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WO1992015676A1 - Therapie genique par les cellules somatiques - Google Patents

Therapie genique par les cellules somatiques Download PDF

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Publication number
WO1992015676A1
WO1992015676A1 PCT/US1992/001890 US9201890W WO9215676A1 WO 1992015676 A1 WO1992015676 A1 WO 1992015676A1 US 9201890 W US9201890 W US 9201890W WO 9215676 A1 WO9215676 A1 WO 9215676A1
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fibroblasts
gene therapy
cells
gene
transduced
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PCT/US1992/001890
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English (en)
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Inder Mohan Verma
Daniel Claude St. Louis
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The Salk Institute For Biological Studies
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Publication of WO1992015676A1 publication Critical patent/WO1992015676A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to gene therapy. More specifically, the present invention relates to somatic cell gene therapy in humans and animals.
  • Retroviral vectors because of their unique structure, modes of replication, and ability to infect a wide variety of cells, including stem cells, are ideally suited to transfer genetic material into somatic cells (Verma, 1985) .
  • pluripotent stem cells in bone marrow have both self - - renewal capacity as well as the ability to give rise to all hematopoietic lineages, they are a popular target for the introduction of functionally active genes (Miller, et al., 1984; Williams, et al., 1984; Keller, et al., 1985; Dick, et al., 1985). Recently, hepatocytes have been used as target cells for introducing functionally active genes (Ledley, et al., 1987; Wolfe, et al., 1987).
  • mice Recently two groups used mouse fibroblasts to introduce and express foreign genes in mice (Selden, et al., 1987; Garver, et al., 1987b).
  • One group implanted mice with a DNA transfected cell line and showed that the recipient mice made the gene product (growth hormone) but maintained the graft only if the mice were immunosuppressed (Selden, et al., 1987).
  • the other group using a chimeric retroviral vector containing the alpha.,-antitrypsin gene, produced a cell line from a transduced cell and then transplanted cells from the line into the peritoneal cavity of nude mice (Garver, et al., 1987b) . In both cases, cell lines were generated that would potentially be tumorigenic in mice. Neither study addresses the issue of cell maintenance in grafted mice without the use of harsh immunosuppressive agents.
  • retroviral-mediated gene transfer can be used to introduce a recombinant human growth hormone gene into cultured human keratinocytes (Morgan, et al., 1987).
  • the transduced keratinocytes secreted biologically active growth hormone into the culture medium.
  • these cultured keratinocytes reconstituted an epidermis that was similar in appearance to that produced by normal cells, but from which human growth hormone could be extracted.
  • transduced fibroblasts are preferably created by infecting fibroblast cells jji vitro with chimeric retroviruses that contain at least one functionally active "replacement gene", i.e., foreign or exogenous genetic material that does not normally occur in fibroblast cells, or if it does, is not expressed by the fibroblast cells in biologically significant concentrations.
  • "replacement gene” can be maintained under the control of the long terminal repeat (LTR) of the retroviral vector and/or under the control of constitutive or inducible exogenous sequences.
  • the transduced fibroblasts are then preferably fixed by culturing them in vitro in an extracellular matrix. Finally, the transduced fibroblasts are implanted subcutaneously in the loose connective tissue of the skin of the individual or animal being treated. To insure rapid vascularization of the implanted fibroblasts, an angiogenic substance such as a fibroblast growth factor is preferably placed in the loose connective tissue along with the implant. Because the fibroblasts are implanted in a highly vascularized compartment of the skin i.e., loose connective tissue of the dermis, the transduced cells, and thus their "replacement" gene products, have direct access to the circulatory system.
  • an angiogenic substance such as a fibroblast growth factor
  • the present invention discloses an alternative strategy for somatic cell gene transfer.
  • the new strategy uses skin fibroblasts that are infected with chimeric retrovirus containing a functionally active endogenous or foreign "replacement" gene. Once infected with the chimeric retrovirus, the transduced fibroblasts are preferably "fixed" in an extracellular collagen matrix, and then implanted in the loose connective tissue of the skin.
  • the transduced fibroblasts and thus their "replacement" gene products, have direct access to the circulatory system.
  • the needed replacement gene products can easily and efficiently be distributed to other parts of the body.
  • the method described herein obviates the need for established cell lines and instead uses fibroblast cells from recipient subjects. Use of a subject's own cells minimizes the possibility of rejection.
  • culturing the cells in an extracellular collagen matrix circumvents the problem of necrosis that would ensue following subcutaneous injection (Bell, et al., 1983).
  • Figure 1 is a schematic drawing of the structural arrangement of the recombinant factor IX retrovirus pAFFIXSVNeo.
  • Figure 2 is a graph that illustrates secretion of human factor IX.
  • Figure 3 is a schematic representation of the protocol used to generate and graft collagen implants into the loose connective tissue of the skin of a mouse.
  • Figure 4 shows the structure of retroviral vectors containing the 9-galactosidase gene, titers of the recombinant retroviruses and expression of / 3-galactosidase activity.
  • Figure 5 shows the RNA transcripts made by the LNL-SLX CMV £-galactosidase and LNL-SLX DHFR 3-galactosidase constructs.
  • the present invention is based on the discovery that transduced skin fibroblasts can be used for somatic cell gene therapy when the transduced fibroblasts are fixed in vitro in an extracellular collagen matrix and implanted in the loose connective tissue of the dermis of a subject to be treated.
  • the discovery makes it possible to overcome several problems that have been encountered when prior art gene therapy methods were used to treat animals or individuals with genetic defects.
  • Such problems include: (1) inefficient expression of the foreign "replacement" genes (Williams, et al., 1984; Joyner, et al., 1985); (2) use of transduced cells that had the potential to be tumorigenic to the animal or individual being treated (Selden, et al., 1987; Garver, et al., 1987b); (3) use of harsh immunosuppressive agents to avoid rejection by the animal or individual being treated (Selden, et al., 1987); (4) necrosis following subcutaneous injection of cells (Bell, et al., 1983); and (5) poor diffusion of the replacement gene product
  • the present invention preferably employs chimeric retroviruses to introduce replacement genes into skin fibroblasts. Because of the high efficiency of retroviral infection and expression in fibroblasts, the present invention essentially eliminates the need to use marker genes to identify transduced cells. This greatly simplifies the overall problem of introducing replacement genes into cells that will be used for gene therapy. Since the invention preferably uses fibroblast cells from recipient individuals, it obviates the need to use potentially tumorigenic cell lines. Use of skin fibroblasts from the subject to be treated minimizes the possibility of rejection, which in turn lessens the need for harsh immunosuppressant drugs.
  • the invention uses transduced fibroblasts that preferably have been fixed in vitro in an extracellular collagen matrix, the problem of necrosis is also minimized.
  • the invention implants the transduced fibroblasts into the highly vascularized loose connective tissue of the dermis, the replacement gene products are easily and efficiently distributed to other parts of the body.
  • LTR long terminal repeat
  • factor IX refers to the blood clotting factor gene or protein of the same name
  • pAFVXM refers to a retroviral construct generated by Kriegler, et al., (1984).
  • pAFVXM is a progenitor construct for the recombinant factor IX retrovirus, pAFFIXSVNeo.
  • a replacement gene of interest (or a cDNA for such a gene) can be linked directly to the 5' LTR in the retrovirus by inserting a BamHI/Hindlll fragment from the gene or clone between the B lll/Hindlll sites of pAFVXM (Anson, et al., 1984);
  • pKoNeo is a neomycin phosphotransferase expression plasmid; when reference is made herein to the Greek letter “psi” ( ⁇ ) , the word psi is sometimes substituted for the symbol ⁇ i the letter “g” is sometimes used herein to signify the symbol for the Greek letter "gamma", -y;
  • MEF means primary mouse embryo fibroblasts
  • Bl/6 refers to an immortalized skin cell line derived from x-ray irradiated skin fibroblasts obtained from C57BL/6J mice;
  • psiFIXNeo means the cell line ⁇ FIXNeo
  • DMEM Dulbecco's modified Eagle's medium, which is substantially the same as Dulbecco-Vogt modified Eagle's medium
  • ELISA means enzyme linked immunoabsorbant assay
  • FGF means fibroblast growth factor.
  • FGF is an angiogenic substance that can be used in the present invention to stimulate vas ⁇ ularization of the implanted fibroblasts;
  • transduction refers to the process of conveying or carrying over, especially the carrying over of a gene from one cell to another by a virus or retrovirus.
  • a retrovirus that carries a gene from one cell to another is referred to as a transducing chimeric retrovirus.
  • An eukaryotic cell that has been transduced will contain new or foreign genetic material (e.g., a replacement gene) in its genome as a result of having been "infected" with the chimeric transducing retrovirus;
  • transfection of eukaryotic cells is the acquisition of new genetic material by incorporation of added DNA; “skin” refers to the body's largest organ.
  • Skin consists of two components, the epidermis and the dermis.
  • the dermis is a relatively inert structure which consists of collagen and other matrix materials.
  • the epidermis lies above the dermis and is separated from it by a basement membrane;
  • fibroblasts refers to flat, elongated connective tissue cells with cytoplasmic processes at each end and an oval, flat nucleus. Fibroblasts, which differentiate into chondroblasts, collagenoblasts, and osteoblasts, form the fibrous tissues in the body, e.g., tendons, aponeuroses, plus supporting and binding tissues of all sorts. Like other cells in the body, fibroblasts carry an entire complement of genetic material. However, only a small percentage of the genes contained in fibroblasts are biologically functional; that is, most of the genes in fibroblasts are not expressed at all or are expressed at such low levels that the proteins they encode are produced in undetectable amounts or at concentrations which are not biologically functional or significant.
  • transduced fibroblasts of the present invention incorporate exogenous genetic material, which they express, thereby producing the gene product encoded by tue incorporated exogenous genetic material;
  • a “promoter” is a specific nucleotide sequence recognized by RNA polymerase, the enzyme that initiates RNA synthesis.
  • the exogenous genes are subject to retroviral control; in such a case, the exogenous gene(s) is transcribed from an endogenous retroviral promoter.
  • retroviral vectors that, in addition to their own endogenous promoters, have exogenous promoter elements which are responsible for the transcription of the exogenous gene(s) .
  • Such exogenous promoters include constitutive and inducible promoters. Constitutive promoters are promoters that control the expression of gene functions that are needed in virtually all cell types.
  • Sustained expression of genes under the control of constitutive promoters occurs under all conditions of cell growth, and does not require the presence of a specific substrate to induce gene expression.
  • expression of genes controlled by inducible promoters is responsive to the presence or absence of an inducing agent. For example, it is possible to make a construct in which there is an additional promoter that is always on, so long as the cell maintains its viability. Alternatively, one can employ a construct modulated by an external factor or cue, and in turn to control the level of exogenous protein being produced by the fibroblasts by activating the external factor or cue.
  • the promoter for a gene which encodes certain constitutive or "housekeeping" functions such as, for example, hypoxanthine phosphoribosyl transferase (HPRT) , dihydrofolate reductase (DHFR) , adenosine dea inase, phosphoglycerol kinase (PGK) , pyruvate kinase, phosphoglycerol utase, and the like, will be continuously expressed.
  • the gene which encodes the metal-containing protein metallothionein is responsive to Cd ** ions. Incorporation of any one of the above-described promoters makes it possible to either continuously produce the protein of interest, or to regulate the production of the proteins produced by the transduced fibroblasts of the invention;
  • **subcutaneously means below the basement membrane of the epidermis (abbreviated as “s.c.”); “i.p.” means intraperitoneally; "skin fibroblasts” are fibroblast cells that are normally found in the dermis portion of the skin;
  • “syngeneic” means isogeneic, i.e., having the same genetic constitution
  • exogenous genetic material means DNA or RNA, either natural or synthetic, that is not naturally found in cells of a particular type; or if it is naturally found in the cells, it is not expressed in these cells in biologically significant levels.
  • a synthetic or natural gene coding for human insulin would be exogenous genetic material to a yeast cell since yeast cells do not naturally contain insulin genes; a human insulin gene inserted into a skin fibroblast cell would also be an exogenous gene to that cell since skin fibroblasts do not express human insulin in biologically significant levels;
  • exogenous genetic material and “foreign” genetic material mean the same thing; and the terms “exogenous” and “foreign”, when used to describe genes or genetic materials, are used interchangeably herein;
  • retroviral vectors are the vehicles used to introduce replacement genes into the skin fibroblasts. The following paragraphs contain some general background information about retroviruses.
  • Retrovirus are RNA viruses; that is, the viral genome is RNA.
  • This genomic RNA is, however, reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells.
  • This integrated DNA intermediate is referred to as a provirus.
  • the retroviral genome and the proviral DNA have three genes: qa ⁇ . pol and env. which are flanked by two long terminal repeat (LTR) sequences.
  • the ⁇ a gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase) ; and the env gene encodes viral envelope glycoproteins.
  • the 5' and 3' LTRs serve to promote transcription and polyadenylation of virion RNAs. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA binding site) and for efficient encapsidation of viral RNA into particles (the ⁇ or psi site). (Mulligan, 1983; Mann et al., 1983; Ver a, 1985.)
  • the various elements required for replication of the retrovirus can be divided into cis- and trans ⁇ acting factors.
  • the trans-actin ⁇ factors include proteins encoded by the viral genome, which are required for encapsidation of viral RNA, entry of virions into cells, reverse transcription of the viral genome, and integration of the DNA form of the virus (i.e., the provirus) into host DNA.
  • the cis-actin ⁇ factors include signals present in the viral RNA which interact with the above-described proteins and other factors during virus replication.
  • murine leukemia virus env gene product is only able to infect rodent cells, which limits the utility of i>2 cell lines.
  • amphotrophic murine retroviruses are able to infect a wide variety of cell types, including human cells.
  • fibroblasts employed herein preferably they will be skin fibroblasts from the animal or individual to be treated with the gene therapy.
  • Fibroblasts from these subjects can easily be obtained by skin biopsy, and then maintained in culture until it is convenient to transduce them.
  • General methods for maintaining fibroblast cells in culture are well known to those skilled in the art of tissue culture. Such methods include culturing the cells in Dulbecco-Vogt modified Eagle's medium with 10% fetal bovine serum. Such known methods can be used by the skilled artisan, without undue experimentation, to culture the fibroblast cells prior to transduction. See generally, the Materials and Methods sections of Palmer, et al., (1987).
  • Exogenous genetic material or genes especially useful in the invention are preferably those genes that encode secretory proteins.
  • Such useful genes include, but are not limited to, genes that encode blood clotting factors such as human factors VIII and IX; hormone genes such as the genes encoding for insulin, parathyroid hormone, luteinizing hormone releasing factor (LHRH) , alpha and beta seminal inhibins, and human growth hormone; enzyme genes; genes encoding cytokines or lymphokines such as interferons, granulocytic macrophage colony stimulating factor (GM-CSF) , colony stimulating factor-1 (CSF-1) , tumor necrosis factor (TNF) , and erythropoietin (EPO) ; genes encoding inhibitor substances such as alpha,-antitrypsin, and genes encoding substances that function as drugs, e.g., genes encoding the diphtheria and cholera toxins.
  • blood clotting factors such as human factors VIII and IX
  • Genes that encode useful "gene therapy” proteins, e.g., many enzyme proteins, that are not normally secreted can be used in the invention if they are “functionally appended” to a signal protein sequence that will "transport” them across the fibroblasts 1 limiting membranes and into the extracellular space.
  • signal sequences are known and can be used by those skilled in the art without undue experimentation.
  • vehicles other than retroviruses to genetically engineer the fibroblasts of the present invention.
  • chimeric retroviruses are the preferred agents used to incorporate new genetic material into the skin fibroblasts. Retroviruses and helper-free replication-defective viral vectors are well known and can be adapted for use in the present invention without undue experimentation.
  • retroviral vectors useful in the method of the present invention will have a cloning site. The presence of such a site makes it possible to introduce exogenous genetic material into the vector and have it expressed by fibroblasts co-cultivated with the recombinant virus.
  • Methods for introducing exogenous genetic material into the retroviral vectors are known and can be used by the skilled artisan without undue experimentation. For example, useful methods are disclosed in the Experimental Section of this specification and in Palmer, et al., (1987).
  • a ⁇ am line which produces a chimeric retrovirus can be constructed as follows: the exogenous gene or cDNA of interest is ligated into a cloning site in the retroviral vector. (Such a vector could also carry a selectable marker such as the Neo gene.) Chimeric retroviruses that carry the exogenous gene or DNA of interest are isolated and transfected into ⁇ am cells. V>am cells that produce the chimeric virus construct are isolated, e.g., as G418 resistant colonies if the chimeric retrovirus carried the Neo gene as a selectable marker.
  • Co-cultivation methods are well known to those skilled in the art and can be used in the present invention without undue experimentation. Generally, the methods can be summarized as follows: On day one, fibroblast cells to be "infected" with chimeric retrovirus are seeded in conventional culture medium at approx. 5 x 10 cells per 60-mm culture dish. On day two, the culture medium is replaced with medium from cells that produce chimeric retrovirus. On day three, the infected fibroblasts are suspended with an enzyme such as trypsin. (Although it would not usually be necessary due to the high efficiency of bulk infection, if the chimeric retrovirus carried a selectable marker, the fibroblast cells would be grown in culture dishes containing selective media.
  • Resistant colonies i.e., those formed from cells that have been transduced by the chimeric retrovirus
  • Resistant colonies would then be scored after an appropriate amount of time (10-12 days) .
  • Fibroblasts from the resistant colonies would contain the new genetic material carried by the transducing chimeric retroviruses.
  • the skin fibroblasts are preferably "fixed” in vitro in an extracellular matrix. See generally, Elsdale, et al., (1972) and Bell, et al., (1979).
  • a preferred method for "fixing" the transduced fibroblasts in vitro in an extracellular matrix is discussed in the Experimental Section of this specification.
  • the fibroblasts are preferably fixed by culturing them in an extracellular matrix composed of collagen (either natural or synthetic) and culture medium. The cells are cultured at about 37'C for about 3 days, during which time the collagen contracts to a tissue-like structure.
  • the "artificial" fibroblast tissue grafts can be implanted into the loose connective tissue in the dermis of the recipient subject. While the extracellular collagen matrix is preferred (since it is easy, inexpensive and effective) , those skilled in the art will realize that other collagen-like materials, both natural and synthetic, could be used to generate the extracellular matrix into which the transduced fibroblasts become fixed.
  • basic fibroblast growth factor along with each graft.
  • the growth factor can be conveniently supplied by first applying it to a piece of sterile sponge, e.g., as gelfoam (Upjohn), which is then implanted in the connective tissue along with each graft.
  • the present invention makes it possible to genetically engineer skin fibroblasts that can secrete a variety of useful gene products (e.g., clotting factors, i munoregulatable factors, hormones and drugs) .
  • useful gene products e.g., clotting factors, i munoregulatable factors, hormones and drugs.
  • the implanted transduced fibroblasts of the present invention can be used in a variety of applications.
  • the implanted fibroblasts can serve as a continuous drug delivery system to replace present regimes that require periodic administration (by ingestion, injection, etc.) of a needed substance (e.g., to provide continuous delivery of insulin) . This would be very useful since it would eliminate the need for daily injections of insulin.
  • fibroblasts can also be used for the production of clotting factors. Hemophiliacs lack a protein called Factor VIII, which is involved in blood clotting. Factor VIII is now administered by injection. Like insulin, it could be made continuously or inducibly by transduced fibroblasts. Similarly, transduced and implanted fibroblasts could also be used to deliver growth hormone.
  • LHRH luteinizing hormone releasing factor
  • seminal and ovarian inhibins are being studied for their ability to regulate fertility. Continuous administration of LHRH results in a sterile individual; yet when administration of the hormone is stopped, fertility returns. Rather than taking LHRH injections or oral medication, one could implant collagen fixed fibroblasts carrying the LHRH gene under the control of a constitutive promoter, and thus provide a continuous supply of the hormone.
  • lymphokines such as GM-CSF can be continuously delivered to boost a subject's immune competence.
  • Such treat ent is especially useful in situations where the subject's immune system has been compromised by disease or treatment, such as in chemotherapy.
  • the amount of replacement gene product delivered to the subject can be controlled by controlling such factors as: (1) the type of promoter used to regulate the replacement gene (e.g., use of a strong promoter or a weak one); (2) the nature of the promoter, i.e., whether the promoter is constitutive or inducible; (3) the number of transduced fibroblasts that are present in the implant; (4) the size of the implant; (5) the number of implants, (6) the length of time the implant is left in place, etc.
  • the type of promoter used to regulate the replacement gene e.g., use of a strong promoter or a weak one
  • the nature of the promoter i.e., whether the promoter is constitutive or inducible
  • the number of transduced fibroblasts that are present in the implant (4) the size of the implant; (5) the number of implants, (6) the length of time the implant is left in place, etc.
  • Mouse primary skin fibroblasts were infected with a recombinant retrovirus containing human factor IX cDNA.
  • Bulk infected cells capable of synthesizing and secreting biologically active human factor IX protein were embedded in collagen and the implant grafted under the epidermis.
  • Sera from the transplanted mice contain human factor IX protein for at least 10-12 days. Loss of immunoreactive human factor IX protein in the mouse sera is not due to graft rejection. Instead, the mouse serum contains anti-human factor IX antibodies, which react with the protein.
  • the recombinant pAFFIXSVNeo is based on a retroviral construct pAFVXM generated by Kriegler, et al. (Kriegler, et al., 1984).
  • a human factor IX cDNA was linked directly to the 5' long terminal repeat (LTR) by inserting a 1.6 kilobase (kb) BamHI/Hindlll fragment from the clone CVI between the BallI and HindiII sites of pAFVXM (Anson, et al., 1984).
  • the entire expression unit from the neomycin phosphotransferase expression plasmid (pKoNeo) was excised by partial Hindlll digestion and inserted into the Hindlll site of the above factor IX viral construct (FIG. 1; in the figure, arrows indicate transcripts that initiate at either the promoter located in the 5* LTR, or the simian virus 40 early promoter located between the two LTRs, and terminate at the polyadenylation signal in the 3' LTR; vertical bars indicate the putative initiation site of transcription; the restriction endonuclease cleavage sites Sstl.
  • Hindlll, BamHI. B ⁇ lll and Clal are diagnostic sites used during the construction of the vector or subsequent characterization of the provirus in the genome of infected cell lines) .
  • Helper free recombinant ecotropic virus in ⁇ 2 cells was generated as described (Miller, et al., 1986; Mann, et al., 1983). The titres of recombinant retrovirus expressed from drug resistant clones were done essentially as described (Miller, et al., 1986).
  • Primary mouse embryo fibroblasts (MEF) were obtained from day 17 embryos of C57BL/6 mice (Todaro, et al., 1963).
  • the BL/6 line is an immortalized skin cell line derived from x-ray irradiated skin fibroblasts obtained form C57BL/6J mice.
  • the skin fibroblast cell line BL/6, and NIH3T3 TK " cells were infected with recombinant retroviruses from the cell line, ⁇ FIXNeo 4, at a multiplicity-of-infection (moi) of 1-2 in the presence of POLYBRENE at 8 ⁇ g/ml; MEF cells were infected at a moi of 5.
  • two artificial tissues containing approximately 4 x 10 infected fibroblasts were grafted into the loose connective tissue of the dermis in the mid-back of a recipient C57BL/6 mouse.
  • a 2-mm piece of gelfoam (Upjohn) containing 2 ⁇ g of basic fibroblast growth factor was inserted into the loose connective tissue along with each graft. Serum samples were drawn at two day intervals and analyzed for the presence of human factor IX by ELISA.
  • Biologically active human factor IX was immunoaffinity purified using A7 antibody (Anson, et al., 1987; Smith, et al., 1987). The amount of biologically active protein was determined by a one step clotting assay using canine factor IX deficient plasma (Goldsmith, et al., 1978). This assay is based on the ability of the sample to decrease the prolonged activated partial thromboplastin time of congenital factor IX-deficient plasma. Purified human factor IX was used as a control.
  • helper-free ⁇ FIXNeo virus produced in the various cell lines ranged from 3 x 10 to 7 x 10 G418 resistant colony forming units per ml when assayed by NIH3T3 TK " cells.
  • all of the virus producing cell lines secreted essentially the same levels of factor IX into the culture media (approx. 200 ng/ml) .
  • All infected and drug resistant cell lines were also found to secrete factor IX into the culture media, albeit at different levels (see FIG.
  • the organization of the integrated recombinant retrovirus in the virus producing cell line was determined by Southern blot analysis of SstI digested genomic DNA isolated from either uninfected or infected FIXNeo 4, NIH3T3 TK " , BL/6, and MEF cells, fractionated by agarose gel electrophoresis, transferred onto a nitrocellulose membrane and hybridized to either a nicktranslated 1.6 kb factor IX cDNA probe, or 1.4 kb Hindlll to BamHI Neo DNA probe; under hybridization conditions, human factor IX cDNA does not hybridize to mouse DNA) .
  • SstI cleaves once in each LTR to generate a 5.1 kb DNA fragment.
  • infected cells displayed a single band of the expected size of approximately 5.1 kb which hybridizes to both the factor IX cDNA and the Neo probe, therefore ruling out any detectable rearrangements. Furthermore, the size of this band in infected NIH3T3 TK " , BL/6, and MEF cells is identical to that found in the virus producing cell line ⁇ FIXNeo 4.
  • RNA blot analysis (of the RNA isolated from FIXNeo 4, infected NIH3T3 TK " , BL/6 and MEF), when hybridized to factor IX probe, shows only one major transcript of the expected size of 5.1 kb, corresponding to full length viral RNA could be detected in the infected cells.
  • Hybridization with Neo probe reveals an additional 2.2 kb transcript that is the predicted size of the mRNA species, the synthesis of which is initiated from the simian virus 40 early promoter and is terminated in the 3' LTR. Ratios of the steady state levels of the 5.1 kb and the 2.2 kb transcripts varied within the different infected cell types. From these results, it is concluded that the ⁇ FIXNeo recombinant retrovirus is properly integrated and expressed in the infected cells.
  • FIG. 2 shows that both rate and extent of antigenic factor IX released into the medium is dependent on the cell type rather than on the relative amounts of the factor IX transcripts. For instance, steady state levels of factor IX transcript in infected NIH3T3 TK " cells is much higher than in BL/6 cells; yet the rate and amount of factor IX secreted in the latter cell type is much higher.
  • infected mouse embryo fibroblasts were cultured in factor IX deficient canine serum obtained from hemophiliac dogs, supplemented with epidermal growth factor (10 ng/ml) and vitamin K (25 ng/ml) .
  • Media harvested after 48 hr incubation was monitored for activity by a one step assay (Goldsmith, 1978) .
  • Conditioned media from MEF cells contained biologically active human factor IX at 210 ng/ml which is similar to the levels seen with ELISA assays;
  • Infected MEF cells and BL/6 cells were cultured in an extracellular matrix, composed of collagen, before grafting.
  • a tumor cell line, BL/6 was chosen in addition to MEF because it has an advantage in growth and vascularization and thus would increase our chances of detecting secreted factor IX in the sera. Attachment of the cells to the collagen resulted in a three-dimensional array of cells stacked on top of one another. After the primary fibroblast cells (MEF) or the tumor cell line BL/6 contracted in the collagen gel, the cells were grafted into the loose connective tissue of the mid-back dermis of a recipient syngeneic C57BL/6 mouse (FIG.
  • the serum levels of the human clotting factor were measured in engrafted mice by ELISA over a 34 day period.
  • the average levels of human factor IX in 3 mice progressively increased from 20 ng/ml at day 2 to a peak of 97 ng/ml 7 days after grafting the BL/6 cells into the mice.
  • the 4 mice grafted with the infected MEF fibroblasts showed a similar pattern of increase in which an average peak of 25 ng/ml of factor IX was detected at day 9. This rise was followed by a rapid decline to near non detectable levels of serum human factor IX at day 16 in both the BL/6 and MEF grafts.
  • Table I reports the amount of antigenic factor IX secreted from cells explanted from grafts; tissue was explanted from the grafts at times indicated in the Table (post implantation) , and were cultured in vitro; when cells were confluent medium was replaced; after 48 hr, levels of secreted factor IX secreted into the culture were assayed by ELISA.
  • nitrocellulose strips were treated with blocking solution for 2 hr followed by 1:100 dilution of naive normal mouse serum; 1:100 dilution of mouse monoclonal anti-factor IX antibody FXC008; 1:100 dilution of serum from mouse harboring grafts containing infected MEF cells drawn at day 7, day 14, day 20, and day 28; and 1:100 dilution of serum from mouse harboring grafts containing infected BL/6 cells drawn at day 7, day 15, day 21 and day 29.
  • mice with MEF grafts After overnight incubation at 37 ⁇ C the strips were washed, incubated wi .th 125I-labeled goat anti.-mouse IgG antibody, and then subjected to autoradiography as described (Glenney, 1986) .
  • the levels of anti-human factor IX IgG antibodies were not detectable in mice with MEF grafts at day 7 to day 21. Slightly higher levels of serum antibodies were detected in mice with BL/6 grafts during this period, presumably because they are releasing more factor IX. Maximum levels of anti-human factor IX antibodies were detected at day 28 in mice with either graft. The mice with BL/6 grafts exhibited the highest level of xeno-antibodies.
  • Grafts are quickly vascularized in the presence of angiogenic factor, fibroblast growth factor, and remain vascularized for at least 28 days. Grafts containing the BL/6 cells grow as aggressive tumors over this period while the size of the grafts containing the MEF cells does not increase over the same period.
  • the clotting factor secreted from the infected cells in the graft is accessible to the circulatory compartment and can easily be detected in serum of the graft recipient. Functional status of the infected cells in the grafts can be measured by monitoring serum levels of human factor IX or by the ability of explanted cells to continue secreting the human protein.
  • C57BL/6 mice recognize the human blood clotting factor as foreign and thus mount a strong humoral immune response against it.
  • the low levels of factor IX can be increased either by making improved vectors capable of generating large amounts of factor IX proteins or, alternatively, by grafting more cells. According to the data presented here, up to 25 ng of factor IX per hr can be generated from an implant containing 4 x 10 cells. In larger animals multiple grafts of up to 10 cells can be easily implanted, increasing the levels of factor IX protein to that required to alleviate the deficiency.
  • the efficiency of the invention delivery system can be further enhanced by such expedients as culturing infected cells in a defined medium (without fetal bovine serum) and applying improved technology for the reconstitution of living skin (Bell, et al., 1983).
  • Retroviral vector LNCdF9L which transduces canine factor IX, has previously been described [Axelrod, et al., Proc. Natl. Acad. Sci. USA £7: 5175-5177 (1990)].
  • the vectors shown in Figure 4 were generated by inserting a 3.1 kBP BamHl fragment containing the entire coding sequence of the 3-galactosidase gene into the Bglll site plasmid LNL-SLX to generate the vector LNL-SLX3gal (in the Figure, the number of NEO colonies examined is in parentheses; expression of j8-galactosidase activity was determined by X-gal staining) .
  • the LNL-SLX vector is a derivative of LNL-XHC [Bender, et al., Virology __: 1639- 1946 (1987)] and contains a new polylinker to increase the number of cloning sites.
  • a 350 bp Hindlll fragment of the mouse dihydrofolate reductase (DHFR) promoter was cloned in the unique Hindlll site of LNL-SLX3gal.
  • a BamHI/Hindlll fragment containing the human intermediate early Cytomegalovirus (CMV-IE) promoter [-522 to +55; Nelson, et al., Mol. Cell. Biol. ⁇ : 4125-4129 (1987)] was cloned in the BamHI/Hindlll site if LNL-SLXSgal.
  • plasmid DNA was transfected into the ecotropic packaging cell line psi-CRE by the calcium phosphate coprecipitation method. The medium was changed 24 hours later; and 48 hours after transfection, the culture medium was harvested and used to infect the amphotropic packaging cell line psi-CRIP in the presence of 8 ⁇ g/ml of POLYBRENE. Single colonies of infected psi-CRIP were isolated by selection in the presence of G418-containing medium and expanded. Recombinant retroviruses were harvested from confluent culture dishes, filtered and used to infect NIH 3T3 cells in the presence of POLYBRENE to determine the viral titers.
  • Beta-galactosidase histochemistry was performed according to Sanes, et al., Embo. J. 5_: 3133-3142 (1986), with minor modifications. Briefly, cultured cells were rinsed with phosphate buffered saline solution (PBS) , pH 7.4, and then fixed for 5 minutes on ice in 2% formaldehyde plus 0.2% glutaraldehyde in PBS.
  • PBS phosphate buffered saline solution
  • the cells were then rinsed 2 times with PBS and overlaid with a solution containing 1 mg/ml of 4-Cl-5-Br-3-indodyl-,9- galactosidase (X-gal) , 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl 2 in PBS, pH 7.4. Incubation was performed at 37 ⁇ C for 2 to 24 hours. To analyze 0-galactosidase activity in the artificial collagen matrix, the fixation was prolonged for 30 minutes on ice.
  • X-gal 4-Cl-5-Br-3-indodyl-,9- galactosidase
  • the canine Factor IX infected syngeneic skin fibroblasts were implanted in nude mice. If transformed mouse fibroblasts producing canine Factor IX are used as an implant, the levels of Factor IX detected in mouse serum are observed to be at their highest at about day 5 post implantation, and by day 10 decline to near basal levels. However, by day 15 the levels of secreted Factor IX begin to increase and continue to increase thereafter. Because the implant contained tumorigenic cells which lead to the formation of palpable tumors, the increase in production of Factor IX reflects cell growth. However, the experiment clearly shows that the reduction of Factor IX levels by day 10 is not due to antibodies against canine Factor IX, since biologically active Factor IX can be detected even after 30-35 days.
  • Clones producing high titre amphotropic recombinant viruses were selected by infecting NIH 3T3 cells, analyzed for ,9-galactosidase activity, and the presence of helper viruses.
  • Figure 4 shows that only a few clones producing greater than 5x10 /ml neo R colonies could be identified, but the resultant recombinant viruses were stably propagated.
  • the initial levels of ,6-galactosidase activity were higher in cells infected with LNL-SLX CMV /S-gal virus (visible after 2 hrs of incubation) as compared to LNL-SLX DHFR /8-gal virus (visible after 8-6 hrs of incubation) .
  • RNA transcripts from cells infected with LNL-SLX CMV 3-gal were identified by the RNA transcripts from cells infected with LNL-SLX CMV 3-gal and the RNA transcripts from cells infected with LNL-SLX CMV 3-gal and the RNA transcripts from cells infected with LNL-SLX CMV 3-gal and the RNA transcripts from cells infected with LNL-SLX CMV 3-gal and the RNA transcripts from cells infected with LNL-SLX CMV 3-gal and
  • LNL-SLX DHFR /3-gal viruses were analyzed.
  • Figure 5 shows that transcripts of the expected size (6.6 kB, 6.1 kB and 3.6 kB) can be detected in virus producing CRIP cells or mouse embryo fibroblasts.
  • the 3.6 kB mRNA represents transcripts initiated from the CMV or DHFR promoter. No detectable levels of /3-galactosidase RNA were observed in uninfected cells.
  • mice H. ⁇ -galactosidase Expression in Mice
  • mouse embryo fibroblasts were infected with either LNL-SLX CMV /3-gal or LNL-LSX DHFR /3-gal viruses.
  • the infected cells were then embedded in a collagen matrix and grafted in mice. After different time intervals, the grafts were explanted and analyzed for the presence of /3-galactosidase positive cells. A minimum of two to three grafts were explanted at each time point.
  • /3-galactosidase positive cells stained blue with X-Gal
  • transduced fibroblasts are preferably created by infecting fibroblast cells in vitro with chimeric retroviruses that contain at least one functionally active "replacement gene", optionally under the additional control of a constitutive or inducible promoter other than the retroviral promoter.
  • replacement genes can be either foreign genetic material that is not found in fibroblast cells, or native genetic material that is found in fibroblast cells but not normally expressed in biologically significant concentrations in these cells. Since the invention uses transduced fibroblasts from the individual or animal to be treated, the possibility of rejection is minimized.
  • the invention implants the transduced fibroblasts in the highly vascularized loose connective tissue of the dermis, the transduced cells, and thus their "replacement" gene products, have direct access to the circulatory system. As a result the needed replacement gene products can easily and efficiently be distributed to other parts of the body. When the gene therapy is no longer needed, the implanted fibroblasts can be conveniently removed.
  • the method of the invention has many important applications for both humans and animals.
  • the method can be used to treat diseases caused by genetic defects, to deliver drugs to individuals and animals, to induce immune response, and to administer birth control hormones.
  • one of ordinary skill can make various changes and modifications to the invention to adapt it to various usages and conditions. As such, these changes and modifications are properly, equitably, and intended to be, within the full range of equivalence of the following claims.

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Abstract

Procédé de thérapie génique par les cellules somatiques utiles notamment dans le traitement de certaines maladies provoquées par des tares génétiques. Selon l'invention, on procède à la transduction de cellules de fibroblastes de sorte qu'elles expriment un gène de 'remplacement' d'intérêt. Ces fibroblastes ayant subi une transduction sont de préférence fixés in vitro dans une matrice extracellulaire, puis ils sont implantés dans le tissu conjonctif lâche de la peau d'un individu ou d'un animal à traiter. Comme les fibroblastes sont implantés dans un compartiment hautement vascularisé de la peau, c'est-à-dire le tissu conjonctif lâche du derme, les cellules ayant subi une transduction ainsi que leurs produits génétiques de 'remplacement' ont accès direct au système circulatoire. Ainsi, les produits génétiques de remplacement nécessaires peuvent être facilement et efficacement répartis dans d'autres parties du corps. Lorsque la thérapie génique n'est plus nécessaire, on peut facilement retirer les fibroblastes implantés.
PCT/US1992/001890 1991-03-08 1992-03-06 Therapie genique par les cellules somatiques WO1992015676A1 (fr)

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FR2704236A1 (fr) * 1993-04-21 1994-10-28 Pasteur Institut Vecteur rétroviral pour la préparation de cellules recombinantes susceptibles d'être implantées in vivo dans un but thérapeutique.
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US5423778A (en) * 1989-12-14 1995-06-13 Elof Eriksson System and method for transplantation of cells
FR2724320A1 (fr) * 1994-09-13 1996-03-15 Transgene Sa Nouvel implant pour le traitement des maladies acquises
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US6326195B1 (en) 1993-04-21 2001-12-04 Institut Pasteur Recombinant retroviral vector
US6068837A (en) * 1993-06-18 2000-05-30 Beth Israel Hospital Association Mesothelial cell gene therapy
US5645829A (en) * 1993-06-18 1997-07-08 Beth Israel Hospital Association Mesothelial cell gene therapy
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