[go: up one dir, main page]

WO1994001129A1 - Utilisation de myoblastes en vue de l'administration prolongee de produits geniques - Google Patents

Utilisation de myoblastes en vue de l'administration prolongee de produits geniques Download PDF

Info

Publication number
WO1994001129A1
WO1994001129A1 PCT/US1992/007791 US9207791W WO9401129A1 WO 1994001129 A1 WO1994001129 A1 WO 1994001129A1 US 9207791 W US9207791 W US 9207791W WO 9401129 A1 WO9401129 A1 WO 9401129A1
Authority
WO
WIPO (PCT)
Prior art keywords
promoter
enhancer
muscle
myoblasts
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1992/007791
Other languages
English (en)
Inventor
Inder Verma
Yifan Dai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Salk Institute for Biological Studies
Original Assignee
Salk Institute for Biological Studies
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Salk Institute for Biological Studies filed Critical Salk Institute for Biological Studies
Priority to JP6503265A priority Critical patent/JPH08501210A/ja
Publication of WO1994001129A1 publication Critical patent/WO1994001129A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the medium comprises a source of essential amino acids, inorganic salts, trace elements and vitamins, as well as other organic components.
  • the following table indicates what has generally been found to be optimum, although as is known in the field, various changes may be made to individual components without deleteriously affecting the growth of the myoblasts.
  • the myoblasts may be transformed in any of a wide variety of ways, including, for example, fusion, transfection, infection, electroporation, ballistics, or the like.
  • the particular method employed for introducing foreign DNA into the myoblast cells is not crucial to the practice of the present invention.
  • Sequences for integration may include DNA associated with a particular muscular defect.
  • myoblasts may be removed from the host, transformed by homologous recombination, and cells cloned and screened for homologous recombination at the site of the defect.
  • the gene of interest is a naturally occurring gene, which is not normally expressed in a myoblast or mature muscle tissue
  • the transcriptional initiation regulatory sequence e.g., promoter with muscle-specific enhancer
  • the myoblasts may then be used for expression of an endogenous gene (native to the host) or heterologous gene, which is normally not expressed in muscle tissue.
  • an endogenous gene native to the host
  • heterologous gene which is normally not expressed in muscle tissue.
  • products such as cytokines, growth factors, colony stimulating factors, interferons, surface membrane receptors, insulin, or the like.
  • the myoblasts may provide for constitutive production of the expression product.
  • a receptor which provides for inducible transcription of a cellular, e.g., cytoplasmic, nuclear, etc., protein.
  • the myoblasts may be induced to produce the desired expression product under the induction of the relevant ligand.
  • Various vehicles or vector constructs may be employed for the transformation of the myoblast cells.
  • replication-defective viral vectors DNA virus or retroviral vectors, which may be introduced into the cells.
  • the vectors will normally be free of any prokaryotic DNA and may comprise a number of different functional sequences, as described above.
  • a marker may be present for selection of cells which contain the vehicle construct. Normally, the marker will allow for positive selection, by providing protection from one or more cytotoxic agents. For example, kanamycin resistance may be employed, where the cells may be selected with G418; dihydrofolate reductase may be employed for resistance to methotrexate, and the like.
  • the marker may be an inducible or non-inducible gene, so that selection may occur under inducing conditions or in the absence of inducing conditions.
  • the vector may also include an origin of replication and such other genes which are necessary for replication in the host.
  • the system comprising the origin and any genes encoding proteins associated with replication may be included as part of a construct.
  • Illustrative replication systems include Epstein-Barr virus.
  • replication defective vehicles may be employed, particularly replication-defective retroviral vectors. These vectors are described by Price et al., Proc. Natl. Acad. Sci. Vol. 84.:156-1650 (1987) and Sares et al., EMBO J. vol. . 5:3133-3142 (1986).
  • the final vehicle construct may have one or more genes of interest. Either a cDNA gene or a chromosomal gene may be employed. Of particular interest is to provide for at least one intron, which may be present in the 5'-non- coding region or in the coding region. It is found that the presence of an intron enhances stability of the messenger RNA.
  • cells may be transformed jln vivo by injection of replication-defective viral vectors, which are infectious.
  • the vectors may be introduced into retroviral producer cells for ecotropic packaging.
  • the cells are then collected, filtered and concentrated by centrifugation and the viral stock may then be injected into a site in vivo. Since it is found that the myoblasts will migrate, relatively few injections into the muscle fibers are required, since the myoblasts will expand into adjacent regions.
  • the injections will be about 10 5 cells per cm3 of muscle tissue.
  • the trauma to the tissue may be substantially minimized by having only a few injections in the region of interest. Particularly, where a patient may have need for extensive treatment, the desirability of having a low number of injections in a particular area is manifest.
  • CMV-IX i.e., LNcdF9L, as described by Axelrod et al., in Proc. Natl. Acad. Sci. USA 82:5173-5177
  • CMV-IX A schematic diagram of CMV-IX is presented in Figure 1.
  • MEaGPcap-IX was made by replacing the Sall-BamHI fragment of the CMV-IX vector (containing the CMV enhancer/promoter) with the 220 bp MCK enhancer- containing fragment, a 650 bp fragment containing the human alpha-globin promoter, and a 55 bp fragment from the Xenopus beta-globin 5 '-untranslated region [see Malone et al., in Proc. Natl. Acad. Sci. USA 8j5: 6077- 6081 (1989)].
  • a schematic diagram of MEaGPcap-IX (or EMaGPcap-IX, wherein the MCK enhancer is incorporated in the reverse orientation) is presented in Figure l.
  • ⁇ cre cells an ecotropic packaging cell line [described by Danos, D. and Mulligan, R.C. in Proc. Natl. Acad. Sci. USA 8.5:6460-6464 (1988)], were transfected with about 20 ⁇ g DNA of the above described three retroviral vectors, respectively, by the calcium phosphate precipitate method [see Chen, C. and Okayama, H. in Mol. Cell. Biol. 2(8) :2745-2752 (1987)]. The G418- resistant colonies were pooled and about ,3 ml of supernatant from these transfected cells were filtered through a 0.45 ⁇ m microfilter and added to AM12 cells, an amphotropic packaging cell line [described by Markowitz, D.
  • Hind leg muscles were taken from 3-4 day old Swiss Webster mice or Balb/C mice, minced and dissociated three times (for about 10-20 minutes for each repetition) in an aqueous solution containing 0.25% trypsin (Gibco) and 100 ⁇ g/ml collagenase.
  • the dissociated cells were seeded on a gelatin-coated dish in Dulbecco's modified Eagle's medium (DMEM) complemented with 20% fetal calf serum and 1% chick embryo extract (inactivated at 65°C for 10 minutes) .
  • Myoblasts were enriched by preplating the cells at 37°C for 20 minutes on a non-gelatin-coated dish to which fibroblasts preferentially adhere.
  • DMEM Dulbecco's modified Eagle's medium
  • Plasma was taken by bleeding the tail vein at times indicated in Figure 2, and an ELISA assay for the presence of dog factor IX in the mouse plasma was carried out [as described by Axelrod et al., in Proc. Natl. Acad. Sci. USA 82:5173-5177 (1990)].
  • IX decreased by day 8 and then increased and remained unchanged for at least 120 days. Up to 20 ng/ml of factor IX can be detected, which is approximately twice the amount detected following one transplantation. The levels of factor IX expression are, therefore, proportional to the number of myoblasts transplanted.
  • the orientation of the enhancer with respect to the CMV promoter was reversed to determine the effect, if any, of the relative orientation of the promoter and enhancer. Higher expression of factor IX was observed when the enhancer was in the same orientation as the promoter.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Des cellules myoblastiques contenant un produit de recombinaison qui comporte un fragment codant un produit génique à administrer sous la commande d'un promoteur/activateur musculo-spécifique sont utiles pour l'administration prolongée de produits géniques à un sujet.
PCT/US1992/007791 1992-07-02 1992-09-15 Utilisation de myoblastes en vue de l'administration prolongee de produits geniques Ceased WO1994001129A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP6503265A JPH08501210A (ja) 1992-07-02 1992-09-15 遺伝子産物の持続的放出のための筋芽細胞の利用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90788792A 1992-07-02 1992-07-02
US907,887 1992-07-02

Publications (1)

Publication Number Publication Date
WO1994001129A1 true WO1994001129A1 (fr) 1994-01-20

Family

ID=25424811

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/007791 Ceased WO1994001129A1 (fr) 1992-07-02 1992-09-15 Utilisation de myoblastes en vue de l'administration prolongee de produits geniques

Country Status (3)

Country Link
JP (1) JPH08501210A (fr)
CA (1) CA2137456A1 (fr)
WO (1) WO1994001129A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013376A1 (fr) * 1993-11-10 1995-05-18 Amgen Inc. Vecteur de therapie genique pour le traitement de l'insuffisance ou des troubles de la production de globules rouges
WO1997033975A1 (fr) 1996-03-12 1997-09-18 Rhone-Poulenc Rorer S.A. Milieu pour la conservation de materiel biologique
US5776747A (en) * 1994-07-20 1998-07-07 Cytotherapeutics, Inc. Method for controlling the distribution of cells within a bioartificial organ using polycthylene oxide-poly (dimethylsiloxane) copolymer
US5843431A (en) * 1994-07-20 1998-12-01 Cytotherapeutics, Inc. Controlling proliferation of cells before and after encapsulation in a bioartificial organ by gene transformation
US6410228B1 (en) 1997-07-14 2002-06-25 Baylor College Of Medicine Method for the identification of synthetic cell- or tissue- specific transcriptional regulatory regions
US6495364B2 (en) 1995-05-23 2002-12-17 Neurotech, S.A. Mx-1 conditionally immortalized cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990015863A1 (fr) * 1989-06-13 1990-12-27 The Board Of Directors Of The Leland Stanford Junior University Isolement, culture et differenciation de cellules musculaires humaines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990015863A1 (fr) * 1989-06-13 1990-12-27 The Board Of Directors Of The Leland Stanford Junior University Isolement, culture et differenciation de cellules musculaires humaines

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MOLECULAR AND CELLULAR BIOLOGY, Volume 10, No. 6, issued June 1990, SMITH et al., "Genes Transferred by Retroviral Vectors into Normal and Mutant Myoblasts in Primary Cultures are Expressed in Myotubes", pages 3268-3271. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 6, No. 7, issued July 1986, BERGSMA et al., "Delimitation and Characterization of cis-Acting DNA Sequences Required for the Regulated Expression and Transcriptional Control of the Chicken Skeletal alpha-Actin Gene", pages 2462-2475. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 8, No. 1, issued January 1988, JAYNES et al., "The Muscle Creatine Kinase Gene is Regulated by Multiple Upstream Elements, Including a Muscle-Specific Enhancer", pages 62-70. *
SCIENCE, Volume 254, issued 06 December 1991, BARR et al., "Systemic Delivery of Recombinant Proteins by Genetically Modified Myoblasts", pages 1507-1509. *
SCIENCE, Volume 254, issued 06 December 1991, DHAWAN et al., "Systemic Delivery of Human Growth Hormone by Injection of Genetically Engineered Myoblasts", pages 1509-1512. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013376A1 (fr) * 1993-11-10 1995-05-18 Amgen Inc. Vecteur de therapie genique pour le traitement de l'insuffisance ou des troubles de la production de globules rouges
US5843431A (en) * 1994-07-20 1998-12-01 Cytotherapeutics, Inc. Controlling proliferation of cells before and after encapsulation in a bioartificial organ by gene transformation
US5858747A (en) * 1994-07-20 1999-01-12 Cytotherapeutics, Inc. Control of cell growth in a bioartificial organ with extracellular matrix coated microcarriers
US5795790A (en) * 1994-07-20 1998-08-18 Cytotherapeutics, Inc. Method for controlling proliferation and differentiation of cells encapsulated within bioartificial organs
US5833979A (en) * 1994-07-20 1998-11-10 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US5840576A (en) * 1994-07-20 1998-11-24 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
EP1983053A2 (fr) 1994-07-20 2008-10-22 Neurotech USA, Inc. Contrôle de la distribution des cellules au sein d'organes bioartificiels
US5853717A (en) * 1994-07-20 1998-12-29 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US5776747A (en) * 1994-07-20 1998-07-07 Cytotherapeutics, Inc. Method for controlling the distribution of cells within a bioartificial organ using polycthylene oxide-poly (dimethylsiloxane) copolymer
US5935849A (en) * 1994-07-20 1999-08-10 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US6392118B1 (en) 1994-07-20 2002-05-21 Neurotech S.A. Mx-1 conditionally immortalized cells
US6495364B2 (en) 1995-05-23 2002-12-17 Neurotech, S.A. Mx-1 conditionally immortalized cells
WO1997033975A1 (fr) 1996-03-12 1997-09-18 Rhone-Poulenc Rorer S.A. Milieu pour la conservation de materiel biologique
US6410228B1 (en) 1997-07-14 2002-06-25 Baylor College Of Medicine Method for the identification of synthetic cell- or tissue- specific transcriptional regulatory regions
US7960536B2 (en) 1997-07-14 2011-06-14 Genetronics, Inc. Synthetic cell-or tissue-specific transcriptional regulatory regions

Also Published As

Publication number Publication date
JPH08501210A (ja) 1996-02-13
CA2137456A1 (fr) 1994-01-20

Similar Documents

Publication Publication Date Title
US5538722A (en) Isolation, growth, differentiation and genetic engineering of human muscle cells
CA2058955A1 (fr) Isolement, croissance et differenciation des cellules musculaires humaines
DE69636122T2 (de) Verfahren zum gentransfer in zielzellen mit einem retrovirus
US5219740A (en) Retroviral gene transfer into diploid fibroblasts for gene therapy
US6867020B2 (en) Method of making glycoprotein exhibiting erythropoiesis regulating and glycoprotein produced by this method
US4868116A (en) Introduction and expression of foreign genetic material in epithelial cells
DE69535560T2 (de) Ein verstärkter, durch viren vermittelter dna-transfer
US5460959A (en) Transduced fibroblasts
TWI239352B (en) Gene transfer method with the use of serum-free medium
AU757816B2 (en) Conditional immortalisation of cells
Naffakh et al. Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts.
JPH10513441A (ja) 哺乳類宿主の結合組織を治療するための遺伝子導入
JPH08511507A (ja) 哺乳動物宿主の結合組織を処置するための遺伝子導入
EP3831945A1 (fr) Hétérodimère protéique et son utilisation
EP0946199B1 (fr) CELLULES SENSIBLES A TGF beta 1 DERIVEES DE LA MOELLE OSSEUSE
EP0381490B1 (fr) Thérapie génique utilisant des cellules du stroma
CA2214385A1 (fr) Procede de culture cellulaire
WO1994001129A1 (fr) Utilisation de myoblastes en vue de l'administration prolongee de produits geniques
Bellini et al. Increases in weight of growth hormone‐deficient and immunodeficient (lit/scid) dwarf mice after grafting of hGH‐secreting, primary human keratinocytes
US6485722B1 (en) Method for selective engraftment of drug-resistant hematopoietic stem cells
Keith et al. Retrovirus mediated transfer and expression of GM-CSF in haematopoietic cells
Hamada et al. A new method of gene transfer into hematopoietic progenitors using liquid culture with interleukin-3 and interleukin-6
KR20020092910A (ko) 세포의 조건부 불멸화

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2137456

Country of ref document: CA