Patent US5994127A successfully refreshed from Google Patents.
US5994127A
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In vivo production and delivery of erythropoietin or insulinotropin for gene therapy
Assignee: Shire Human Genetics Therapies Inc
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Filing: 1994-11-04
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Granted: 1999-11-30
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Expires: 2014-11-04
Abstract
Abstract The present invention relates to transfected primary and secondary somatic cells of vertebrate origin, particularly mammalian origin, transfected with exogenous genetic material (DNA) which encodes erythropoietin or an insulinotropin [e.g., derivatives of glucagon-like peptide 1(GLP-1)], methods by which primary and secondary cells are transfected to include exogenous genetic material encoding erythropoietin or an insulinotropin, methods of producing clonal cell strains or heterogenous cell strains which express eruthropoietin or an insulinotropin, methods of gene therapy in which the transfected primary or secondary cells are used, and methods of producing antibodies using the transfected primary or secondary cells. The present invention includes primary and secondary somatic cells, such as fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, other somatic cells which can be cultured and somatic cell precursors, which have been transfected with exogenous DNA encoding EPO or an insulinotropin, which is stably integrated into their genomes or is expressed in the cells episomally.
Inventors
- 👤 Richard F Selden
- 👤 Douglas Treco
- 👤 Michael W. Heartlein
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 23:43
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/87; C12N15/90; C12N15/902; C12N15/907; A; A61; A61K; A61K47/00; A61K47/50; A61K47/69; A61K47/6901; A61K48/00; A61P; A61P3/00; A61P3/06; A61P3/08; A61P3/10; A61P37/00; A61P37/02; A61P39/00; A61P39/06; A61P43/00; A61P5/00; A61P5/06; A61P5/18; A61P7/00; A61P7/02; A61P7/04; C07; C07K; C07K14/00; C07K14/435; C07K14/475; C07K14/505; C07K14/575; C07K14/605; C07K14/61; C12N15/63; C12N15/79; C12N15/85; C07K2319/00; C07K2319/01; C07K2319/02; C12N2510/00; C12N2510/02; C12N2800/00; C12N2800/10; C12N2800/108; C12N2830/00; C12N2830/001; C12N2830/002; C12N2830/80; C12N2830/85; C12N2840/00; C12N2840/20; C12N2840/44
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
70 words
An erythropoietin delivery composition comprising: 1) transfected primary or secondary cells of vertebrate origin which express erythropoietin upon introduction into a mammal, said transfected primary or secondary cells having stably integrated into their genomes: a) exogenous DNA which encodes erythropoietin; and b) exogenous DNA sufficient for expression of the exogenous DNA of a) in the transfected primary or secondary cells; and 2) means for administering said cells to said mammal.
Claim 2
Dependent Claim
70 words
An erythropoietin delivery composition comprising: 1) transfected primary or secondary cells of vertebrate origin which express erythropoietin upon introduction into a mammal, said transfected primary or secondary cells having stably integrated into their genomes: a) exogenous DNA which encodes erythropoietin; and b) exogenous DNA sufficient for expression of the exogenous DNA of a) in the transfected primary or secondary cells; and 2) means for administering said cells to said mammal.
Claim 3
Dependent Claim
38 words
The delivery composition of claim 1, wherein the cells are selected from the group consisting of fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, hepatocytes, and the progenitors thereof.
Claim 4
Dependent Claim
15 words
The delivery composition of claim 1, wherein the cells are from a clonal cell strain.
Claim 5
Dependent Claim
20 words
The delivery composition of claim 1, wherein the cells are a mixed population of two or more different cell strains.
Claim 6
Dependent Claim
20 words
The delivery composition of claim 1, wherein the transfected cells are provided in admixture with untransfected primary or secondary cells.
Claim 7
Dependent Claim
12 words
the delivery composition of claim 1, wherein the transfected cells are fibroblasts.
Claim 8
Dependent Claim
13 words
The delivery composition of claim 1 wherein the cells are of mammalian origin.
Claim 9
Dependent Claim
17 words
The delivery composition of claim 1 wherein the cells additionally include exogenous DNA encoding a selectable marker.
Claim 10
Dependent Claim
19 words
The delivery composition of claim 1 wherein the cells, in their untransfected state, do not make or contain erythropoietin.
Claim 11
Dependent Claim
13 words
The delivery composition of claim 7 wherein the cells are of human origin.
Claim 12
Independent Claim
157 words
A method of producing a delivery composition comprising a clonal cell strain of transfected secondary cells of vertebrate origin adapted for introduction into a mammal and expression of erythropoietin upon said introduction into a mammal, ad means for administering said cells to said mammal, said method comprising the steps of: a) producing a mixture of cells of vertebrate origin containing primary or secondary cells; b) transfecting The primary or secondary cells produced in (a) with a DNA construct comprising: i) exogenous DNA encoding erythropoietin; and ii) exogenous DNA sufficient for expression of the exogenous DNA in i) in the primary or secondary cells; thereby producing transfected primary or secondary cells which express erythropoietin; c) culturing a transfected primary or secondary cell which expresses the erythropoietin produced in (b), under conditions appropriate for propagating the transfected cell, thereby producing a clonal cell strain of transfected secondary cells, and (d) providing means for administering said cells to said mammal.
Claim 13
Independent Claim
157 words
A method of producing a delivery composition comprising a clonal cell strain of transfected secondary cells of vertebrate origin adapted for introduction into a mammal and expression of erythropoietin upon said introduction into a mammal, ad means for administering said cells to said mammal, said method comprising the steps of: a) producing a mixture of cells of vertebrate origin containing primary or secondary cells; b) transfecting The primary or secondary cells produced in (a) with a DNA construct comprising: i) exogenous DNA encoding erythropoietin; and ii) exogenous DNA sufficient for expression of the exogenous DNA in i) in the primary or secondary cells; thereby producing transfected primary or secondary cells which express erythropoietin; c) culturing a transfected primary or secondary cell which expresses the erythropoietin produced in (b), under conditions appropriate for propagating the transfected cell, thereby producing a clonal cell strain of transfected secondary cells, and (d) providing means for administering said cells to said mammal.
Claim 14
Dependent Claim
41 words
The method of claim 11 wherein the primary or secondary cells are selected from the group consisting of: fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, hepatocyte cells and the progenitors thereof.
Claim 15
Dependent Claim
20 words
The method of claim 11 wherein in step (b) the DNA construct additionally comprises DNA encoding an exogenous selectable marker.
Claim 16
Dependent Claim
60 words
The method of claim 11 wherein, in step (b), the primary or secondary cells are transfected with the DNA construct by combining the primary or secondary cells and the DNA construct and subjecting the resulting combination to electroporation under conditions which result in production of at least one primary or secondary cell having exogenous DNA stably integrated into genomic DNA.
Claim 17
Dependent Claim
31 words
The method of claim 11 wherein in step (b) the primary or secondary cells are transfected with the DNA construct by microinjecting the DNA construct into the primary or secondary cells.
Claim 18
Dependent Claim
45 words
The method of claim 11 wherein in step (b), the primary or secondary cells are transfected with the DNA construct by a method selected from the group consisting of: calcium phosphate precipitation, modified calcium precipitation, fusion methodologies, receptor mediated transfer, microprojectile bombardment and polybrene precipitation.
Claim 19
Dependent Claim
31 words
The method of claim 11 wherein in step (b), the exogenous DNA is introduced into genomic DNA by homologous recombination between DNA sequences present in the DNA construct and genomic DNA.
Claim 20
Dependent Claim
15 words
The method of claim 12 wherein the primary or secondary cells are of mammalian origin.
Claim 21
Dependent Claim
28 words
The method of claim 14 wherein electroporation is carried out at an electroporation voltage of between 250 and 300 volts and a capacitance setting of approximately 960 μFarads.
Claim 22
Dependent Claim
15 words
The method of claim 18 wherein the primary or secondary cells are of human origin.
Claim 23
Independent Claim
186 words
A method of producing a delivery composition comprising a clonal cell strain of secondary fibroblasts of mammalian origin adapted for introduction into a mammal and expression of erythropoietin upon said introduction into a mammal, and means for administering said fibroblasts to said mammal, comprising the steps of: a) providing primary fibroblasts of mammalian origin; b) producing a population of secondary fibroblasts from the primary fibroblasts provided in (a); c) combining the secondary fibroblasts with a DNA construct comprising: i) exogenous DNA encoding erythropoietin; and ii) exogenous DNA sufficient for expression of the exogenous DNA of i) in the fibroblasts; d) maintaining the combination produced in (c) under conditions which result in transfection of the DNA construct into the secondary fibroblasts, thereby producing a mixture of transfected secondary fibroblasts and non-transfected secondary fibroblasts; e) isolating a transfected secondary fibroblast produced in (d); f) culturing the transfected secondary fibroblast isolated in (e) under conditions appropriate for production of a clonal population of transfected secondary fibroblasts which express the exogenous DNA encoding erythropoietin; and g) providing means for administering said clonal population of transfected secondary fibroblasts to said mammal.
Claim 24
Independent Claim
186 words
A method of producing a delivery composition comprising a clonal cell strain of secondary fibroblasts of mammalian origin adapted for introduction into a mammal and expression of erythropoietin upon said introduction into a mammal, and means for administering said fibroblasts to said mammal, comprising the steps of: a) providing primary fibroblasts of mammalian origin; b) producing a population of secondary fibroblasts from the primary fibroblasts provided in (a); c) combining the secondary fibroblasts with a DNA construct comprising: i) exogenous DNA encoding erythropoietin; and ii) exogenous DNA sufficient for expression of the exogenous DNA of i) in the fibroblasts; d) maintaining the combination produced in (c) under conditions which result in transfection of the DNA construct into the secondary fibroblasts, thereby producing a mixture of transfected secondary fibroblasts and non-transfected secondary fibroblasts; e) isolating a transfected secondary fibroblast produced in (d); f) culturing the transfected secondary fibroblast isolated in (e) under conditions appropriate for production of a clonal population of transfected secondary fibroblasts which express the exogenous DNA encoding erythropoietin; and g) providing means for administering said clonal population of transfected secondary fibroblasts to said mammal.
Claim 25
Dependent Claim
20 words
The method of claim 21 wherein the secondary fibroblasts of step (d) are transfected with the DNA construct by electroporation.
Claim 26
Dependent Claim
27 words
The method of claim 21 further comprising maintaining the clonal population produced in step (f) for sufficient time and under appropriate conditions for at least 20 doublings.
Claim 27
Dependent Claim
32 words
The method of claim 22 wherein in step (d) the electroporation is carried out at an electroporation voltage of between 250 and 300 volts and a capacitance setting of approximately 960 μFarads.
Claim 28
Dependent Claim
66 words
An erythropoietin delivery composition comprising: 1) a clonal population of transfected secondary vertebrate fibroblasts which express erythropoietin upon introduction into a mammal, said transfected secondary fibroblasts having stably integrated into their genomes: a) exogenous DNA which encodes erythropoietin; and b) exogenous DNA sufficient for expression of the exogenous DNA of a) in the transfected secondary fibroblasts; and 2) means for administering said fibroblasts to said mammal.
Claim 29
Dependent Claim
66 words
An erythropoietin delivery composition comprising: 1) a clonal population of transfected secondary vertebrate fibroblasts which express erythropoietin upon introduction into a mammal, said transfected secondary fibroblasts having stably integrated into their genomes: a) exogenous DNA which encodes erythropoietin; and b) exogenous DNA sufficient for expression of the exogenous DNA of a) in the transfected secondary fibroblasts; and 2) means for administering said fibroblasts to said mammal.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2002-11-29 | 2003-05-31 | 2003-11-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2006-11-29 | 2007-05-31 | 2007-11-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2010-11-29 | 2011-05-31 | 2011-11-30 | $7700.00 | Window Closed / Lapsed |