WO2000017375A2 - Procede de traitement de l'hemophilie par therapie genique in vivo a l'aide de vecteurs retroviraux - Google Patents

Procede de traitement de l'hemophilie par therapie genique in vivo a l'aide de vecteurs retroviraux Download PDF

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WO2000017375A2
WO2000017375A2 PCT/EP1999/007384 EP9907384W WO0017375A2 WO 2000017375 A2 WO2000017375 A2 WO 2000017375A2 EP 9907384 W EP9907384 W EP 9907384W WO 0017375 A2 WO0017375 A2 WO 0017375A2
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fviii
level
retroviral vector
biologically active
protein
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WO2000017375A3 (fr
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Thierry Vandendriessche
Marinee K. L. Chuah
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Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
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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
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • 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
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10344—Chimeric viral vector comprising heterologous viral elements for production of another viral vector

Definitions

  • the invention relates to a gene transfer system more in particular to pseudotyped retroviral vectors, allowing stable expression of biologically active proteins at therapeutical, physiological or supraphysiological level, useful for in vivo gene therapy.
  • the present invention relates particularly to a method to treat haemophilia using pseudotyped retroviral vectors encoding coagulation factors, such as coagulation factor VIII (FVIII) or coagulation factor IX (FIX).
  • coagulation factor VIII FVIII
  • FIX coagulation factor IX
  • Haemophilia is characterized by spontaneous and prolonged bleeding in the joints, muscle and internal organs. It is potentially life-threatening and is often associated with disabling arthropathy resulting from the recurring joint bleeding episodes.
  • Haemophilia A and B are congenital X-chromosome linked coagulation disorders, due to a deficiency in coagulation factor VIII (FVIII) or factor IX (FIX) respectively, which are normally expressed in the liver.
  • Haemophilia A occurs in 1 in 10,000 whereas haemophilia B affects 1 in 30,000 males.
  • FVIII has no intrinsic enzymatic activity and functions as a cofactor to accelerate the activation of factor X by activated FIX in the presence of calcium and phospholipids.
  • the coagulation cascade leads to the localized generation of thrombin and conversion of fibrinogen to insoluble fibrin polymers, which in conjunction with platelet aggregation maintains hemostasis.
  • Retroviral vector-mediated transduction of primary cells with the gene encoding coagulation FVIII or FIX offers the potential of long-term gene expression in haemophilia A or haemophilia B patients, respectively and hence phenotypic correction of the bleeding disorder.
  • previous reports on ex vivo transduction of hematopoietic stem/progenitor cells with FVIII-retroviral vectors followed by reimplantation in normal (Hoeben et al., 1992) or haemophilic mice (Evans and Morgan, 1998) did not yield detectable FVIII protein and the haemophilic phenotype could not be corrected.
  • An alternative gene transfer approach consists of using conjugates composed of the FVIII cDNA, adenoviral particles and transferrin as targeting ligand (Zatloukal et al., 1994).
  • conjugates composed of the FVIII cDNA, adenoviral particles and transferrin as targeting ligand
  • adenovirus-augmented gene delivery By combining receptor-mediated uptake of the FVIII gene via the transferrin receptor with adenovirus-augmented gene delivery, primary mouse fibroblasts were transfected with a B-domain (Toole et al., 1986) deleted FVIII expression plasmid.
  • B-domain Toole et al., 1986
  • low levels of FVIII could be detected for only one day after intrasplenic administration (Zatloukal et al., 1994). Since the use of these conjugates led to transient expression in vitro and in vivo, it is unlikely that this approach will be useful for the treatment of haemophilia A.
  • a further 10-100-fold increase in production would be required to reach a clinically beneficial range.
  • a novel procedure has been developed for mediating direct in vivo gene transfer into hematopoietic cells (Nelson et al., 1997). The procedure involves injection of irradiated retroviral producer cells, that produce retroviral vectors containing the human FIX cDNA, into the femoral bone marrow cavity in rabbits without preconditioning. The emergence of vector-marked cells in multiple peripheral blood hematopoietic lineages was detected 1 week post- injection and persisted until the animals were sacrificed up to 20 months later. Vector-marked cells were also detected in different hematopoietic tissues including bone marrow, spleen, thymus, and lymph node.
  • FIX protein was expressed in granulocytes, isolated 14 months after the procedure indicating that hematopoietic stem or early progenitor cells had been transduced in vivo. However, the FIX protein could not be detected in the plasma.
  • WO 98/00542 discloses a non-invasive in vivo gene therapy for haemophilia A using high-titer retroviral vectors.
  • These FVIII retroviral vectors contain an intron upstream of the B-domain (Toole et al., 1986) deleted FVIII cDNA.
  • Using these vectors it was shown that moderate levels of human FVIII can be expressed long-term in juvenile rabbits and in juvenile dogs following direct intravenous injection of high-titer retroviral vectors.
  • the normal physiological level of FVIII necessary in humans 200 ng/ml
  • the present invention provides a method to treat haemophilia A and/or B, using a gene transfer system or vector, more in particular a pseudotyped retroviral vector, to obtain a physiological level of FVIII and/or FIX over a long period of time.
  • a gene transfer system or vector more in particular a pseudotyped retroviral vector
  • This is the first time that stable, long-term therapeutic levels of FVIII or FIX and phenotypic correction of the bleeding disorder by gene therapy in a clinically relevant haemophilia A or B animal model such as haemophilia A or B mice have been demonstrated.
  • a genetic disease can surprisingly be corrected by direct in vivo retroviral treatment such as retroviral injection.
  • an intron-based Moloney murine leukemia virus retroviral vector comprising a B-domain (Toole et al., 1986) deleted human FVIII cDNA (designated as MFG-FVIIIDB) was pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G).
  • VSV-G vesicular stomatitis virus G glycoprotein
  • the invention is not limited to this specific pseudotyping, but other proteins such as the amphotropic 4070A amphotropic envelope, the Gibbon ape leukemia virus envelope (Miller et al., 1991 ) , the 10A1 envelope (Miller and Chen, 1996), the Sendai virus glycoprotein F (Spiegel et al., 1998), or the RD114 feline endogenous virus envelope (Cosset et al., 1995) may also be used for pseudotyping. Then the pseudotyped vector is concentrated by centrifugation.
  • proteins such as the amphotropic 4070A amphotropic envelope, the Gibbon ape leukemia virus envelope (Miller et al., 1991 ) , the 10A1 envelope (Miller and Chen, 1996), the Sendai virus glycoprotein F (Spiegel et al., 1998), or the RD114 feline endogenous virus envelope (Cosset et al., 1995) may also be used for pseudotyping. Then the pseudotyped vector
  • concentration such as ultracentrifugation (Yee et al., 1994, Miyanohara et al., 1995, Sekhar et al., 1996) precipitation (Sekhar et al., 1996), filtration (Kotani et al., 1994, Sekhar et al., 1996), lyophilization (Kotani et al., 1994) and/or chromatography may also be used to concentrate the vector. This high titer vector preparation is then injected, optionally with one or more transduction additives, into new-born recipients.
  • Retroviral vectors derived from retroviruses see RNA Tumor Viruses, 2 nd Edition, Cold Spring Harbour Laboratory, 1985) including for example onco-retroviruses (e.g. Moloney murine leukemia virus) as well as spumaviruses (e.g.
  • human foamy virus Russell and Miller, 1996) and lentiviruses (Naldini et al., 1996) such as Human Immunodeficiency Virus, Feline Immunodeficiency Virus (Poeschla et al., 1998), Simian Immunodeficiency Virus or Equine Infectious Anemia Virus, which do not require cell division for stable transduction, can also be used for gene therapy of haemophilia A or B, or for other genetic diseases.
  • lentiviruses such as Human Immunodeficiency Virus, Feline Immunodeficiency Virus (Poeschla et al., 1998), Simian Immunodeficiency Virus or Equine Infectious Anemia Virus, which do not require cell division for stable transduction, can also be used for gene therapy of haemophilia A or B, or for other genetic diseases.
  • Direct injection of high-titer retroviral vectors may also be useful for delivering proteins deficient in other diseases, for example in familial hypercholesterolemia, lysosomal storage disorders (Gaucher's disease) and other hepatic diseases or diseases resulting from plasma protein deficiencies.
  • the invention concerns a gene transfer system capable to sustain a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.
  • a gene transfer system capable to sustain by in vivo gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.
  • the invention also concerns a gene transfer system capable to sustain by in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.
  • a viral vector capable to sustain by in vivo or in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.
  • the invention relates to a retroviral vector capable to sustain by in vivo or in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.
  • An important aspect of the invention is a pseudotyped retroviral vector capable to sustain in vivo a therapeutic, physiological or supraphysiological level of a biologically active protein for at least 30 days, preferentially at least
  • a further aspect of the invention concerns said pseudotyped retroviral vector in which the biologically active protein is derived form the group of coagulation factors.
  • Another aspect of the invention is a pseudotyped retroviral vector capable to sustain in vivo a theurapeutic human coagulation factor VIII level in blood plasma and/or other bodily fluids .
  • the invention concerns a pseudotyped retroviral vector capable to sustain in vivo a human coagulation factor VIII level in blood plasma and/or other bodily fluids of at least 100 ng/ml, preferentially at least 130 ng/ml, more preferentially at least 200 ng/ml and this for at least 30 days, preferentially at least 100 days up to at least 440 days or more.
  • Another aspect of this invention is a method to treat haemophilia A and/or B, using said vectors or gene transfer system encoding coagulation factors such as FVIII and/or FIX respectively.
  • Still another aspect of the invention is the use of said vector(s) or gene transfer system to prevent induction of inhibitory and/or neutralising antibodies directed against said biologically active protein, in particular coagulation FVIII and/or FIX.
  • To the invention also belongs the use of a gene transfer system or a vector according to the invention for the manufacture of a medicament to treat haemophilia A and/or B. Definitions
  • Biologically active protein means any protein, polypeptide or peptide having a biological function.
  • Said protein can be, amongst others, an enzyme, a cytokine, a signalling molecule, a transcription factor, a cofactor or a structural protein and the like.
  • Biological activity of factor VIII refers to a function or set of functions performed by the polypeptide or fragments thereof in a biological system or in an in vitro system.
  • Gene transfer system means any method allowing delivery of genetic information or material in cells.
  • the system comprises a (non) viral vector allowing to deliver in one or more cells a biologically active protein.
  • In vivo gene therapy means direct injection, or other delivery method known to the people skilled in the art into an animal or a patient of a vector encoding a biologically active protein resulting in gene transfer of the recipient's cells in vivo.
  • Physiological level of a biologically active protein means the average level of said protein as determined in a healthy individual. The physiological level of
  • Supraphysiological level of a biologically active protein means a level of said protein that is significantly higher than the physiological level.
  • Moderate haemophilia A or B is characterized by functional FVIII or respectively FIX levels ranging from 1 to 5 percent (as measured with a functional assay, compared to the normal physiological level).
  • Mild haemophilia A or B is characterized by functional FVIII or respectively
  • FIX levels ranging from 6 to 40 percent (as measured with a functional assay, compared to the normal physiological level).
  • Therapeutic levels of FVIII or FIX means a level that converts severe to moderate haemophilia A or B, respectively; or preferably severe and moderate to mild haemophilia A or B, respectively; or more preferably severe, mild and moderate haemophilia A or B respectively, to normal in humans or in a clinically relevant haemophilia A or B animal model such as haemophilia A or B mice or dogs.
  • Levels below one percent FVIII ( ⁇ 2ng/ml) or FIX ( ⁇ 50 ng/ml) in humans or haemophilia A or B mice or dogs are defined as non-therapeutic.
  • Therapeutic levels for any other disease caused by an insufficient level and/or activity of a biologically active protein means a production of said biologically active protein in animal models or in human patient, suffering from said disease, that leads to a less severe form of said disease or leads to a correction of the disease.
  • Pseudotyped retroviral vector means a vector that comprises part of the genome of a retrovirus in a viral particle that comprises preferably a modified envelope or an envelope from another type of virus or from another retroviral strain than said retrovirus.
  • Vector construct refers to a nucleic acid construct which carries and is capable of directing the expression of a nucleic acid molecule of interest.
  • Factor VIII is a non-enzymatic cofactor found in blood in an inactive precursor form. Precursor factor VIII is converted to the active cofactor, factor Villa, through limited proteolysis at specific sites by plasma proteases, notably thrombin and factor IXa.
  • FVIII means antibodies that specifically inhibit the FVIII activity, as measured by the Bethesda assay and expressed in Bethesda units (BU). 1 BU is defined as the amount of antibody that leads to a 50% reduction in FVIII activity. A plasma with an activity higher than 0.5 BU is considered to contain inhibitory antibodies.
  • Inhibition of induction of neutralising and/or inhibitory antibodies means that no such antibodies can be detected using an Elisa test (i.e. level ⁇ 1 ng/ml using Mab18 as control) and/or the Bethesda assay (i.e. activity ⁇ 0.5 BU/ml).
  • Tail-clipping was performed 2 months post-injection except for mice #5 and #6 (3 months).
  • mice #9 to #13, in Table I Five out of 13 mice injected with VSV 293 -FVIII vector (#9 to #13, in Table I) and all control mice injected with PBS (9 out of 9) did not yield detectable FVIII.
  • Fig. 2 Reciprocal correlation between FVIII activity and inhibitor titer in transient FVIII expressors.
  • Functional FVIII expression, (#, H ) and inhibitory antibody titer (O, D ) of mouse #7 (circles) and #8 (squares) (see Table I) were shown at different intervals post-injection of VSV 293 -FVIII.
  • FIG. 3 Modulation of infectivity of VSV 293 -FVIII.
  • Viral vector producer cells were grown in the presence (+) or absence (-) of tetracycline (Tet), to repress or induce VSV-G expression, respectively, and incubated with (+) or without (- ) neutralizing VSV-G specific Mab prior to transduction of COS-7 cells in vitro or injection in FVIII-deficient neonates.
  • FVIII activity was determined in the 24 hr-conditioned medium of the transduced COS-7 cells and the relative transduction efficiencies were determined by FVIII-specific PCR (B) (lanes 2- 5).
  • PCR was also performed on DNA from liver (lane 7), spleen (lane 8) and lungs (lane 9) of FVIII-deficient recipient mice injected with the same inactivated concentrated vector preparations. Positive control derived from FVIII-containing cells (lane 10) and molecular weight (MW) marker corresponding to the Smart Ladder (lanes 1 & 6) (Eurogentec, Belgium) are included and FVIII (1.1 kb)-specific fragments are indicated.
  • Fig. 4 Analysis of gene transfer efficiency by quantitative PCR in liver, spleen and lungs (A) and in testis, heart, brain, kidney, stomach and intestine (B).
  • the average vector copy number per diploid genomic equivalent was determined by comparison with a serially diluted linear standard (correlation coefficient r 2 -- 0.98) ranging from 0.5 to 0 (negative control) copies per diploid genomic equivalent.
  • Organs from individual mice were indicated (#1 , #2, #5, #7 from Fig.1 and Table I).
  • Mouse #1 and #2 were not sacrificed but a liver biopsy was taken instead and decreasing amounts of target DNA were used as template: 200 ng (a), 100 ng (b) and 50 ng (c).
  • a constant amount of total DNA (200 ng) was maintained in the standard and in the liver samples from mouse #1 and #2 by adding spleen DNA from a FVIII-deficient mouse.
  • FVIII 1.1 kb
  • control b-actin 0.2 kb
  • the MW marker corresponds to the Smart Ladder.
  • Fig. 5 Analysis of gene transfer efficiency by quantitative PCR in liver, spleen and lungs of non-expressor (#10, #11 , #13, from Table I) and transient expressor mice (#7). Bands corresponding to the amplified FVIII or control 2-acftn-specific fragments are indicated (1.1 kb, 0.2 kb, respectively). The same standard was used as described in the legend of Fig. 4.
  • the MW marker corresponds to the Smart Ladder for the FVIII-PCR and the 1 kb ladder for ⁇ -actin.
  • Fig. 6 Expression analysis of FVIII mRNA by RT-PCR in transduced organs derived from either FVIII-expressor mice (#1 , #2, #5 from FigJ and Table I) or a PBS-injected FVIII-deficient mouse as negative (-) control. RNA samples with (+) or without (-) RT as controls were shown to exclude genomic DNA amplification and FVIII -specific RT-PCR products were indicated (0.7 kb).
  • the non-pseudotyped MFG-FVIIIDB vector (Dwarki et al., 1995) was produced and titrated as previuosly described (Chuah et al. 1998).
  • subconfluent 293GPG packaging cells were transduced successively with conditioned medium containing the MFG-FVIIIDB vector in the presence of polybrene (8 microgram/ml) and tetracycline (1 microgram/ml ).
  • the 293GPG cell line expresses VSV-G in a conditional tetracycline-regulated manner.
  • VSV-G expression was induced leading to syncytia formation and production of VSV-G pseudotyped vector particles, whereas VSV-G expression was repressed by addition of tetracycline, allowing normal cell growth. Individual clones were obtained by limiting dilution.
  • a random-primed vector- specific probe was derived by PCR as described (Chuah et al., 1998) using the MFG-FVIIIDB plasmid as target and primers spanning a 418-bp region within the packaging sequence (5'-GGGCCAGACTGTTACCACTCCC-3' and 5'-GCGCCTAGAGAAGGAGTGAGGG-3'). Additional controls consisted of serially diluted viral vector supematants with known functional titer based on VSV-G pseudotyped vectors containing a neomycine resistance gene (LXSN) (Miller and Rosman, 1989).
  • LXSN neomycine resistance gene
  • Functional titer was determined by transduction of NIH-3T3 cells as previously described (Chuah et al., 1995) and expressed in G418-resistant colony forming units per ml (cfu/ml). To exclude the presence of rearranged proviral sequences, producer clones were subjected to Southern blot analysis (Chuah et al., 1995). Following expansion of the highest producer clone in the presence of tetracycline, semiconfluent cells were seeded in a 10-tray cell-factory (Nalge Nunc Inc.). When the plates were 80-90% confluent, VSV-G protein expression was induced by growing the cells in medium without tetracycline.
  • the supernatant was harvested at 24 or 48 hr interval over 2 weeks, frozen immediately on dry ice prior to storage at - 80°C and filtered using a 0.45 ⁇ m filter before use. Viral concentration was carried out by centrifugation (Bowles et al., 1996) and titer of concentrated vector preparations and yield was determined by RNA dot blot analysis as described above.
  • the titer achieved represents a significant 10 6 -fold increase in viral titer compared to non-pseudotyped, non- concentrated first-generation FVIII retroviral vectors (Chuah et al., 1995; Hoeben et al., 1990; Israel and Kaufman, 1990).
  • FVIII-deficient mice containing a disruption of the murine FVIII gene in exon- 17 were backcrossed with C57BI/6 mice over 5 generations. Genotyping and phenotypic characterisation of the offspring was performed as described (Bi et al., 1995) and confirmed that all FVIII-deficient mice used contained the disrupted murine FVIII gene.
  • Two to three days old homozygous female and hemizygous male FVIII-deficient littermates 14 mice in total
  • nine FVIII-deficient littermates were injected with PBS as control.
  • Biologically active human FVIII was quantified in citrated plasma samples in triplicate from mice by measuring the FVIII-dependent generation of factor Xa from factor X using a chromogenic assay (Coatest FVIII, Chromogenix,
  • FVIII-deficient mice was spiked with human plasma derived FVIII
  • Human FVIII-specific antibodies were detected by ELISA as previously described (Connelly et al., 1998) with some modifications. Plates were coated with 3 U/ml human plasma-derived FVIII (Octapharma, Langenfeld, Germany). Serially diluted monoclonal mouse anti-human FVIII antibody (Mab 18) specific for the light chain was used as a control (Gilles et al., 1993). The lowest concentration of this antibody that could still be detected was 0.5 ng/ml. The human FVIII inhibitory antibody titer in the serum samples that scored positive in ELISA was determined with a Bethesda assay as described (Kasper et al., 1975).
  • Inhibition of FVIII activity by serially diluted mouse plasma was measured using a functional FVIII Coatest (Chuah et al., 1995, 1998).
  • One Bethesda Unit (BU) was defined as the amount of antibody that leads to a 50% reduction in FVIII activity.
  • the functional FVIII activity in plasma was completely and specifically inhibited with polyclonal anti-FVIII antibodies from a haemophilia patient and with a monoclonal anti-human FVIII antibody (Mab18) (Gilles et al., 1993). Expression of FVIII was transient in 2 of the 13 (15%) and non-detectable in 5 of the 13 (38%) animals receiving the high-titer vector (Fig. 1 , Table 1). Six of the seven animals with transient or no expression of human FVIII developed FVIII inhibitors as measured by ELISA and Bethesda assays (7 to 350 BU/ml), continued to bleed following tail-clipping and died within a few hours after injury (Table 1 ). Functional human FVIII or human FVIII inhibitory antibodies could not be detected in any of the FVIII-deficient control animals injected with PBS (Table 1) which all died following tail-clipping whereas control C57BI/6 mice survived the tail-clipping.
  • PCR polymerase chain reaction
  • Genomic DNA was extracted from different organs by phenol-chloroform extraction. To determine relative transduction efficiencies in the various organs, PCR was performed on 200 ng of DNA using primers specific for the FVIII cDNA spanning the B-domain deletion (5'-GATGAGAACCGAAGCTGG- 3' and 5'-GTCAAACTCATCTTTAGTGGGTGC-3') and ⁇ -actin specific primers.
  • PCR was performed using AmpliTaq Gold (Perkin Elmer) by denaturation for 10 min at 95°C, followed by 30 cycles for FVIII (and 28 cycles for ⁇ -actin ) of 1 min at 95°C, 1 min at 59°C, 2 min at 72°C and a final extension for 5 min at 72°C, yielding a 1.1 kb FVIII (B-domain deleted)- specific PCR product and a 0.2 kb ⁇ -actin specific product.
  • Serially diluted DNA obtained from a producer clone containing 5 integrated FV7//-proviral copies was used as a standard to calculate the average vector copy number per diploid genomic equivalent in the various organs.
  • a constant amount of DNA (200 ng) was maintained in the standard by adding spleen DNA from a FVIII-deficient mouse. Amplified products were separated by gel electrophoresis on 1.5% agarose gels. The intensities of the PCR-amplified vector-specific FV7//-fragment relative to the standard were quantified with a Stratagene Eagle Eye II and NIH Image 1.61/ppc software after background subtraction and ⁇ -actin normalization.
  • RT-PCR reverse transcriptase PCR
  • Viral vector producer cells were grown in the presence or absence of tetracycline, to repress or induce VSV-G expression, respectively.
  • the conditioned medium was concentrated 1000-fold by centrifugation and incubated at 4°C for at least 30 min with or without 10% (v/v) anti-VSV-G Mab (11 , 1-2 mg/ml ascites) (Lyles et al., 1982) prior to transducing COS-7 cells (10 5 cells/ml) or injection into FVIII-deficient neonates.
  • In vitro transductions were performed by centrifuging vector and target cells at 2600 rpm for 1 hr (32°C) with polybrene (8 mg/ml).
  • Cosset FL Takeuchi Y, Battini JL, Weiss RA, Collins MK: High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol 69:7430-6, 1995
  • the human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate. Mol Cell Biol 16: 4264-72, 1996
  • Miller AD Chen F: Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry. J Virol 70: 5564- 71 , 1996 Miller AD, Rosman GJ: Improved retroviral vectors for gene transfer and expression. Biotechniques 7:980-2, 1989

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Abstract

L'invention concerne un système de transfert génique, de préférence des vecteurs rétroviraux pseudotypés, permettant une expression stable de protéines actives biologiquement aux niveaux thérapeutique, physiologique ou supraphysiologique. L'invention concerne notamment une technique de traitement de l'hémophilie faisant appel à ces vecteurs.
PCT/EP1999/007384 1998-09-23 1999-09-21 Procede de traitement de l'hemophilie par therapie genique in vivo a l'aide de vecteurs retroviraux WO2000017375A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64681/99A AU6468199A (en) 1998-09-23 1999-09-21 Method to treat haemophilia by (in vivo) gene therapy with retroviral vectors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP98203203 1998-09-23
EP98203203.9 1998-09-23

Publications (2)

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WO2000017375A2 true WO2000017375A2 (fr) 2000-03-30
WO2000017375A3 WO2000017375A3 (fr) 2000-07-27

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002034296A1 (fr) * 2000-08-30 2002-05-02 Jiahui Xia Nouvel agent de therapie genique permettant de traiter l'hemophilie b et son procede de preparation
WO2005052171A2 (fr) * 2003-10-30 2005-06-09 Oxford Biomedica (Uk) Limited Vecteurs
US7541044B2 (en) 2004-01-09 2009-06-02 Oxford Biomedica (Uk) Limited Administration of 5T4 antigen and immune response of cells expressing 5T4 and CEA antigens
EP3155098A4 (fr) * 2014-06-11 2018-01-03 Howard, Tom E. Réparation de mutations et induction de tolérance du facteur viii et adnc, compositions, procédés et systèmes associés
US10272163B2 (en) 2012-12-07 2019-04-30 The Regents Of The University Of California Factor VIII mutation repair and tolerance induction
US11185573B2 (en) 2004-12-06 2021-11-30 Haplomics, Inc. Allelic variants of human factor VIII

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WO1998000542A2 (fr) * 1996-07-03 1998-01-08 Chiron Corporation Procedes d'administration d'excipients d'apport de genes recombines dans le traitement de l'hemophilie
WO1998053063A2 (fr) * 1997-05-16 1998-11-26 Leuven Research & Development Vzw Transduction de cellules de mammiferes utilisee en therapie genique

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1998000542A2 (fr) * 1996-07-03 1998-01-08 Chiron Corporation Procedes d'administration d'excipients d'apport de genes recombines dans le traitement de l'hemophilie
WO1998053063A2 (fr) * 1997-05-16 1998-11-26 Leuven Research & Development Vzw Transduction de cellules de mammiferes utilisee en therapie genique

Non-Patent Citations (6)

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Title
CHUAH ET AL: "BONE MARROW STROMAL CELLS AS TARGETS FOR GENE THERAPY OF HEMOPHILIA A" HUMAN GENE THERAPY, vol. 9, no. 3, 10 February 1998 (1998-02-10), pages 353-365, XP002090778 cited in the application *
DWARKI V J ET AL: "GENE THERAPY FOR HEMOPHILIA A: PRODUCTION OF THERAPEUTIC LEVELS OF HUMAN FACTOR VIII IN VIVO IN MICE" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 92, no. 4, 14 February 1995 (1995-02-14), pages 1023-1027, XP000569659 cited in the application *
KREUZ ET AL: "FACTOR VIII INHIBITORS IN PATIENTS WITH HEMOPHILIA A: EPIDEMIOLOGY OF INHIBITOR DEVELOPMENT AND INDUCTION OF IMMUNE TOLERANCE FOR FACTOR VIII" SEMINARS IN THROMBOSIS AND HEMOSTASIS, vol. 21, 1995, pages 382-389, XP002098129 cited in the application *
MIYANOHARA ET AL: "EFFICIENT IN VIVO TRANSDUCTION OF THE NEONATAL MOUSE LIVER WITH PSEUDOTYPED RETROVIRAL VECTORS" GENE THERAPY, vol. 2, 1995, pages 138-142, XP002098128 cited in the application *
VANDENDRIESSCHE THIERRY ET AL: "Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA AUG. 31, 1999, vol. 96, no. 18, 31 August 1999 (1999-08-31), pages 10379-10384, XP002136830 ISSN: 0027-8424 *
YEE J -K ET AL: "A GENERAL METHOD FOR THE GENERATION OF HIGH-TITER, PANTROPIC RETROVIRAL VECTORS: HIGHLY EFFICIENT INFECTION OF PRIMARY HEPATOCYTES" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 91, September 1994 (1994-09), pages 9564-9568, XP000674728 cited in the application *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002034296A1 (fr) * 2000-08-30 2002-05-02 Jiahui Xia Nouvel agent de therapie genique permettant de traiter l'hemophilie b et son procede de preparation
US7361639B2 (en) 2000-08-30 2008-04-22 Jiahui Xia Gene therapy agent for Haemophilia B and its preparation method
WO2005052171A2 (fr) * 2003-10-30 2005-06-09 Oxford Biomedica (Uk) Limited Vecteurs
WO2005052171A3 (fr) * 2003-10-30 2005-07-28 Oxford Biomedica Ltd Vecteurs
US7541044B2 (en) 2004-01-09 2009-06-02 Oxford Biomedica (Uk) Limited Administration of 5T4 antigen and immune response of cells expressing 5T4 and CEA antigens
US11185573B2 (en) 2004-12-06 2021-11-30 Haplomics, Inc. Allelic variants of human factor VIII
US10272163B2 (en) 2012-12-07 2019-04-30 The Regents Of The University Of California Factor VIII mutation repair and tolerance induction
US11083801B2 (en) 2012-12-07 2021-08-10 Haplomics, Inc. Factor VIII mutation repair and tolerance induction
EP3155098A4 (fr) * 2014-06-11 2018-01-03 Howard, Tom E. Réparation de mutations et induction de tolérance du facteur viii et adnc, compositions, procédés et systèmes associés

Also Published As

Publication number Publication date
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WO2000017375A3 (fr) 2000-07-27

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