EP0378576B1
Expired / Lapsed
EP
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Transduced fibroblasts and uses therefor
Assignee: Whitehead Institute for Biomedical Research
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Filing: 1988-09-08
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Granted: 1995-01-18
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Expires: 2008-09-08
Abstract
Abstract Fibroblasts 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 fibroblasts. Such fibroblasts are useful in delivering the encoded polypeptide or protein, such as an enzyme, a hormone or a drug, to an individual who has had a graft or implant of the transduced cells.
Inventors
- 👤 Richard C. Mulligan
- 👤 James M. Wilson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 06:46
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/635; A; A61; A61K; A61K48/00; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/65; C12N15/79; C12N15/85; C12N15/86; A61K38/00; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13041; C12N2740/13043; C12N2740/16011; C12N2740/16041; C12N2740/16043
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
87 words
Fibroblasts (e.g. human fibroblasts) in conjunction with (or bound to) a supporting matrix, the fibroblasts having been transduced with a recombinant helper-free retrovirus and incorporating genetic material of interest comprising DNA or RNA which; (a) does not normally occur in fibroblasts, (b) normally occurs in fibroblasts but which is not normally expressed at biologically significant levels, (c) normally occurs in fibroblasts but which has been modified so that it is expressed in the transduced fibroblasts, or (d) can be modified to be expressed in the transduced fibroblasts.
Claim 2
Dependent Claim
16 words
Fibroblasts according to claim 1 wherein the supporting matrix comprises microcarrier beads e.g. collagen-coated microcarrier beads.
Claim 3
Dependent Claim
36 words
Fibroblasts according to claim 1 or claim 2 wherein the incorporated genetic material further comprises material encoding at least one selectable marker, for example a dominant selectable marker, e.g. a gene encoding an antiobiotic resistance determinant.
Claim 4
Dependent Claim
13 words
Fibroblasts according to claim 3 wherein the marker is a neomycin resistance determinant.
Claim 5
Dependent Claim
53 words
Fibroblasts according to any one of the preceding claims wherein the incorporated genetic material encodes a therapeutic protein, for example; (a) a hormone, e.g. human parathyroid hormone or a fertility-regulating hormone, (b) an enzyme, or (c) a drug; the hormone, enzyme or drug not normally being made at biologically significant levels in fibroblasts.
Claim 6
Dependent Claim
46 words
Fibroblasts according to any one of the preceding claims, wherein the retrovirus is a recombinant amphotropic retrovirus having a genome comprising the long terminal repeat sequences, tRNA binding site and Psi packing site derived from amphotropic Moloney murine leukemia virus, and the genetic material of interest.
Claim 7
Dependent Claim
22 words
Fibroblasts according to claim 6 wherein the retroviral genome further comprises a eukaryotic promoter which can be modulated by an external cue.
Claim 8
Dependent Claim
291 words
Fibroblasts according to claim 6, wherein the retrovirus is any of pRO (pRO being vectors in which the gene of interest contains all the sequences necessary for transcription (i.e. promoter/enhancer, coding sequence with or without introns, and poly adenylation signal) in an orientation in which transcription of the gene is in a direction opposite to that of normal retroviral transcription), pEM (pEM being a vector in which the entire coding sequence for gag , pol and env of the wild type virus is replaced with the gene of interest, the 5' flanking sequence, 5' LTR, 400 bp of contiguous sequence (up to the BAMHI site) and 3' flanking sequence and LTR being from pZIP, while the cla site 150 bp upstream from the 3' LTR is linkered with BAMHl to form the other half of the BamHI cloning site present in the vector; the Hind III/ Eco Rl fragment of pBR322 forming the plasmid backbone) and pIP (pIP being a vector capable of expressing a single gene driven from an internal promoter, the 5' section of the vector, including the 5' flanking sequences, 5' LTR, and 1400 bp of contiguous sequence (up to the xho site in the gag region) being wild type Moloney Leukemia virus with a Sac II linker cloned into the Hae III restriction site immediately adjacent to the ATG of the gag gene, the 3' section of the vector, including the 3' flanking sequences, 3' LTR and 3' contiguous sequence (up to the cla I site in the env coding region) being from pZIP modified in that 1) the cla I site is linked to Bam HI and 2) a small sequence in the 3' LTR spanning the enhancer (from Pvu II to Xba I) is deleted).
Claim 9
Independent Claim
65 words
A method of making the fibroblasts of any one of claims 1 to 8, comprising the steps of: (a) contacting cultured fibroblasts with media containing amphotropic helper-free recombinant retrovirus having a recombinant genome comprising the genetic material of interest, and (b) maintaining the cultured fibroblasts and the media containing the infectious recombinant retrovirus under conditions appropriate for infection of the fibroblasts by the recombinant retrovirus.
Claim 10
Dependent Claim
23 words
A method according to claim 9 further comprising the step of culturing the fibroblasts of step (b) under conditions appropriate for their growth.
Claim 11
Dependent Claim
21 words
Fibroblasts according to any one of claims 1 to 8 for use in therapy, e.g. as a continuous drug delivery system.
Claim 12
Dependent Claim
27 words
Use of fibroblasts according to any one of claims 1-8 for the manufacture of a medicament for use in therapy, e.g. as a continuous drug delivery system.
References Cited (Backward Citations)
No citation records indexed.
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