EP1578897A2 - Technique permettant d'induire une tolerance fonctionnelle a des produits de transfert genique - Google Patents

Technique permettant d'induire une tolerance fonctionnelle a des produits de transfert genique

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Publication number
EP1578897A2
EP1578897A2 EP00967159A EP00967159A EP1578897A2 EP 1578897 A2 EP1578897 A2 EP 1578897A2 EP 00967159 A EP00967159 A EP 00967159A EP 00967159 A EP00967159 A EP 00967159A EP 1578897 A2 EP1578897 A2 EP 1578897A2
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EP
European Patent Office
Prior art keywords
cells
stem cells
gene
vector
egfp
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.)
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Application number
EP00967159A
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German (de)
English (en)
Inventor
Goran K. Andersson
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Biotransplant Inc
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Biotransplant Inc
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Filing date
Publication date
Application filed by Biotransplant Inc filed Critical Biotransplant Inc
Publication of EP1578897A2 publication Critical patent/EP1578897A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes

Definitions

  • the invention relates to methods for reducing the immune response to gene transfer products. More particularly, the methods include the establishment of mixed molecular hematopoietic chimerism by the transplantation of hematopoietic stem cells that have been transduced by a gene therapy vector, followed by the transplantation of somatic cells also transduced by the same gene therapy vector.
  • the effector cells of the immune system that are involved in mounting a functional immune response include the CD4 + T helper cells, CD8 + T killer cells and B-lymphocytes.
  • the latter cell type produces antibodies to the gene therapy vector itself as well as antibodies that effectively neutralize neoantigens expressed from the transgene or neoantigens produced as a result of the activity of the molecule encoded by the transgene.
  • CD8 + cytotoxic T cells become activated and efficiently destroy vector-transduced cells. This MHC class I restricted cytotoxic T cell-mediated killing of genetically modified cells appears to be a major hurdle for the maintenance of targeted cells (Riddell et al. 1 996. Nat. Med., 2:21 6-223).
  • BMC bone marrow cells
  • ⁇ -GT porcine ⁇ -1 ,3 galactosyltransferase
  • the present invention relates to methods of treating an animal, such as a human patient, so as to achieve immunological tolerance to somatic cells and genetically engineered vectors containing and expressing foreign genes so as to facilitate the treatment of disease conditions in said animal by transplanting said cells or injecting said vectors.
  • immunological tolerance is the predominant mechanism by which tolerance is induced, although other currently undefined processes for, or mechanisms of, tolerance induction may occur.
  • the present invention relates to a process for pretreating an animal that is to receive one of a vector encoding a therapeutic polypeptide or recombinant cells comprising one of said vector or a polynucleotide encoding said therapeutic polypeptide comprising treating said animal with hematopoietic cells transduced with a member selected from the group consisting of said vector or said polynucleotide.
  • the procedure of the invention can then be followed by introducing into said animal, such as, for example, a human patient in need thereof, of a sample of somatic cells expressing one or more of the same foreign or therapeutic genes as used to transduce the hematopoietic stem cells or administering to said patient a sample of a vector containing one or more foreign genes for subsequent expression in a target cell or cells.
  • somatic cells may be in the form of a cell suspension, or possibly a solid tissue mass as is common in cases of transplantation.
  • the methods of the present invention are ideally suited to inducing immunological tolerance in an animal, especially a human patient, receiving gene therapy by administering to the recipient of the therapy a myeloreductive procedure, such as administration of immunosuppressive agents, followed by administration of hematopoietic stem cells transgenically altered so as to express the neoantigens, or foreign antigens, that are the subject of the gene therapy procedure, thereby lessening the immunological response to the subsequent exposure of the recipient to the foreign gene products.
  • a myeloreductive procedure such as administration of immunosuppressive agents, followed by administration of hematopoietic stem cells transgenically altered so as to express the neoantigens, or foreign antigens, that are the subject of the gene therapy procedure, thereby lessening the immunological response to the subsequent exposure of the recipient to the foreign gene products.
  • Such a process may optionally utilize an immunosuppressive regimen subsequent to administration of said stem cells, such as hematopoietic stem cells, but prior to actual transplant
  • the type of gene therapy contemplated for use with the methods disclosed herein will commonly comprise administration of some type of genetically engineered cells, or vectors, possibly cells drawn from the recipient and genetically modified so as to contain one or more new genes for subsequent expression after reinsertion into the recipient.
  • the immunological tolerance generated by the methods of the invention are designed to achieve tolerance to the genes utilized in gene therapy by earlier expression of said genes as part of the genome of stem cells, such as hematopoietic stem cells.
  • a vector is utilized as part of a gene therapy procedure (i.e., an in vitro gene delivery system, e.g., retrovirus supernatant, DNA-liposome complexes, DNAs and RNAs, both sense and antisense), such as for the insertion of transgenes into somatic cells to be used as part of the gene therapy regimen, or as a stand-alone vector modified so as to be targeted to a specific tissue, such as cancerous tissue
  • stem cells such as hematopoietic stem cells
  • diseases include atopic diseases and autoimmune diseases.
  • Figure 1 is a bar graph showing percentages of EGFP expression in different lineages of all mice analyzed.
  • Therapeutics may often involve the need to provide a patient with genetically engineered cells having specific genes inserted which are effective in alleviating a disease condition existing in the recipient of such cells.
  • a major disadvantage of such therapy is that the genes present in the cells, when expressed as proteins essential to such therapy, succeed not only in ameliorating the disease condition but also in eliciting an unwanted, possibly dangerous, immune response.
  • the cells themselves, if derived from a different organism, possibly from a different species, will often likewise elicit an unwanted immunological response.
  • vectors are to be injected into an animal, such as a human patient, having a disease such as cancer and wherein said vectors specifically bind to, and insert themselves into, the cancerous cells, thereby introducing genes that will either destroy the cancerous cells, or sensitize them to subsequently administered anti-cancer agents, such vectors will also likely produce an unwanted and detrimental immune response.
  • the use of foreign genes in cells and vectors to express products useful in combating disease where said cells and vectors must be introduced into an animal, such as a human patient in need thereof has the concomitant effect of producing immunological problems.
  • the present invention therefore relates to the use of therapeutic genes whose expression product acts to alleviate or ameliorate said detrimental immune response.
  • neoantigen means an antigen, or antigenic determinant, or epitope, found in an animal, such as a human patient, following gene therapy but not endogenous to said patient.
  • transgenically modified cells such as transgenic somatic cells
  • said treatment is carried out on a human patient, as part of a regimen of gene therapy, and, upon expression of the products of said transgenes, said products are subsequently exposed to the immune system of the recipient patient with resultant mounting of an immune response to such expression products.
  • One aspect of the present invention provides a method for treating a subject having a need for a molecular therapeutic agent comprising: (a) administering a myeloreductive treatment to the subject, (b) introducing stem cells, such as hematopoietic stem cells, containing at least one gene transfer product such that mixed molecular hematopoietic stem cell chimerism can be induced in the subject, and (c) introducing into the subject non-hematopoietic stem cells (i.e. somatic cells) containing the same gene transfer product.
  • the present invention relates to a process for pretreating an animal that is to receive one of a vector encoding a therapeutic polypeptide or recombinant cells comprising one of said vector or a polynucleotide encoding said therapeutic polypeptide comprising treating said animal with hematopoietic cells, especially hematopoietic stem cells, transduced with a member selected from the group consisting of said vector or said polynucleotide.
  • the process of the present invention could be preceded by a myeloreductive treatment, including an immunosuppressive regimen.
  • the pretreatment process recited according to the present invention could be followed by a myeloreductive treatment, including an immunosuppressive regimen, separate, if not altogether different, from any myeloreductive treatment, or immunosuppressive regimen, preceding the pretreatment.
  • the present invention relates to a method of inducing immunological or functional tolerance for a foreign or therapeutic gene product in an animal receiving, or to receive, genetically different somatic cells or gene therapy vectors comprising said foreign gene, comprising:
  • the somatic cells utilized herein may be genetically different from the cells of the recipient or, in fact, may have been genetically altered or otherwise genetically engineered to express antigenic structures different from those of the recipient.
  • somatic cells may be utilized simply to replace or supplement cells otherwise present in the recipient but genetically different because derived from a different organism or may be somatic cells deliberately genetically altered or modified so as to facilitate various types of gene replacement therapies.
  • the methods herein are also effective where the recipient is to receive gene therapy vectors expressing one or more genes deemed foreign by the recipient.
  • said somatic cells or vectors may be introduced to the recipient by almost any feasible form of administration, such as injection or infusion of such cells or vectors.
  • said foreign antigen may be termed an alloantigen or xenoantigen or other designation of a foreign gene as known and used by those skilled in the art.
  • some type of disease correction treatment involving either the introduction into the animal, especially where said animal is a human patient, a sample of autologous somatic cells transduced ex vivo with a vector containing one or more foreign genes, or the introduction of an in vivo gene delivery system, e.g., retrovirus supernatant, DNA-liposome complexes, DNAs or RNAs, both sense and antisense.
  • such foreign gene is commonly a therapeutic gene, of which there may be one or more than one in the same somatic cell or vectors introduced into the animal, or human patient, will commonly be transgenes.
  • transgenes are understood herein as being genes introduced into a cells by techniques of genetic engineering under circumstances where said genes are not endogenous to the recipient cells.
  • Such transgenes may therefore include genes derived from similar cells but where such genes are different alleles of the same genes, such as different forms of the same genes derived from different cells of the same organism, or different organisms of the same species, or from organisms of a different species, such alleles not being found in the recipient cells.
  • transgenes For example, if a gene coding for insulin in swine were inserted into the genome of cells from a human, such genes would be transgenes and such cells would be transgenic cells.
  • Such transgenes also include totally different genes, such as genes found only in cells of a species different from the species of the recipient cells, such as a gene coding for a plant enzyme being inserted into an animal cell.
  • therapeutic gene means a gene, such as a foreign gene or gene autologous to, but lacking or non-functioning in, the patient or recipient, that is being used for purposes of gene therapy.
  • the object of the present invention is to inhibit adverse immune responses to transplantation, be that by transplantation of organs or as a result of gene therapy that produces proteins found to be foreign by the recipient.
  • the term “inhibit” is intended to mean prevention, or inhibition, or reduction in severity, or induction of tolerance to, or reversal of graft rejection.
  • graft as used herein means any and all transplantation, including, but not limited to, allograft and xenograft transplantation. Such transplantation may by way of example include, but not be limited to, transplantation of cells, bone marrow, tissue, solid-organ, bone, etc.
  • immune response(s) is intended to mean immune responses dependent upon T cell activation and proliferation which includes both cellular effects and T cell dependent antibodies which may be elicited in response to, by way of example and not limitation: (i) grafts, (ii) graft versus host disease, and (iii) autoantigens resulting in autoimmune diseases, which by way of example include but are not limited to rheumatoid arthritis, systemic lupus, multiple sclerosis, diabetes mellitus, etc.
  • the myeloreductive treatment that is part of the method of the present invention includes treating the subject with an immunosuppressive regimen, prior to the introduction of the donor stem cells, in an amount sufficient to prevent rejection of the donor stem cells.
  • donor stem cells will commonly be hematopoietic stem cells.
  • Such immunosuppressive regimens can include a treatment of the subject which inactivates and/or depletes host T-lymphocytes and/or natural killer (NK) cells in the subject.
  • the immunosuppressive regimen can include treatment with T cell-depleting anti-CD4 and/or CD8 antibodies, such as anti-thymocyte globulin (ATG), OKT3 (Orthoclone OKT3 monoclonal antibody, Ortho Pharmaceutical Corp. Raritan, NJ .), LO-CD2a, and humanized-LO-CD2a (US patents 5,730,979 and 5,951 ,983 - the entire specifications of which is incorporated herein by reference) .
  • Humanized-LO-CD2a is an antibody that can be produced by recombinant technology. This latter antibody is particularly useful in reducing the density of the CD2 + lymphocytes and inhibiting natural killer (NK) cell activity.
  • NK cell activity represents a non-MHC restricted cytotoxic mechanism implicated in graft-versus-host disease.
  • many such antibodies can be used for this purpose, both naturally occurring and monoclonal, as well as wholly synthetic antibody molecules prepared by recombinant techniques.
  • Such antibodies may be derived from many different species, including, but in no way limited to, human, rat, murine, porcine, and bovine, and also may include chimeric, humanized antibodies (as well as active fragments and derivatives of such antibodies).
  • derivative means a chimeric or humanized antibody, single chain antibody, bispecific antibody or other such antibody which binds to the same epitope (or a portion thereof) as recognized by any antibody effective for use in the methods disclosed herein (such as, for example, LO-CD2a).
  • fragment means a portion of an antibody, by way of example such portions of antibodies shall include but not be limited to CDR, Fab, or such other portions, which bind to the same epitope or any portion thereof as recognized by any antibody useful in the methods disclosed herein (such as, for example, LO-CD2a) .
  • antibody includes polyclonal, monoclonal antibodies as well as antibody fragments, derivatives as well as antibodies prepared by recombinant techniques, such as chimeric or humanized antibodies, single chain or bispecific antibodies which bind to the same epitope or a portion thereof as recognized by the polyclonal and monoclonal antibody antibodies useful for practicing the present invention (such as, for example, the LO-CD2a antibody already mentioned).
  • molecules includes by way of example and not limitation, peptides, oligonucleotides or other such compounds derived from any source which mimic the antibody or bind to the same epitope or a portion thereof as the antibody fragment or derivative thereof.
  • the immunosuppressive regimen useful in the present invention can further include physical methods designed to suppress the immune response, including treatment with irradiation, either sub-lethal whole body irradiation or thymic irradiation, or both.
  • Such substances can be utilized singly or in any combination as desired by the researcher or clinician and based on the needs, susceptibilities and overall state of health of the individual recipient (such as a human patient receiving treatment).
  • agents may be used at dosage levels that may be related to each other or at totally unrelated dosage levels, depending on the requirements of the particular treatment involved and the overall state of health of the recipient.
  • the myeloreductive treatment includes treating the subject, prior to introduction of the donor stem cells, with a cytoreductive agent, e.g., cyclophosphamide.
  • a cytoreductive agent e.g., cyclophosphamide.
  • the conditioning regimen includes administration of T cell inactivating antibodies, e.g., MEDI-507 (BioTransplant Incorporated, Charlestown, MA) and thymic irradiation.
  • the method of the present invention also includes a myeloreductive step wherein an immunosuppressive regimen is utilized and comprises treatment with an immunosuppressive agent selected from the group consisting of macrolide immunosuppressant, azathioprine, steroids, co- stimulatory blocking agents, or any of these either alone or in combination and, when in combination, either in equal dosages or in any separate varying dosages.
  • any of the myeloreductive methods disclosed herein can be used either separately or in combination, including both chemical and physical methods.
  • radiation can be used in combination with chemical agents within the methods disclosed herein.
  • the myeloreductive treatment may comprise treatment with both thymic radiation and T cell inactivating antibodies.
  • steroids are employed as an immunosuppressive agent
  • said steroids are preferably selected from the group consisting of prednisone and methyl prednisolone.
  • Co-stimulatory blocking agents available for use in the methods of the present invention are preferably selected from the group consisting of anti-CD40 ligand antibodies and CTLA4-lg fusion proteins.
  • the myeloreductive treatment utilized in the methods of the present invention may also comprise treatment with a cytoreductive agent, such as cycloheximide.
  • a cytoreductive agent such as cycloheximide.
  • the overall purpose of the myeloreductive step as utilized in the present invention is to provide at least a temporary suppression of the overall immune response and to prepare the transplantation recipient, or gene therapy patient, for the subsequent administration of stem cells carrying the foreign gene or genes and expressing the particular product or products (the latter most commonly being some type of protein).
  • the myeloreductive treatment involves the administration of an immunosuppressive antibody, such as humanized-LO-CD2a, such molecule (given alone or in combination with other immunosuppressive agents suggested by the present disclosure and including any effective (within the methods of the present invention) antibody or fragment or derivative thereof or molecule of the type hereinabove described) may be administered in vivo in accordance with the present invention to inhibit the activation and proliferation of T-cells, and decrease the density of CD2 expression on the cell surface and thereby reduce the number of CD2 + T lymphocytes.
  • an immunosuppressive antibody such as humanized-LO-CD2a
  • such antibodies are administered to prevent and/or inhibit immune response and thereby inhibit T cell activation and proliferation.
  • An antibody or fragment or derivative thereof or molecule of the type herein above described may be administered ex vivo in accordance with the present invention to decrease the density of CD2 + expression on the cell surface and thus reduce the number of CD2 + cells of the donor cells.
  • such antibodies or fragments or derivatives thereof or molecules would be infused into donor bone marrow prior to transplantation to prevent the onset of graft versus host disease upon transplantation.
  • the antibody or fragment or derivative thereof or molecule will be administered in a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier there may be mentioned normal saline solution, buffers, etc.
  • Such pharmaceutical carriers are well known in the art and the selection of a suitable carrier is deemed to be within the scope of those skilled in the art from the teachings contained herein.
  • the antibody or other molecule for use in the present invention may be administered in vivo intravenously or by intramuscular administration, etc.
  • an antibody or other molecule of the present invention is administered in vivo in an amount effective to inhibit graft rejection.
  • an effective amount for purposes of this disclosure shall mean that amount of immunosuppressive agent capable of producing the desired effect, e.g., the inhibition of graft rejection or inhibition of the activation of T-cells.
  • the agent is an antibody
  • the latter is administered in an amount of at least 1 mg. It is to be understood that lower amounts could be used.
  • the herein above described amounts may be reduced for subsequent treatments, if any. Thus the scope of the invention is not limited by such amounts.
  • the techniques of the present invention for inhibiting the activation of T-cells may be employed alone or in combination with other techniques, drugs or compounds for inhibiting the activation of T-cells or inhibiting graft rejection or graft versus host disease.
  • a major advance offered by the present invention is the utilization of a subsequent step (following myeloreduction) whereby stem cells, such as hematopoietic stem cells, are administered to the transplant recipient, or gene therapy patient, and wherein such cells contain and express a foreign gene, or genes, such that said foreign gene, or genes, are also present in the biological material to be transplanted or are present in a vector to be administered to a patient as part of a gene therapy regimen and where the product of such gene, or genes, will be deemed foreign by the recipient's immune system .
  • stem cells used in the methods of the present invention are hematopoietic stem cells they are preferably CD34 + (i.e., exhibit the
  • CD34 antigen on their surfaces said antigen being a marker for hematopoietic progenitor cells.
  • Such cells may be recovered by procedures known in the art.
  • the donor stem cells may be allogeneic, autologous, syngeneic or xenogeneic stem cells.
  • the donor stem cells are provided as bone marrow cells, mobilized peripheral blood cells, cord blood cells, or pluripotent stem cells.
  • the donor stem cells in some instances, can be expanded ex vivo for transplantation.
  • the donor stem cells are xenogeneic stem cells, i. e. , from a different species than the subject
  • the donor species is preferably a swine, i.e. a miniature swine.
  • the miniature swine is inbred at the swine MHC (the swine major histocompatibility complex (MHC) is denoted swine leukocyte antigen (SLA) and consists of multiple loci) .
  • MHC the swine major histocompatibility complex
  • SLA swine leukocyte antigen
  • the subject is a human and the donor stem cells are from the same human.
  • Prior tolerization of the recipient to immunogens should be obtained by pre-delivery of the gene encoding the molecule by using gene transfer of hematopoietic stem cells or other cells with tolerance inducing capacity.
  • the gene(s) that is(are) to be introduced into the transplant recipient expresses a highly immunogenic molecule, thereby more effectively inducing tolerance.
  • the method can include a further step of treating the subject with an immunosuppressive regimen, after introduction of the donor hematopoietic stem cells, in an amount sufficient to prevent a graft versus host rejection mediated by the donor stem cells, preferably hematopoietic stem cells.
  • the present invention also relates to a method of inducing immunological tolerance in an animal receiving genetically different somatic cells or gene therapy vectors, comprising:
  • the somatic cells utilized herein may be genetically different from the cells of the recipient or, in fact, may have been genetically altered or otherwise genetically engineered to express antigenic structures different from those of the recipient.
  • somatic cells may be utilized simply to replace or supplement cells otherwise present in the recipient but genetically different because derived from a different organism or may be somatic cells deliberately genetically altered or modified so as to facilitate various types of gene replacement therapies.
  • the methods herein are also effective where the recipient is to receive gene therapy vectors expressing one or more genes deemed foreign by the recipient, as previously recited for procedures not utilizing step (c) above. Also as previously recited the methods of the present invention will commonly be followed by some type of gene replacement therapy involving the introduction of somatic cells or gene therapy vectors.
  • the stem cells and/or somatic cells are both derived from the same animal.
  • the method will be directed for the correction of genetic deficiencies of somatic cells.
  • the invention is directed toward the induction of immunological tolerance in a recipient, e.g., a human, to genetically altered cells or tissues obtained from a second individual.
  • a recipient e.g., a human
  • hematopoietic stem cells from either the second individual or from the recipient will be subjected to the gene delivery. More preferably CD34 + hematopoietic stem cells would be utilized for this gene therapeutic method
  • the method is used to alleviate unwanted immune responses associated with atopic diseases as well as autoimmune diseases.
  • the method described in the current invention is particularly useful in gene transfer approaches for the treatment of genetic diseases where the etiology of the disease is caused by the absence or lack of function of a given molecule and when corrective treatments result in inhibitory immune responses.
  • the method is useful in gene transfer treatments of several genetic diseases e.g.
  • cystic fibrosis muscular dystrophy, hemophilia A or B, familial hypercholesterolaemia, haemoglobinopathies, thalassaemias/sickle cell anaemia, Gaucher's disease, ⁇ ,-antitrypsin deficiency, inherited emphysema, chronic granulomatous disease, Fanconi's anemia as well as other inherited genetic diseases including certain immunodeficiencies e.g. adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID).
  • immunodeficiencies e.g. adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID).
  • ADA adenosine deaminase
  • the methods of the present invention find use in the treatment of such diseases as cystic fibrosis (CF).
  • Cystic fibrosis is an inherited disease with high fatality of exocrine glands affecting the pancreas, respiratory system, and apocrine glands.
  • CF is the major cause of severe chronic lung disease in children.
  • the defective gene in CF patients encodes a nonfunctional cAMP-activated chloride channel, denoted the cystic fibrosis transmembrane conductance regulator (CFTR).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the ongoing clinical trials that are attempting to eliminate the disease by gene delivery of the CFTR gene have only met with limited success (Alton et al. 1 998. Gene Therapy, 5:291 -292).
  • the principal cells targeted by gene transfer with the CFTR gene are respiratory epithelial cells. Besides the difficulty of targeting sufficient numbers of respiratory cells in the nasal epithelium, the respiratory tract is highly immunocompetent and immune responses directed against cells expressing CFTR is likely to be significant.
  • Rozmahel et al have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient in the endogenous protein.
  • expression of the endogenous CFTR gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event.
  • Animals homozygous for the gene replacement failed to show either improved intestinal pathology or survival when compared to mice completely lacking CFTR. The authors concluded from their data that the failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice.
  • the methods of the present invention solve this problem by facilitating enhanced expression of the CFTR gene.
  • autologous CFTR knockout mice hematopoietic stem cells are transduced by gene transfer vectors designed to express the CFTR gene and reintroduced into the CFTR knockout mice. Thereafter, gene transfer of the mouse CFTR gene is accomplished by administration to mouse airway epithelia of any of the gene transfer agents that have been used in clinical studies e.g., adenovirus vectors, adeno-associated viruses, cationic lipids.
  • the present invention relates to tolerance induction to dystrophin, as described further below.
  • the present invention relates to tolerance induction methodology as applied to suicide cancer therapies.
  • Such methodologies include the ability to perform effective repeat administrations of vectors which may be desirable in gene transfer protocols aimed at treating cancer.
  • the therapeutic gene carried by the vectors is frequently the suicide-gene herpes simplex virus thymidine kinase (HSV-TK) .
  • HSV-TK suicide-gene herpes simplex virus thymidine kinase
  • the method does not result in the development of antibodies to both the vector and to the HSV-TK transgene, thereby adversely affecting the outcome of the treatment.
  • the present invention relates to a method for treating cancer in an animal comprising:
  • the immunogenic molecules expressed by the gene transfer vectors of the present invention can be peptides, polypeptides, carbohydrates, nucleic acids, and lipids.
  • the methods according to the present invention employ conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Immunology eds Coligan et al. John Wiley and Sons; Current Protocols in Molecular Biology eds Ausubel et al. John Wiley and Sons.
  • Gene transfer products include gene transfer vectors, either DNA or RNA, the products expressed by the gene transfer vector, for example protein or peptide or polypeptide encoded by the genes contained within the vector. Also included are RNA molecules either antisense or sense. Also included are products that are the result of the enzymatic activity of any of the gene products encoded by the gene transfer vector.
  • the methods of the present invention contemplate the genetic alteration of cells that will be able to present the immunogenic molecules and effectively induce T cell tolerance.
  • these cells are derived from the hematopoietic lineage.
  • the method described in the invention is not restricted to any particular cell type. Based on current knowledge the most appropriate cell type would be a hematopoietic stem cell.
  • the present invention also relates to vectors which include polynucleotides intended for expression by the cells to be introduced into the recipient as well as polynucleotides essential to the particular form of gene therapy being attempted on the recipient.
  • host cells either those intended for transplantation as a form of therapy, or stem cells, such as hematopoietic stem cells to be transgenically altered so as to express the antigens of the somatic cells intended for subsequent transplantation or therapy, are genetically engineered with vectors by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with suitably engineered vectors containing the transgenes of interest (the nature and structure of which will depend on the particular type of transplantation or therapy intended) and will therefor commonly be expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, or any other suitable structure, all of which are well known to those of skill in the relevant art.
  • the vector may itself be the object of the gene therapy, such as where a vector is targeted for a particular destination, such as cancer cells existing within a tumor, or more diffuse, such as in a form of leukemia, and said vector will itself generate an immune response from the recipient that is in addition to any response generated by the genes inserted into said vector and intended for transducing the target cells.
  • the methods of the present invention find use in ameliorating any immune response to said vector as well as to the expressed products of the transgene, or transgenes, it carries.
  • the engineered host cells such as the somatic cells intended for therapy, and the stem cells, such as hematopoietic stem cells, intended for use in the methods disclosed herein, can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention.
  • the culture conditions such as temperature, pH and the like, utilized in preparing the transgenic stem cells for the methods disclosed herein, are commonly those previously used with the host cell selected for transplantation or therapy, and will be apparent to those skilled in the relevant art.
  • the polynucleotides intended for expression by the stem cells used according to the present invention may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • expression vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40, plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies, as well as any other vector so long as it is replicable and viable in the host.
  • the appropriate DNA sequence for transgenically modifying the stem cells according to the present invention may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site by procedures known to those skilled in the relevant art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence, or promoter sequence, including enhancer sequences, to direct mRNA synthesis.
  • promoter sequence including enhancer sequences
  • promoters there may be mentioned: retroviral long terminal repeats (LTR) or SV40 promoter (known to control expression of genes in eukaryotic cells, especially the stem cells used herein.
  • LTR retroviral long terminal repeats
  • SV40 promoter known to control expression of genes in eukaryotic cells, especially the stem cells used herein.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate stem cell, such as a hematopoietic stem cell, possibly but not necessarily derived from the same animal, such as a human patient, on whom the methods of the present invention are to be applied, to permit the stem cell to express the transgenic protein.
  • an appropriate stem cell such as a hematopoietic stem cell, possibly but not necessarily derived from the same animal, such as a human patient, on whom the methods of the present invention are to be applied, to permit the stem cell to express the transgenic protein.
  • Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector.
  • Gene therapy or drug delivery via gene transfer entails the creation of specialized vectors each vector being applicable only to a particular disease.
  • a vector cloning system be available which consistently maintains the necessary safety features yet permits maximal flexibility in vector design. Subtle changes in gene position, or in the specific combination of regulatory sequence(s) with the gene of interest, can lead to profound differences in vector titer or in the way that transferred genes function in target cells.
  • Transgene constructs useful in transforming or transducing hematopoietic stem cells comprise a vector, such as a plasmid or viral vector, into which a sequence for expression has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • vectors can include adenoviruses, herpes virus vectors and, preferably, retrovirus vectors.
  • Adenovirus genomes are linear, double-stranded DNA molecules of about 36 kbp in length.
  • adenovirus The well-characterized molecular genetics of adenovirus render it an advantageous vector for gene transfer.
  • the knowledge of the genetic organization of adenoviruses allows substitution of large fragments of viral DNA with foreign sequences.
  • recombinant adenoviruses are structurally stable and no rearranged viruses have been observed after extensive amplification so that these viruses are highly useful as delivery vehicles for introducing desired genes into eukaryotic cells.
  • methods have been developed that permit use of adenovirus vectors specific for different cell types. [See: U.S. Patent 5,756,086] .
  • retroviral vectors are also extremely useful for transfecting actively dividing cells, such as cultured hematopoietic stem cells.
  • Retroviral vectors are also useful as agents to mediate retroviral- mediated gene transfer into eukaryotic cells. Such vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Retroviral vectors useful in the methods of the present invention are, for example, contained in U.S. Patent 5,672,51 0.
  • the retroviral vector could a derivative of pPBM1 9 (See: Banerjee et al. ( 1 997) Xenotransplantation 4: 1 74- 1 85, expressing the ovarian cancer BRCA1 gene). Other examples are described in lerino et al ( 1 999) Transplantation 67: 1 1 1 9-1 1 28.
  • plasmids or vectors may be used so long as they are replicable and viable in the stem cells to be used as hosts and are suitable for gene therapy.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Eukaryotic promoters include Cytomegalovirus (CMV) immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the relevant art.
  • transgenic constructs into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation.
  • Appropriate cloning and expression vectors for use with eukaryotic hosts, as well as means for transfecting the cells, are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1 989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a CMV early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the neomycin resistance gene and dihydrofolate reductase (dhfr) genes, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • the promoter sequence should provide cis-acting regulatory elements sufficient to target transcription and expression of the transgene to the appropriate cell lineage or tissue where the therapeutic effects are desired.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Mammalian expression vectors suitable for use with stem cells will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Utilization of the methods of the present invention are in no way restricted to the use of any particular gene delivery system .
  • the preferred system would be one that effectively delivers and introduces the transgene(s) into non-cycling cells with subsequent long-term sustained transgene expression and biological activity resulting from the expression of such transgene(s), e.g., lentivirus expression system.
  • transgene(s) e.g., lentivirus expression system.
  • tolerogenic protein(s) appears to be required to induce effective functional tolerance in mice (Fraser et al. 1 995. J. Immunol., 1 54: 1 587-1 595; Schumacher et al. 1 996. Transplantation., 62:831 -836; Zambidis et al. 1 997. Mol. Med., 3:21 2-224) .
  • the method presented in the invention is not restricted to induction of tolerance to any particular molecule(s) expressed as a result of gene transfer.
  • Genetically transferred molecules include molecules encoded by the gene introduced by gene transfer and that result from the activity of such genes.
  • Induction of immunological tolerance by bone marrow molecular chimerism is a methodology that can be utilized to effectively eliminate immune responses towards gene therapy vectors or the therapeutic transgene product as described in the present invention disclosure. Applied to clinical gene therapy protocols this methodology should improve the effectiveness and safety of gene therapy. Conventional gene therapy protocols do not involve a pre-tolerization step to induce immunological tolerance or neutrality to the gene therapy products. As described here, host immune responses directed against transgene products recognized as foreign by the recipient of gene therapy modified cells represents a major obstacle to long-term persistence of such genetically corrected cells in vivo (McCarthy. 1 996. Lancet 347:31 4; Verma and Somia. 1 997. Nature 389:239-242) .
  • Example 1 comprises the development of an animal model to demonstrate the induction of tolerance to a transgene-encoded marker protein that is known to be immunogenic, i. e., enhanced green fluorescent protein (EGFP), by using a tumor rejection model of EGFP transduced EL-4 lymphoma cells.
  • EGFP enhanced green fluorescent protein
  • Example 1 takes advantage of the immunogenicity of EGFP, as the immunogenicity of a protein correlates directly with its ability to induce immunological tolerance (Bachmann et. al. 1 997. J. Immunol., 1 58:5106-51 1 1 ) .
  • the potential for inducing tolerance to the transgene product is also maximized and the need for tolerance induction is at its greatest.
  • the development of proof of principle using the mouse as an animal model facilitates development of additional mouse models for treatment of genetic and autoimmune disease as outlined in Examples 2 and 3.
  • the technique builds on re-educating the immune system and exploiting the phenomenon of immune tolerance by establishing mixed molecular chimerism and transplantation of hematopoietic stem cells.
  • EGFP is a commonly used marker protein in gene transfer protocols to measure the efficiency of transduction (Prasher et al. 1 992. Gene., 1 1 1 :229-233; Chalfie et al. 1 994. Science., 263:802-805; Zhang et al. 1 996. Biochem. Biophys. Res. Commun., 227:707-71 1 ; Ramiro et al. 1 998. Hum. Gene. Ther., 9: 1 1 03-1 109; Tsien. 1 998. Annu. Rev. Biochem., 67:509-544) . Strong immune responses to EGFP have been reported (Stripecke et al. 1 999.
  • Retroviral transductions with monocistronic retroviral vectors expressing EGFP is also achieved using the mouse as a model system .
  • the widely used EL-4 T cell lymphoma cell line (Gorer. 1 950. Br. J. Cancer., 4:372- 379; Klein and Klein. 1 964. J. Natl. Cancer Inst.
  • EGFP expression is monitored by fluorescent activated cell sorting (FACS) in peripheral blood mononuclear cell (PBMC) preparations with the expected lack of tumor development, readily monitored by palpation as described (see below and Stripecke et al. 1 999. Gene Therapy, 6: 1 305-1 31 2). Concomitantly, these samples are analyzed for the presence of neutralizing anti-EGFP antibodies.
  • FACS fluorescent activated cell sorting
  • PBMC peripheral blood mononuclear cell
  • C57BL/6 (Ly5.1 ) congenic hematopoietic stem cells
  • VSV-G-EGFP and ampho-EGFP supernatants such cells are transplanted into C57BL/6J recipient mice conditioned with myeloablative treatment for optimal engraftment.
  • a myeloablative conditioning regimen was chosen in this experiment to obtain proof of principle of tolerance induction to EGFP. It should be emphasized that aggressive myeloablative treatments prohibit the use of this approach in preparing patients for gene therapy. However, when proof of principle is achieved, non-myeloablative or myeloreductive conditioning regimens are performed in subsequent groups of mice as outlined in Example 3 in this patent application.
  • Minimal or non- myeloablative conditioning regimens in which high dose whole body irradiation (WBI) has been replaced with lower dose WBI or treatment with chemical drugs, coupled with treatment with anti-T cell antibodies and thymic irradiation have been developed.
  • WBI whole body irradiation
  • These procedures allow relatively low levels of allogeneic donor stem cell engraftment which were sufficient for the establishment of donor specific tolerance to organ or tissue grafts in murine, pig, and non-human primate models (Sharabi and Sachs, 1 989. J. Exp. Med., 1 69:493-502; Huang et al. 2000. J. Clin. Invest., 1 05: 1 73-1 81 ; Kimikawa et al.
  • mice Following engraftment of EGFP transduced hematopoietic stem cells, approximately four weeks post-bone marrow transplantation, mice were bled and EGFP expression was determined in different hematopoietic lineages (see below). After an additional 4 weeks (week 8 post-bone marrow transplantation), mice are bled again and the lineage analysis repeated. When mice have recovered from the bleeding procedure (3 months post-transplantation), subcutaneous implantation of EGFP transduced EL-4 cells is performed as described for the first control group.
  • mice expressing readily detectable EGFP in the peripheral blood mononuclear cells the expected tumor development, EGFP expression and anti-EGFP antibodies is analyzed as described above. In any given individual mouse, the exact time frame for tolerance induction to EGFP and the minimal level of stem cell derived EGFP expression is determined experimentally.
  • the expected outcome in the control group of animals infused with EL-4-EGFP cells is a transient expression of EGFP coinciding with the emergence of anti-EGFP antibodies and the CTL-dependent destruction of EL-4-EGFP cells and no tumor growth.
  • This retrovirus construct has an insert consisting of the EGFP cDNA and internal ribosome entry site (IRES) as described below and expresses EGFP under the control of Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR).
  • IRS internal ribosome entry site
  • MoMLV Moloney murine leukemia virus
  • LTR long terminal repeat
  • Nucleotides 5- 1 61 7 nucleotides 2- 1 61 4 of plasmid pLN
  • a Mlu I INot I fragment containing the open reading frame (ORF) of EGFP was made by PCR of plasmid pEGFP-1 (CLONTECH accession # U55761 ) with oligonucleotides as follows: Sense-5' TTTACGCGTGTCGCCACCATGGTGAGCAAGGGC 3'
  • a Not I ISal I fragment containing BiP (the 5'untranslated (5'-UT) sequence of the immunoglobulin heavy chain binding protein also referred to as GRP 78 (nucleotides 376-586 of accession # M 1 9645) was available by digesting another retroviral vector., the two fragments above were ligated at the Not I sites, the Sal I site was blunt ended to join the retroviral vector at a blunt ended- Cla I site (the Sal ⁇ ICIa I junction is at nucleotide 2596).
  • Nucleotides 3148-5688- correspond to base pairs 2924-5464 of pLN.
  • This retrovirus construct has an internal ribosome entry site (IRES)- EGFP insert as described below, and expresses EGFP under the control of Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR).
  • IRES internal ribosome entry site
  • MoMLV Moloney murine leukemia virus
  • LTR long terminal repeat
  • a Nco I -Not I EGFP fragment was obtained by restriction digest from the plasmid pEGFP-1 (CLONTECH, Palo Alto, CA).
  • the EMC and EGFP fragments were ligated together in a Xho I and Not I digested shuttle plasmid (pcDNA3 Invitrogen, SanDiego CA) from which the EMC- EGFP fragment was subsequently released by digestion with Xho I and a downstream Eco Rl .
  • Nucleotides 5-1 61 7 nucleotides 2-1 61 4 of plasmid pLN (accession # M28245, Miller and Rosman, 1 989 BioTechniques 7: 980- 990) .
  • Amphotropic retrovirus (derived from PA31 7 packaging cell line, licensed from the Fred Hutchinson Cancer Center (Seattle, WA)), containing construct pPBM25 were collected from confluent Roller Bottles (Corning) in Dulbecco's Modified Eagles Medium (DMEM), supplemented with 5 % fetal bovine serum (FBS) at 32 °c. Viral titers were determined as described (Limon et al 1 997. Blood 90:331 6-3321 ). Supernatant was negative for replication competent retrovirus as determined by S + L " focus assay (ViroMed Inc., Camden, NJ) and as described previously (Banerjee et al. 1 997. Xenotransplantation 4: 1 61 -1 73) .
  • VSV-G vesicular stomatitis virus
  • the vector containing construct pGBiP pseudotyped with envelope glycoprotein G of vesicular stomatitis virus (VSV-G) was prepared using the Pantropic Retroviral Expression System (CLONTECH, Palo Alto, CA) according to manufacturer's instructions. Briefly, packaging cells were grown in DMEM supplemented with 1 0% FBS. The 293GP cells from CLONTECH were transduced with supernatant containing construct pGBiP. They were enriched by FACS from 1 3 % to 75 % EGFP + and expanded.
  • mice 1 0 female congenic B6.SJL-Ptprc a Pep3 b /BoyJ (Ly5.1 ) stock # 00201 4 - (Jackson Laboratory, Bar Harbor ME)
  • Donor B6-Ly5.1 mice were injected intraperitoneal (i.p.), at day -7 with 5-FU dissolved in sterile saline (0.1 ml/1 0 g b.wt.) at a dose of 1 50 mg/kg.
  • Mice were euthanized at day 0 and bone marrow cells (BMCs) were harvested from femurs, tibias and humeri by crushing in a sterilized mortar and pestle in sterile 1 X HANK's Buffered Saline Solution (1 X HBSS).
  • BMCs were separated from bone and tissue fragments by filtration through a sterile metal strainer. Cell clumps were dispersed by passing cells through a 22 gauge needle. Cells were counted and cultured as described below.
  • Mouse Bone Marrow media is as follows: Iscove's Modified Dulbecco's Medium (IMDM, cat# 51471 -78P; JRH Biosciences, Lenexa, KS) is supplemented with 1 5% FBS (HyClone, Logan UT, cat# SH30071 .03), beta -mercaptoethanol (0.1 mM), Gentamycin (0.02 mg/ml) and 1 00 ng/ml recombinant murine Stem Cell Factor (rmSCF) and 50 ng/ml each recombinant human Thrombopoietin (rhTPO), interleukin 6 (rhlL-6), and rmFlt3-ligand. All recombinant cytokines were obtained from R&D Systems (Minneapolis, MN) Donor mouse bone marrow transduction
  • Bone marrow cells prepared as described above from donor
  • mice C57BL/6 (B6.SJL-Ptprc a Pep3 /BoyJ) (B6-Ly5.1 ) female mice were transduced on RetroNectin® coated plates using monocistronic retroviral vectors, either EPA25.1 6 or VSV-G-GBiP, carrying the EGFP cDNA (see below) .
  • RetroNectin® (Takara Shuzo Co., Ltd) stock solution is prepared at 1 mg/ml in dH 2 O, sterile filtered through 0.22 ⁇ m MILLEX-GV filter (MILLIPORE) .
  • MILLEX-GV filter MILLIPORE
  • cells were harvested from the 6-well plate, centrifuged and resuspended in the same volume of supernatant as above, adjusted to contain the cytokines from MBM medium.
  • the final concentration of cells during transduction with mock and EPA 25.1 6 was 3 x 1 0 6 cells /ml; for transduction with VSV-G-GBiP, the concentration was 4 x 1 0 6 cells/ml.
  • the medium plus any floating cells was centrifuged and the pellet of cells resuspended in a fresh aliquot of supernatant + cytokines for two additional hits over a 48 hr period.
  • transduction After transduction, cells were harvested from the coated wells by gently scraping, washed once in 1 X HBSS and then resuspended in 1 X HBSS at a concentration of 5 x 1 0 6 cells/ml. These transduced cells were infused in lethally irradiated recipient mice (see below) .
  • FCM Flow Cytometry
  • the cells were infused (transplanted) in restrained C57BL/6J female recipient mice via the tail vein.
  • the recipient animals received 0.2 ml infusion by tail vein injection. Both donor and recipient mice were female to avoid sex-related immune responses to minor histocompatibility antigens.
  • FCM Flow cytometry antibodies-- all antibodies were obtained from BD PharMingen (San Diego CA) unless otherwise noted. Antibody staining was performed in 96-well plate, sample is 20-30 ⁇ l whole mouse blood. Fc block (anti-CD1 6/32, clone 24G2) was prepared in house; samples were blocked with 2 ⁇ g/well for 5-1 0 minutes at room temperature. Samples were stained with the following antibodies: a) Donor/host analysis- PE anti-mouse CD45.1 Isotype PE mouse lgG2a. Biotin anti-mouse CD45.2 revealed with StrepAvidin-PerCP (PCP) Isotype control Biotin mouse lgG2a. b) lymphoid- T cells: PCP-anti-mouse CD3e, isotype PCP hamster IgG .
  • Pan B cells PE anti-mouseCD45R/B220. Isotype PE-rat lgG2a. c) myeloid - PE-anti mouse CD 1 1 b , isotype PE- rat lgG2b After staining and washing, whole blood was lysed with PharmLyseTM for 10 min at 4°C. Acquisition and analysis was performed on a Becton Dickinson FACScan with Cell Quest software.
  • the experiment consisted of 4 groups of 1 0 (or 1 2 in the case of Group 4) recipient mice. Animals in each group were either given (1 ) no irradiation or bone marrow cells (BMCs), (2) 1 0 Gy TBI followed by mock transduced BMCs, (3) 1 0 Gy TBI followed by BMCs transduced using the amphotropic EPA25.1 6 vector and (4) 10 Gy TBI followed by BMCs transduced using the pseudotyped VSV-G-GBiP vector.
  • the different groups are divided into 2 sub-groups of 5 mice whereby one is implanted with EL-4 cells (see below 8) and the other monitored for long-term EGFP expression (up to 9 months) from transduced hematopoietic stem cells.
  • the groups of animals used in this experimental approach are described below (Table 1 ).
  • Transduced cells were analyzed by flow cytometry on day 0 and also plated for CFU analysis on day 0, as described in the art.
  • the high- titer VSV-G-GBiP supernatant greatly improved the transduction efficiency. There was a correspondingly higher percentage of EGFP + colony-forming cells.
  • the fluorescence intensity of GBiP transduced cells was higher than in EPA25-1 6 transduced cells.
  • the supernatants had a minor, negative effect on cell expansion and CFU- forming capacity compared to mock (medium alone). Results are summarized in Table 2.
  • mice The average level in Group 3 mice was 1 4 fold lower, but all mice had positive staining compared to the mock transduced Group.
  • the low level of staining in Group 3 mice (except mouse 23) limited the ability to accurately assess by FCM the lineages of the EGFP + cells.
  • the lineage profile in transplanted mice were similar to a control animal (Table 4) .
  • EGFP expression was detected in all major lineages analyzed. In the Group 4 mice, similar percentages of EGFP expression was observed in B (42%) and myeloid lineages (50%) .
  • mice In the single mouse (#23) analyzed from the EPA group (Group 3), EGFP expression was skewed toward myeloid cells (59%) .
  • the EL-4 cell line (ATCC # TIB-39) used here as a tumor model was originally established from a lymphoma induced in a C57BL mouse by 9, 1 0-dimethyl-1 ,2-benzanthracene (Gorer. 1 950. Br. J. Cancer., 4:372- 379; Klein and Klein. 1 964. J. Nat/. Cancer Inst., 32:547). Following sub-cutaneous implantation of syngeneic EL-4 tumor cells into C57BL/6 mice tumors develop. However, if the cells are transduced with a retrovirus vector expressing the immunogenic EGFP transgene product prior to their administration, an effective immune response is raised and the EL-4-EGFP cells are rejected from the animal and no tumors develop.
  • EL-4 cells were transduced with VSV-G-GBiP or alternatively with amphotropic virus denoted EPA25.1 6 that express EGFP (1 0 6 cells for 4 hours at 37°C in the presence of polybrene (8 ⁇ g/ml).
  • Single cell clones were generated by MoFlo sorting on day 4.
  • a total of 75 clones were expanded after selection of clones with EGFP expression ranging from 1 0-500 times the background of autofluorescence. The growth rates of all the clones were indistinguishable. All clones were shown to express stable levels of EGFP.
  • EL-4 clones expressing high and low level of EGFP was further selected and will be used for s.c, implantation into C57BL/6 mice.
  • the animals are anesthetized using Isoflurane inhalation and injected subcutaneously in the hind flank with either 5 x 1 0 4 wild-type EL-4 tumor cells or 5 x 1 0 4 EGFP transduced EL-4 tumor cells in 50 ⁇ L volumes. Tumor development is followed by palpation and subsequently measured using calipers. Animals are euthanized when tumors exceed an average diameter of 1 5 mm. Assessment and measurement of tumors is according to methods established in tumor biology and tumor immunology.
  • Immunological tolerance to EGFP is also assessed by skin grafting of C57BL/6-EGFP transgenic skin grafts. Mice from each of Group 1 to 4 are subjected to this skin grafting procedure. In mice from Group 1 and 2 B6-EGFP skin grafts are rejected whereas in Group 3 and 4 mice the B6- EGFP skin grafts are accepted because tolerance to EGFP is induced. Furthermore, rejection of B6-EGFP skin grafts in Group 1 and 2 mice serves as a control that the myeloablative conditioning treatment these mice achieved did not result in a general immunodeficiency.
  • C57BL/6- EGFP C57BL/6-TgN(ACTbEGFP) 1 0sb
  • EGFP expression in these mice is under the control of a chicken beta-actin promoter and cytomegalovirus enhancer.
  • DMD Duchenne muscular dystrophy
  • the condition is typically lethal and inherited in a recessive manner and caused by a defective dystrophin gene.
  • Dystrophin is a cytoskeletal protein expressed in skeletal and cardiac muscles.
  • Recent attempts to treat DMD have used adenovirus and adeno- associated virus vectors (Hartigan-O'Connor and Chamberlain 2000. Microsc. Res. Tech 48: 223-238).
  • the feasibility of correcting DMD has also for many years been investigated using different approaches in the dystrophin-deficient mdx mice model. These mice differ only from C57BL/1 0 (B1 0) mice in the lack of dystrophin expression (Hoffman et al. 1 987.
  • the feasibility of the proposed method is substantiated by the partial restoration of dystrophin expression in the affected muscle that was achieved following stem cell transplantation (Gussoni et al. 1 999. Nature 401 : 390-394) .
  • This partial restoration is attributed to the myogenic capacity of hematopoietic cells and that transplantation of wild- type bone marrow from normal mice allowed induction of tolerance to dystrophin.
  • the dystrophin expression was long lasting and without evidence of anti-dystrophin immune responses (Gussoni et al. 1 999. Nature 401 : 390-394).
  • hematopoietic progenitor stem cell transplantation of dystrophin transduced cells into mdx recipient mice conditioned with minimal or non-myeloablative treatment for optimal engraftment is performed.
  • the nucleotide sequence of murine Dystrophin is available in the public domain and methods to isolate the cDNA, generate gene transfer vectors and methods to detect Dystrophin expression is available for those skilled in the art.
  • Dystrophin cDNA is inserted into VSV-G-based retrovirus vector.
  • a further application of the current invention disclosure is treatment of autoimmune disease where a major autoantigen has been identified.
  • myelin basic protein MBP
  • MBP myelin basic protein
  • the murine experimental autoimmune encephalomyelitis EAE is used as a model for inflammatory autoimmune disorders of demyelinating primary central nervous system, and has been frequently used as an animal model for human MS (Zamvil and Steinman 1 990. Annu. Rev. Immunol 8: 579-621 ) .
  • EAE can be induced in the SJL/J mouse model strain by immunization with MBP (Wekerle 1 993.
  • hematopoietic progenitor stem cell transplantation of MBP transduced cells into SJL recipient mice conditioned with minimal or non- myeloablative treatment for optimal engraftment is performed.
  • the nucleotide sequence of murine MBP is available in the public domain and methods to isolate the cDNA, generate gene transfer vectors and methods to detect MBP expression is available for those skilled in the art.
  • MBP cDNA is inserted into VSV-G-based retrovirus vector.
  • EGFP in C57BL/6 mice are applied to primate models (Baboon, Rhesus, and Cynomolgus monkey) . Immunogenicity of EGFP has been documented in Rhesus models (Alexander et al. 1 999. AIDS. Res. Hum. Retrovirus 1 5: 1 1 -21 ; Johnson, R.P., Mol. Ther. (1 (5):S7, 201 8 (2000)).
  • neomycin phosphotransferase has been demonstrated by introduction of neo in Rhesus hematopoietic stem cells (Heim et al. 2000. Mol. Ther. 1 : 533-544) .
  • Primate hematopietic progenitor cells are mobilized by administration of recombinant human granulocyte-colony stimulating factor (rhG-CSF) administered subcutaneous (s.c.) daily for 5 days at a concentration of 1 0 ⁇ g/kg followed by either two consecutive leukapheresis of 2.5 times the blood volume on day 6 as described (Donahue et al. 1 996. Blood.
  • rhG-CSF granulocyte-colony stimulating factor
  • the donor stem cells may be allogeneic, syngeneic, xenogeneic or autologous. In preferred embodiments for induction of tolerance to gene therapy products, the donor progenitor stem cells are autologous.
  • the degree of progenitor enrichment is determined by colony- forming unit progenitor assays as described in the art.
  • Enriched CD34 + cells are transduced with VSV-G (see above) or retrovirus with Gibbon ape leukemia virus (GaLV) envelopes expressing EGFP.
  • GaLV retrovirus was produced in TE FLY packaging cells carrying the GaLV envelope expressed from the FBdelPGSAF plasmid as described (Cosset et al. 1 995. J. Virol. 69: 7430-7436) .
  • the procedure for the retroviral transduction and infusion of hematopoietic stem cells is described in detail in Example 3.
  • Alternative gene delivery methods including Herpes virus, Adeno-associated virus or naked DNA delivery may also be used to target the progenitor stem cell population.
  • EGFP-transduced primate CD34 + progenitor stem cells are infused in recipients that have received either myeloablative, myeloreductive or non-myeloablative conditioning regimen as described above.
  • Successful gene marking of EGFP is determined by flow cytometry (FCM) of blood samples taken on a weekly to monthly basis.
  • FCM flow cytometry
  • FCM and PCR in multiple hematopoietic lineages
  • animals are challenged with injection of autologous somatic cells genetically modified to express EGFP.
  • the preferred somatic cell types are those that are readily challenged by the recipient's immune system and are not immuno-privileged somatic cells.
  • mice and control animals are otherwise as described for the mouse experiment (see above) .
  • Animals receiving a tolerance inducing BMT of EGFP-transduced CD34 + progenitor stem cells subsequently accept any autologous cell type that express EGFP without any immunological or other adverse effects.
  • animals that challenged primarily and exclusively with somatic cells other than hematopoietic cells will largely reject such cells.
  • anti-EGFP CTL responses and anti- EGFP antibody responses are detected by immunological methods established in the art.
  • the ability to perform effective repeat administrations of vectors is desirable in gene transfer protocols aimed at treating malignancies.
  • suicide gene transfer for different tumors and for graft versus host disease (GVHD) has shown promising anti-cancer effects both in animal models as well as in clinical trials (Singhal and Kaiser. 1 998. Surg. Oncol. Clin. Nam., 7:505-536; Tiberghien ( 1 998) Current Opin. Hematolog., 5: 478-482).
  • the therapeutic gene carried by the vectors is the suicide-gene herpes simplex virus thymidine kinase (HSV-TK) .
  • ganciclovir a nucleoside analogue that is only metabolized by cells expressing HSV-TK
  • the development of antibodies to both the vector and to the HSV-TK transgene product has been reported and adversely affects the outcome of the treatment.
  • pre-existing anti-HSV-1 antibodies may affect the outcome of the treatment (Herrlinger et al. 1 998. Gene Therapy., 5:809-81 9).
  • transplantable tumor cells preferably pre-B leukemic or T cell lymphomic transduced with retroviral vectors expressing HSV-TK and the E. coli lacZ gene as a marker are injected intraperitoneally (i.p. ) .
  • ganciclovir is administered and eradication of transduced tumor cells is measured by a conventional beta- galactosidase assay.
  • the development of, or the pre-existence of anti- HSV-1 antibodies is likely to inhibit the efficiency of ganciclovir to eliminate transduced cells. Therefore, in a second group of animals, transduction of BM cells prior to the i.p. injection is performed in order to establish tolerance to HSV-TK and thereby produce more efficient elimination of HSV-TK expressing cells by the ganciclovir administration.
  • the patients subjected to this Phase II clinical trial were selected based on less extensive disease. Such patients may represent the optimal target population for current gene therapy approaches.
  • a previous phase I trial using patients with severe extensive metastatic cancer that met with partial success in tumor reduction, only minimal anti-BRCA1 Ab response was observed (Tait et al. ( 1 997) Clin. Cancer. Res. , 3: 1 959-1 968) .
  • the distinction in success rate between the Phase I and Phase II trials may be caused by the higher immunocompetence in the Phase II patients. This was indicated by several criteria analyzed including development of anti-BRCA1 Abs, high serum albumin levels and high WBC counts (Tait et al. 1 999. Clin. Cancer. Res., 5: 1 708-1 71 4) .
  • the high immunocompetence should make patients with less severe cancer more suitable for tolerance induction to the tumor suppressor BRCA 1 as described below in this example.
  • the protocol is as follows, hematopoietic stem cells are transduced with a retrovirus vector expressing BRCA1 .
  • the retroviral vector could be a derivative of pPBM 1 9 (Banerjee et al. ( 1 997) Xenotransplantation 4: 1 74-1 85) expressing the ovarian cancer BRCA 1 gene.
  • the procedure for the transduction and infusion of hematopoietic stem cells is described in lerino et al (1 999) Transplantation 67: 1 1 1 9-1 1 28. Subsequently, after several weeks to be determined experimentally but not longer than 1 6 weeks, i.p. injections of the vector are performed as described (Tait et al. ( 1 999) Clin. Cancer. Res. , 5: 1 708-1 714) .
  • BM aspirates are harvested and transduced separately before the conditioning regimen.
  • the initial BM aspirates (harvested from the iliac crest) are transduced in the weeks preceding the conditioning treatment and cryopreserved until infused.
  • the final BM harvest (from the humerus through an open incision or the iliac crest) is collected and transduced during the week of the conditioning regimen before irradiation.
  • CD34 + cells are isolated and transduced.
  • a patient conditioned with the non- myeloablative conditioning regimen are transplanted with transduced CD34 + BM cells only.
  • a patient conditioned with the myeloablative regimen receive both CD34 + and CD34 " transduced BM cells to further ensure engraftment and reduce the potential risk of BM aplasia.
  • CD34 + and CD34 " cells are enriched from the low-density FICOLL gradient fraction of BM by positive and negative selection, respectively using an immunoadsorption column.
  • CD34 separation is performed using an anti-CD34 Ab (from commercially available sources) on magnetic beads (Miltenyi, Auburn, CA) .
  • CD34 cells are further depleted of T cells using anti-T cell antibodies.
  • the culture conditions before and during transductions of CD34 + or CD34 cells are performed by initial pre-stimulation in StemSpan medium (Stem Cell Technologies, Seattle, WA) supplemented with 300 ng/ml recombinant human stem cell factor (hSCF) (R&D Systems, Minneapolis, MN), 300 ng/ml human Flt-3 ligand (hFlt-3L, R&D Systems), 1 00 ng/ml human thrombopoietin (hTPO, R&D Systems) and 1 00 ng/ml human interleukin-6 (hlL-6 R&D Systems).
  • Retronectin ® coated dishes (PanVera Corporation, Madison, Wl) .
  • Cultured BM cells undergo three viral exposures (each for 8 hr) over a 4- day period using a supernatant containing amphotropic recombinant virus in the presence of Polybrene ® (6 ⁇ g/ml, Sigma, St. Louis, MO ) and growth factors as above.
  • supernatant containing the retrovirus is centrifuged with the BM cells at 800 x g for 1 hr at room temperature.
  • Colony-forming unit (CFU) assays are performed on all transduced BM cells to assess the efficiency of transduction in vitro by determining the frequency of EGFP positive green colonies. Positive colonies are also assessed by RT-PCR.
  • CFU Colony-forming unit
  • the patient receives Ofloxacin (R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ), 50 mg intravenously (i.v.) daily, as prophylactic antibiotic treatment during the neutropenic period, and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Novartis) from day 0 to day 1 4 at a dose of 5 ⁇ g/kg/day to shorten the period of radiation-induced neutropenia.
  • Ofloxacin R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the two conditioning regimens used to prepare the recipients for BMT are as follows: (a) for the non-myeloablative regimen, the patient receives whole body irradiation of 3 Gy on day -3 and thymic irradiation of 7 Gy on day -1 from a cobalt-60 source. Anti-thymocyte globulin (Pharmacia Upjohn, Kalamazoo, MIO at 50 mg/kg i.v. is administered on days -3,-2, and -1 .
  • Cyclosporin A ( CsA, Novartis, Basel, Switzerland) is started on day 0 at a dose of 1 5-25 mg/kg/day by i.v or intramuscular (i.m.) injection to maintain a mean plasma trough level > 200 ng/ml, and was continued for 28 days.
  • Transduced autologous BM cells (cryopreserved and/or freshly transduced CD34 + only are infused on day 0 as a single dose (total number of cells infused ranges from 1 -50 x 1 0 6 ) .
  • the patient receives whole body irradiation of 4.5 Gy on days -2 and -1 .
  • CsA is administered as in the non-myeloablative regimen.
  • Recombinant human megakaryocyte growth and differentiating factor (Amgen, Thousand Oaks, CA) is given at a dose of 2.5 ⁇ g/kg/day subcutaneously from day 0 to day 9 in order to decrease the platelet transfusion requirement.
  • Transduced autologous BM cells are infused on days 0, 1 , and 2 (total number of cells infused ranges from 1 -600 x 1 0 6 ).
  • Transduced cells infused on days 0 and 1 are cryopreserved, and cells infused on day 2 are freshly prepared BM cells.
  • Preparative regimen for infusion of somatic cells expressing BRCA1 gene At times of up to 1 6 weeks following transplantation of the stem cells the patient receives a surgically implanted peritoneal catheter to administer infusions of vector (as described above) as well as to retrieve daily samples of peritoneal fluid for analysis. Ovarian cancer patients receive four daily i.p. injections of the vector for three cycles, 4 weeks apart. Patient peritoneal fluid and plasma are analyzed extensively by PCR, western blot and chemical and hematological tests. The dose of retroviral supernatant is 1 00 ml daily times 4 ( 1 0 9 -1 0 10 viral particles).

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Abstract

L'invention concerne des techniques permettant d'induire une tolérance fonctionnelle à des produits exprimant des transgènes dans des cellules somatiques. Ces techniques consistent à introduire des cellules souches dans un récipient, telles que des cellules souches hématopoïétiques transgéniquement modifiées de façon à exprimer au moins un néoantigène, cette procédure étant éventuellement précédée d'une procédure myéloréductive. Le but de ces techniques est d'induire une tolérance aux antigènes lorsqu'ils seront ultérieurement exprimés par des cellules ou des vecteurs associés à une thérapie génique.
EP00967159A 1999-10-01 2000-09-29 Technique permettant d'induire une tolerance fonctionnelle a des produits de transfert genique Withdrawn EP1578897A2 (fr)

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AU2003245752A1 (en) * 2002-06-28 2004-01-19 Bio Transplant, Inc. Process for promoting graft acceptance by depletion of hematopoietic stem cells
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US20100313284A1 (en) * 2007-11-01 2010-12-09 Deltacell B.V. Means and methods for eliciting an immune response
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