claim
16 words
Transduced endothelial cells of claim 31, further comprising DNA which encodes at least one selectable marker.
Claim 41
Dependent Claim
31 words
Transduced endothelial cells of claim wherein said genetic material of interest encodes a protein or polypeptide selected from the group consisting of a hormone, a receptor, an enzyme and a polypeptide.
Claim 42
Dependent Claim
27 words
Transduced endothelial cells of claim 31, wherein said genetic material of interest encodes human parathyroid hormone, tissue plasminogen activator, Factor VIII, low density lipoprotein receptor or beta-galactosidase.
Claim 43
Dependent Claim
34 words
Transduced human endothelial cells having genetic material of interest integrated therein, the transduced human endothelial cells having been engineered to express the polypeptide or protein encoded by said genetic material of interest in vivo.
Claim 44
Dependent Claim
34 words
Transduced human endothelial cells having genetic material of interest integrated therein, the transduced human endothelial cells having been engineered to express the polypeptide or protein encoded by said genetic material of interest in vivo.
Claim 45
Dependent Claim
75 words
Transduced human endothelial cells of claim 35, wherein said genetic material of interest is selected from the group consisting of: DNA which is present in and expressed by normal endothelial cells; DNA which does not normally occur in endothelial cells; DNA which normally occurs in endothelial cells but is not expressed in them at levels which are biologically significant; and any DNA which can be modified so that it can be expressed in endothelial cells.
Claim 46
Dependent Claim
18 words
Transduced human endothelial cells of claim 36, additionally comprising DNA which encodes at least one dominant selectable marker.
Claim 47
Dependent Claim
34 words
Transduced human endothelial cells of claim 36 in which the genetic material of interest encodes a protein or polypeptide selected from the group consisting of: a hormone, a receptor, an enzyme, and a polypeptide.
Claim 48
Dependent Claim
28 words
Transduced human endothelial cells of claim 38 wherein said genetic material of interest encodes human parathyroid hormone, tissue plasminogen activator, Factor VIII, low density lipoprotein receptor, or beta-galactosidase.
Claim 49
Dependent Claim
22 words
Cultured endothelial cells containing integrated DNA encoding a selected protein not made by naturally-occurring endothelial cells and DNA encoding a selectable marker.
Claim 50
Dependent Claim
22 words
Cultured endothelial cells containing integrated DNA encoding a selected protein not made by naturally-occurring endothelial cells and DNA encoding a selectable marker.
Claim 51
Dependent Claim
35 words
Transduced endothelial cells that have been engineered to express genetic material of interest integrated therein, wherein said genetic material of interest was reverse transcribed from RNA which was provided as part of a retroviral vector.
Claim 52
Dependent Claim
35 words
Transduced endothelial cells that have been engineered to express genetic material of interest integrated therein, wherein said genetic material of interest was reverse transcribed from RNA which was provided as part of a retroviral vector.
Claim 53
Dependent Claim
36 words
Transduced human endothelial cells that have been engineered to express genetic material of interest integrated therein, wherein said genetic material of interest was reverse transcribed from RNA which was provided as part of a retroviral vector.
Claim 54
Dependent Claim
36 words
Transduced human endothelial cells that have been engineered to express genetic material of interest integrated therein, wherein said genetic material of interest was reverse transcribed from RNA which was provided as part of a retroviral vector.
Claim 55
Independent Claim
105 words
A method of making transduced endothelial cells which express integrated genetic material of interest encoding at least one protein of interest or at least one polypeptide of interest, comprising the steps of: a) contacting endothelial cells with media containing an infectious recombinant retrovirus having a recombinant RNA genome comprising sequence corresponding to the DNA of interest wherein said retrovirus was derived from a retroviral vector selected from the group consisting of α-SGC and MFG; and b) maintaining said endothelial cells and the media containing the infectious recombinant retrovirus under conditions appropriate for infection of the endothelial cells by recombinant retrovirus, thereby producing transduced endothelial cells.
Claim 56
Independent Claim
105 words
A method of making transduced endothelial cells which express integrated genetic material of interest encoding at least one protein of interest or at least one polypeptide of interest, comprising the steps of: a) contacting endothelial cells with media containing an infectious recombinant retrovirus having a recombinant RNA genome comprising sequence corresponding to the DNA of interest wherein said retrovirus was derived from a retroviral vector selected from the group consisting of α-SGC and MFG; and b) maintaining said endothelial cells and the media containing the infectious recombinant retrovirus under conditions appropriate for infection of the endothelial cells by recombinant retrovirus, thereby producing transduced endothelial cells.
Claim 57
Dependent Claim
15 words
A method of claim 43 wherein said infection of the endothelial cells occurs in vitro.
Claim 58
Independent Claim
63 words
A method of producing a confluent layer of endothelial cells lining the inner surface of a synthetic prosthetic vessel, comprising applying to the inner surface of the vessel endothelial cells comprising incorporated genetic material encoding an endothelial cell mitogen, under conditions appropriate for endothelial cells proliferation wherein said genetic material comprises a retroviral vector selected from the group consisting of α-SGC and MFG.
Claim 59
Independent Claim
63 words
A method of producing a confluent layer of endothelial cells lining the inner surface of a synthetic prosthetic vessel, comprising applying to the inner surface of the vessel endothelial cells comprising incorporated genetic material encoding an endothelial cell mitogen, under conditions appropriate for endothelial cells proliferation wherein said genetic material comprises a retroviral vector selected from the group consisting of α-SGC and MFG.
Claim 60
Dependent Claim
14 words
A method of claim 45, wherein said genetic material encodes endothelial cell growth factor.
Claim 61
Dependent Claim
42 words
A method of producing a prosthetic vessel having endothelial cells which express incorporated genetic material of interest on the inner surface of said vessel, comprising lining the inner surface of said vessel with endothelial cells produced by the method of claim 44.
Claim 62
Dependent Claim
19 words
Transduced endothelial cells of claim 30 wherein said vector further comprises a portion of a gag gene coding sequence.
Claim 63
Dependent Claim
38 words
Transduced endothelial cells of claim 48 wherein said gag coding sequence comprises a splice donor site and a splice acceptor site, wherein said splice acceptor site is located upstream from an insertion site for said gene of interest.
Claim 64
Dependent Claim
40 words
Transduced endothelial cells of claim 49 wherein said vector further comprises a gag transcriptional promoter functionally positioned such that a transcript of a nucleotide sequence inserted into said insertion site is produced, wherein said transcript comprises gag 5' untranslated region.
Claim 65
Dependent Claim
14 words
Transduced endothelial cells of claim 50 wherein said endothelial cells are human endothelial cells.
Claim 66
Dependent Claim
75 words
Transduced endothelial cells of claim 51 in which the genetic material of interest is selected from the group consisting of: DNA which is present in and expressed by normal endothelial cells; DNA which does not normally occur in endothelial cells; DNA which normally occurs in endothelial cells but is not expressed in them at levels which are biologically significant; and any DNA which can be modified so that it can be expressed in endothelial cells.
Claim 67
Dependent Claim
22 words
Transduced endothelial cells of claim 50, wherein the genetic material of interest encodes a hormone, a receptor, an enzyme or a polypeptide.
Claim 68
Dependent Claim
27 words
Transduced endothelial cells of claim 52 wherein the genetic material of interest encodes human parathyroid hormone, tissue plasminogen activator, Factor VIII, low density lipoprotein receptor, or beta-galactosidase.
References Cited (Backward Citations)
WO1989005575A1
Cited
US5399346A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2000-10-06 | 2001-04-07 | 2001-10-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-10-06 | 2005-04-07 | 2005-10-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-10-06 | 2009-04-07 | 2009-10-07 | $7700.00 | Window Closed / Lapsed |