WO1994028151A1 - Therapy genique s'appliquant a l'hemophilie - Google Patents

Therapy genique s'appliquant a l'hemophilie Download PDF

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Publication number
WO1994028151A1
WO1994028151A1 PCT/GB1994/001114 GB9401114W WO9428151A1 WO 1994028151 A1 WO1994028151 A1 WO 1994028151A1 GB 9401114 W GB9401114 W GB 9401114W WO 9428151 A1 WO9428151 A1 WO 9428151A1
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construct
promoter
nucleic acid
construct according
sequence
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PCT/GB1994/001114
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English (en)
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Geoffrey Goldspink
Christine Lee
Edward Tuddenham
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Royal Free Hospital School Of Medicine
Medical Research Council
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Priority to EP94915659A priority Critical patent/EP0698107A1/fr
Priority to JP7500375A priority patent/JPH09501306A/ja
Publication of WO1994028151A1 publication Critical patent/WO1994028151A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/30—Vector systems having a special element relevant for transcription being an enhancer not forming part of the promoter region
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to gene therapy, and to DNA vectors for the use in such treatment.
  • Genetic diseases which have been the subject of preliminary clinical trials include cystic fibrosis (CF) and adenosine deaminase (ADA) deficiency.
  • CF cystic fibrosis
  • ADA adenosine deaminase
  • Haemophilia A is an X-linked genetic disease caused by a defect in the gene coding for the blood clotting protein, Factor VIII.
  • the incidence of haemophilia is about 1 in 5,000 of the male births.
  • Sufferers from haemophilia are unable to clot blood properly at the site of wounds.
  • this poses for the treatment of open cuts the inability to clot blood properly causes damage to joints and to internal tissues, eg muscles.
  • Factor VIII Treatment of haemophilia A is possible by the administration of Factor VIII.
  • Factor VIII preparations had to be prepared by concentration of blood donations which was problematic in that the preparations could be contaminated with infectious agents such as Hepatitis B virus, Hepatitis C virus or HIV.
  • the gene for Factor VIII has been cloned (see for example Vehar et al, Nature, 1984, 312;337) and this has allowed the production of recombinant Factor VIII. Although this allows supplies of Factor vm protein which are of higher purity than blood concentrates, the exogenous supply of Factor VIII to a patient still means that repeated doses are required throughout the lifetime of a patient, which is inconvenient and expensive.
  • haemophilia include haemophilia B, caused by a defect in the gene coding for factor IX.
  • the present invention seeks to address the above mentioned problems by providing a DNA construct for gene therapy of haemophilia and other blood clotting disorders.
  • a class of muscle specific promoter sequences which can be linked to a gene encoding a functional human blood clotting protein carrying a signal sequence.
  • this enables the functional protein to be exported from the muscle cell, thus permitting delivery of the protein, via the bloodstream, to a desired site of action.
  • the use of a muscle specific promoter provides a steady constitutive level of expression which allows an effective amount of protein to be produced. The level of expression can be enhanced by stimulation of the muscle cells. This enables the output of the engineered gene product to be increased mimicking the natural situation with regard to clotting factor production (flight and fright response).
  • the present invention provides a recombinant nucleic acid construct comprising:
  • nucleic acid encoding a functional blood clotting protein
  • the elements (i)-(iii) being operably linked to provide for expression of the functional protein.
  • the myosin heavy chain promoter is one which will enable selective expression of the construct in muscle cells. This means that the construct will not function to any significant extent, if at all, in other cells types, eg liver or epithelial cells, in comparison to the levels of expression in muscle cells, especially skeletal muscle cells.
  • Suitable promoters include the ⁇ -myosin and ⁇ -myosin heavy chain promoters.
  • a preferred mammalian myosin heavy chain promoter is the ⁇ -myosin heavy chain promoter.
  • the human, pig and rabbit forms of this promoter have been obtained and these are set out below.
  • the promoter region of the human and porcine ⁇ -myosin heavy chains are shown numbered upstream from the presumed start of transcription.
  • the rabbit sequence can be obtained as described in Cribbs et al, J. Biol. Chem. 264; 1989, 10,672-10,678. Alternatively, it may be made synthetically based upon the sequence shown.
  • the other mammalian forms of these promoters can be obtained in an analogous manner.
  • fragments of the human promoter sequence given below can be synthesised and used as probes to probe a genomic DNA library made from humans or another species of mammal.
  • the probe will be used under conditions which will enable homologous sequences to hybridize. Suitable hybridization conditions can be determined by reference to standard manuals, eg. Sambrook et al, Molecular Cloning (1989, Cold Spring Harbor, N.Y).
  • the human ⁇ -myosin heavy chain promoter is set out in Seq. ID No.3. There is a "TATA" box at nucleotides 871-876 of the sequence and the start of transcription has been determined to be at the G nucleotide at position 906.
  • the porcine (pig) ⁇ -myosin heavy chain promoter is set out in Seq. ID No.4.
  • the "TATA" box region is highly homologous to the human sequence of Seq. ID No.3, and is at positions 822-827 of Seq. ID No.4.
  • the start of transcription has been determined to be at the A nucleotide at position 845.
  • the rabbit B-myosin heavy chain promoter is set out in Seq. ID No.5.
  • a "TATA" box is found at positions 653-658 and transcription starts a short distance downstream of this. The start of transcription from all the above promoters may be confirmed or determined by standard techniques such as SI mapping or primer extension.
  • a promoter region from the "TATA box" (usually found about 30 nucleotides from start of transcription) upstream to include sequence elements responsible for specificity of expression in muscle cells.
  • the myosin heavy chain promoter will desirably include at least all the nucleotides upstream (5') of the TATA box to about the 400th, eg 500th, 600th, 700th, 800th, 900th or further nucleotide 5' to the start of transcription. It is also preferred to include the TATA box together with the native sequence downstream of the TATA box to at least the start of transcription.
  • the promoter is modified to remove regions which control specificity of expression in different types of muscle cells. Desirably, this is achieved by truncating the promoter at about between 500 to 1000, preferably about 700 to 900, for example about 750 to 850 nucleotides upstream from the start of transcription of the myosin gene from which the promoter is derived.
  • ⁇ -myosin heavy chain promoters truncated in this way have the ability to be expressed in a variety of muscle types (eg. skeletal and cardiac).
  • the promoter may be modified to delete or alter specific regions responsible for this level of tissue specificity. For example, site-directed mutagenesis could be used to modify the sequence of regions of the promoter, and such promoters tested using constructs of the invention for their ability to express genes in both skeletal and cardiac muscle cells.
  • the invention also includes modified myosin heavy chain promoter sequences which are capable of selectively hybridizing to the naturally-occurring sequences.
  • a promoter sequence capable of selectively hybridizing to a naturally-occurring myosin heavy chain promoter sequence' will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the promoter region or fragment thereof over a region of at least 20, preferably at least 30, for instance 40, 60, 100 or 500 or more contiguous nucleotides.
  • Such promoter regions are included within the scope of the invention and are regarded as mammalian myosin heavy chain promoters. Desirably, these modified sequences still retain the tissue specificity of their natural counterparts.
  • a construct according to the invention is DNA, it may also be RNA or modified nucleic acid.
  • the nucleic acid may contain modifications in its backbone and possibly additions at either the 5' or 3', or both, ends of the molecule (in the case of linear, as opposed to circular, constructs). This may assist in prolonging the life of the DNA when taken up by muscle cells which may enhance the potency of the construct.
  • Known modification to DNA molecules include the provision of methylphosphonate and phosphorothioate backbones, and addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the signal sequence may be any mammalian signal sequence which can be made to export protein from muscle cells. We have found that the 19 amino acid factor VIII signal sequence set out in Seq. ID. No.1 will enable expressed proteins to be exported from muscle cells. DNA encoding this sequence, for example the DNA sequence of Seq. ID. No.1 may be used in the construct of the invention. Other examples of signal sequences which may be used include the signal sequences of insulin-like growth factors I (Jansen et al, Nature, 1984, 306;609-611) or II (Bell et al, Nature, 1984, 310:775-777). Other signal sequences may be obtained and tested for their ability to drive the export of the functional protein from muscle cells.
  • the signal sequence is capable of being cleaved from the functional protein during or following the export process so that the functional protein will appear in its native form within the vascular compartment.
  • Blood clotting proteins include coagulant and anti-coagulant proteins involved in physiological haemostatic mechanisms.
  • the nucleic acid encoding Factor VIII protein is preferably the human Factor VIII cDNA or an active fragment thereof.
  • the amino acid and DNA sequence may be obtained by reference to Wood et al (Nature, 1984, 312;330-337.)
  • the entire coding region of the cDNA from nucleotide 1 to nucleotide 7040 (as specified by Wood et al, ibid) may be used.
  • a modified cDNA lacking the B domain amino acids 712-1648 of Wood et al, ibid
  • the factor VIII gene contains a signal sequence to direct export of the factor VIII protein from a cell, and it is preferred that the signal sequence of the construct is the native factor VIII signal sequence.
  • factor IX the deficiency of which causes haemophilia B
  • factor IX the deficiency of which causes haemophilia B
  • the coding sequence for factor IX may be obtained by reference to Anson et al, Nature, 1985, 315;683-685.
  • Factor VII may also be expressed in a construct of the invention.
  • the factor VII sequence may be obtained by reference to Hagen et al, Proc. Natl. Acad. Sci., 1986, 83; 2412-2416.
  • Further blood clotting proteins which are preferred include Von Willebrand factor (the gene for which may be obtained by reference to Mancuso et al, J. Biol. Chem, 1989, 264; 19514-27), Factor X (Leytus et al, Biochemistry, 1986, 25; 5098-5102), Factor XI (Asakai et al, Biochemistry, 1987, 26; 7221-28), Factor V (Jenny et al, Proc. Natl. Acad. Sci.
  • Genes, in the form of genomic DNA, cDNA or engineered mini genes, encoding these and other blood clotting proteins may be used to produce constructs according to the present invention.
  • Engineered mini genes include cDNA sequences into which one or more introns have been introduced or genomic DNA sequences which have been modified to remove one or more introns.
  • “Operably linked” refers to a juxtaposition wherein the muscle specific regulatory element and nucleic acid encoding the signal sequence and the functional protein are in a relationship permitting them to function in their intended manner.
  • a promoter sequence according to the invention "operably linked" to the coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter and any other control sequences.
  • the construct may also contain a poly-adenylation signal operably linked 3' to the nucleic acid encoding the functional protein.
  • a poly-adenylation signal operably linked 3' to the nucleic acid encoding the functional protein.
  • the 3'-UT region will be about from 50 to 1000 base pairs.
  • Such a sequence may be, for example, the 3'-UT sequence of the gene which is being expressed.
  • it may be a 3'-UT sequence from a muscle specific gene, including the (8-myosin heavy chain gene itself. It will also be possible to use other mammalian or viral 3'-UT sequences.
  • the constructs of the invention may also contain an enhancer for the promoter.
  • Suitable enhancer elements include a myosin light chain enhancer sequence.
  • One such sequence is the 900 base pair myosin light chain enhancer element as described by Donoghue et al, Genes and Development, 1988: 2; 1779-1790.
  • the enhancer element may be inserted into a construct of the invention either 3' or 5' of the promoter and gene which are to be expressed. It is preferred that the enhancer is 3' to the gene and poly-adenylation signal.
  • the constructs according to the invention may be incorporated into vectors, for example, plasmid, virus or phage vectors provided with an origin of replication.
  • the vector may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a vector for mammalian cells.
  • the construct is incorporated into a plasmid vector, since it has been found that covalent closed circle (CCC) plasmid DNA can be taken up directly by muscle cells but that the DNA does not integrate into the genomic DNA of the cells.
  • CCC covalent closed circle
  • a further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of vectors according to the invention.
  • the cells will be chosen to be compatible with the vector and may for example be bacterial, yeast, insect or mammalian. While it is possible for the nucleic acid constructs of the invention to be administered alone it is preferable to present them as pharmaceutical formulations.
  • the formulations of the present invention comprise a nucleic acid construct according to the invention, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients.
  • the carrier or diluent will preferably be such that the composition is suitable for injection into skeletal muscle of a patient, for uptake of the construct or vector by the skeletal muscle cells.
  • Suitable liposomes include, for example, those comprising the positively charged lipid (N[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), those comprising dioleoylphosphatidylethanolamine (DOPE), and those comprising 3j8[N-(n',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol).
  • DOTMA positively charged lipid
  • DOPE dioleoylphosphatidylethanolamine
  • DC-Chol 3j8[N-(n',N'-dimethylaminoethane)-carbamoyl]cholesterol
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use.
  • Injection solutions and suspensions may be prepared extemporaneously from sterile powders, granules and tablets of the kind previously described.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question.
  • sterile pyrogen-free aqueous and non-aqueous solutions are preferred.
  • the invention also provides constructs, vectors and compositions according to the invention for use in a method of treatment of a mammal, including man.
  • the invention also provides a method of treatment of a human or animal subject, which subject is suffering from a deficiency in a functional protein, which comprises administering to the skeletal muscle of said subject an effective amount of a construct or vector according to the invention encoding said functional protein.
  • suitable dose are in the range of from about 10 ⁇ g to about 10 mg of DNA per kg of muscle tissue, eg. from about 100 ⁇ g to about 5 mg, eg. from about 1 mg to about 2.5 mg per kg of muscle tissue.
  • the DNA may be administered in a single dose or in divided multiple doses. If the DNA is administered in divided doses, the doses may be administered to different muscle tissues in the body. The doses may be administered sequentially, eg. at daily, weekly or monthly intervals, or in response to a specific need of the patient, eg. following injury or other physical trauma.
  • Preferred routes of administration are oral delivery and injection, typically intramuscular injection. Injection of the vaccine composition into the skeletal muscle of the human or animal subject is particularly preferred. Another mode of delivery of a vaccine composition according to the invention is by a biolistic or "particle gun" method.
  • a rabbit ⁇ -Myosin heavy chain promoter of 781 bp (Fig.1 and Cribbs et al., J Biol Chem, 1989, 264:10672-10678) was cloned into the Hind m site of the plasmid vector pUC 19, with the 5' end of the promoter facing away from the poly cloning site of the vector, leading to the clone pPR.
  • Clone pPRPAS-E9 was obtained by inserting a mouse myosin light chain enhancer element of 900 bp (Donoghue et al, Genes & Development, 1988, 2:1779-1790) into the Bam HI site of pPRPAS.
  • a cDNA sequence from blood clotting factor VIII spanning nucleotides 1-7040 was finally inserted into the Sal I site, thus leading to pPRF8PAS-E9.
  • the construct spans approximately 11,500 bp.
  • Large scale preparations were produced by transfection into competent E Coli (SURE Cells, Stratagene) and standard purification methods. Digests with specific restriction enzymes give rise to the following DNA fragment sizes [kb]:
  • the ⁇ -cardiac promoter was tested in bandshift assay (gel retardation assays) with nuclear protein extracts from various skeletal muscle tissues from rabbit.
  • bandshift assay gel retardation assays
  • the binding pattern of nuclear proteins to specific promoter regions could not detect a difference between soleus and tibialis anterior, making both muscles a candidate for gene expression driven by the cardiac promoter.
  • Mouse myoblasts from the cell line C2 were grown to 80% confluence (approx 1.5 ⁇ 10 6 cells) and transfected with 20 ⁇ g DNA of a chimeric plasmid, containing the CAT reporter gene drive by the ⁇ -cardiac promoter.
  • the same myoblasts were cotransfected with 10 ⁇ g pCH101, a ⁇ -gal reporter construct driven by a viral promoter. 24 hours after transfection differentiation was imitated by reducing the serum in the culture media from 10% fetal calf serum to 4% horse serum. Cells were harvested 1, 3, 5, 7 and 9 days after differentiation onset.
  • myosin light chain enhancer sequence has been identified.
  • Transfections of C2 myoblasts and cotransfections with pCH101 were carried out as described above for the ⁇ -cardiac promoter/CAT gene construct.
  • CAT assays revealed high activity of the construct in all differentiation stages.
  • the excision of the enhancer and subsequent transfection with a construct containing only promoter and CAT gene revealed a significantly lower expression compared to the full construct.
  • the enhancer element proved to upregulate expression in the chosen cell system.
  • Example 4 The construct of Example 1 was transfected into a myoblast cell culture system (C2 cells). 20 ⁇ g of plasmid DNA was incubated with myoblast monolayers cultured in 10% foetal bovine serum-DMEM in the presence of 2mM CaCl 2 for 16-24 hours. 48 hours after transfection the medium was changed to 4% horse serum-DMEM to induce fusion and differentiation of myoblasts to myotubes. Culture supernatant was harvested and stored for testing. Nine days after transfection all muscle cells were harvested and total RNA isolated by standard methods.
  • factor VIII construct Expression of the factor VIII construct was tested at the level of transcription. Using a reverse transcription polymerase chain reaction, factor VIII mRNA was identified, using primers to exon 14 so demonstrating the presence of factor VIII mRNA in transfected cells. Although a nested PCR was performed for clarity of result, the expected product was demonstrable after a single round of amplification.
  • Example 5 A construct analogous to the factor VIII construct of Example 1 was made except that the expression cassette contains 2.46kb factor VII cDNA (sequence as given by Hagen et al Proc. Natl. Acad. Sci, 1986: 83: 2412-2416) from nucleotide 1 to nucleotide 2426, cloned into the cassette Sal I site. This construct contains some 5' and 3' untranslated regions of factor VII.
  • the apparent discrepancy between the VII: Ag assay and VII clotting assay may be accounted for by the modifications made to VII: Ag assay when being used for analysis of the supernatant.
  • the clotting assay was able to treat the supernatant as any other plasma sample. Control supernatant had a VII activity of 4u/dl.
  • the demonstration of biological activity of supernatant in a one stage clotting assay demonstrates effective post translational terminal gamma carboxylation of transfected factor VII protein.
  • Example 4 50 ⁇ g of the factor VII construct of Example 4 in a hypertonic solution (20% sucrose final concentration) was injected directly into quadriceps muscles in two sites. At daily intervals, from day 1 to 7, and at 14 and 21 days, injected mice were sacrificed and bled by intracardiac puncture. Plasma samples were then assayed for VII: Ag(human VII: Ag is not cross reactive with murine VII: Ag in this assay system). All animal work was performed in duplicate. One animal (day 5 sacrifice) showed a 4 fold increase in VII: Ag compared to background. This result was confirmed on repeat testing.
  • Example 7 A B-domainless factor VIII is constructed.
  • the B domain of the factor VIII gene is inhibitory to the expression of factor VIII. Eliminating this domain improves gene expression.
  • the construct is made using analogous methods to those described in Example 1.
  • MOLECULE TYPE DNA (genomic) (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
  • MOLECULE TYPE DNA (genomic)
  • GGACTCTGTG CAAGAGGGCC CAGGCTGGTC CCAGGGGGCA GGGGCCGAGG GCAGCAGTGT 480
  • GGACTGGGTG CAGGTGGGGG TGGGGACGCC CTGCTGCCCC ATATATACAG CCCCTGACCA 840
  • MOLECULE TYPE DNA (genomic)
  • GTCCGCGCCC CTTAGCGAAC AGCTCTCCCT CCAGCTGCCC CCCTCCAGCC CCTGGTTCTG 180

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Abstract

L'invention concerne un produit de recombinaison de l'acide nucléique comprenant: (i) un promoteur de chaînes lourdes de la myosine mammifère; (ii) l'acide nucléique codant une séquence de signaux; et (iii) l'acide nucléique comprenant une protéine fonctionnelle de coagulation du sang; les éléments (i)-(iii) étant liés de manière fonctionnelle pour produire l'expression de la protéine fonctionnelle. Le produit de recombinaison est utile dans le traitement de maladies telles que l'hémophilie et est administré dans les cellules des muscles. L'activité du promoteur peut être augmentée par l'exercice ou la stimulation des cellules des muscles.
PCT/GB1994/001114 1993-05-20 1994-05-20 Therapy genique s'appliquant a l'hemophilie WO1994028151A1 (fr)

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EP94915659A EP0698107A1 (fr) 1993-05-20 1994-05-20 Therapy genique s'appliquant a l'hemophilie
JP7500375A JPH09501306A (ja) 1993-05-20 1994-05-20 血友病の遺伝子療法

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GB939310441A GB9310441D0 (en) 1993-05-20 1993-05-20 Gene therapy

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997011190A2 (fr) * 1995-09-19 1997-03-27 Pharmadigm, Inc. Adn de recombinaison contenant un element regulateur a specificite musculaire pour des immunisations ou pour des therapies geniques
WO1998049333A2 (fr) * 1997-04-25 1998-11-05 University College London Cassette d'expression genetique eucaryote et ses utilisations
WO1999054491A1 (fr) * 1998-04-20 1999-10-28 Children's Hospital Medical Center Utilisation de promoteurs a chaine lourde de la myosine murine en therapie genique et dans la production d'animaux transgeniques

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2198870A4 (fr) * 2007-08-27 2011-08-03 Univ Nagoya Nat Univ Corp Activateur pour le promoteur du facteur vii de la coagulation du sang et son utilisation

Citations (2)

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WO1997011190A3 (fr) * 1995-09-19 1997-06-26 Paradigm Biosciences Inc Adn de recombinaison contenant un element regulateur a specificite musculaire pour des immunisations ou pour des therapies geniques
US5795872A (en) * 1995-09-19 1998-08-18 Pharmadigm, Inc. DNA construct for immunization
AU710756B2 (en) * 1995-09-19 1999-09-30 Inflabloc Pharmaceuticals, Inc. DNA construct comprising a muscle specific regulatory element for immunization or gene therapy
WO1998049333A2 (fr) * 1997-04-25 1998-11-05 University College London Cassette d'expression genetique eucaryote et ses utilisations
WO1998049333A3 (fr) * 1997-04-25 1999-01-28 Royal Free Hosp School Med Cassette d'expression genetique eucaryote et ses utilisations
WO1999054491A1 (fr) * 1998-04-20 1999-10-28 Children's Hospital Medical Center Utilisation de promoteurs a chaine lourde de la myosine murine en therapie genique et dans la production d'animaux transgeniques

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GB9310441D0 (en) 1993-07-07
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