WO1998019540A1 - Procede de selection in vivo de cellules hematopoietiques primitives - Google Patents

Procede de selection in vivo de cellules hematopoietiques primitives Download PDF

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Publication number
WO1998019540A1
WO1998019540A1 PCT/US1996/017660 US9617660W WO9819540A1 WO 1998019540 A1 WO1998019540 A1 WO 1998019540A1 US 9617660 W US9617660 W US 9617660W WO 9819540 A1 WO9819540 A1 WO 9819540A1
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cells
hematopoietic
antifolate
nonmodified
subject
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PCT/US1996/017660
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English (en)
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Brian P. Sorrentino
Raymond L. Blakely
H. Trent Spencer
James Allay
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Saint Jude Children's Research Hospital
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Priority to AU76057/96A priority Critical patent/AU7605796A/en
Priority to US09/297,749 priority patent/US6500421B1/en
Priority to PCT/US1996/017660 priority patent/WO1998019540A1/fr
Publication of WO1998019540A1 publication Critical patent/WO1998019540A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
    • C12N9/003—Dihydrofolate reductase [DHFR] (1.5.1.3)
    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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

Definitions

  • This invention relates generally to selection of hematopoietic cells. Specifically, this invention relates to selection of hematopoietic progenitor and hematopoietic stem cells using a mutant dihydrofolate reductase in combination with a nucleoside transport inhibitor and an antifolate.
  • Retroviral-mediated gene transfer is a potential therapeutic strategy for a number of diseases that affect the hematopoietic system.
  • the early hematopoietic cells including hematopoietic progenitor cells and hematopoietic stem cells (HSC) are desirable targets for gene therapy.
  • the hematopoietic stem cell is especially desirable for gene therapy because it can contribute progeny to all hematopoietic lineages and can support hematopoiesis throughout the lifetime of an animal. Despite these attractive features, primate HSCs remain relatively refractory to genetic modification.
  • Dihydrofolate reductase is a ubiquitous cellular enzyme that catalyzes the generation of tetrahydrofolate, a necessary cofactor for purine and pyrimidine biosynthesis.
  • Antifolate drugs such as methotrexate (MTX) are powerful inhibitors of DNA synthesis by virtue of their strong binding to the active site of DHFR.
  • MTX methotrexate
  • the discovery that single amino acid substitutions in the active site of DHFR could disrupt drug binding and thereby confer antifolate resistance (Simonsen et al. "Isolation and expression of an altered mouse dihydrofolate reductase cDNA.” Proc. Natl. Acad. Sci. U S A 80:2495 (1983)) raised the possibility that mutant DHFRs could potentially be used as drug resistance genes.
  • DHFR genes were the first drug resistance genes to be transferred to primary hematopoietic cells.
  • DHFR as an in vivo selectable marker
  • evidence for in vivo selection has been equivocal using this experimental system.
  • MTX-treated mice containing the murine L22R (leucine to arginine substitution at codon 22) variant of DHFR there appeared to be an enrichment of vector-transduced CFU-S cells following MTX treatment. (Corey et al.
  • the present invention overcomes these problems by disclosing a method which allows for effective elimination of unmodified hematopoietic cells which do not contain a transferred DHFR.
  • the method thereby allows the modified hematopoietic progenitor and stem cells containing a modified DHFR to form a large proportion of the hematopoietic cells after reconstitution.
  • the present method utilizes a nucleoside transport inhibitor to sensitize the non-modified hematopoietic cells to the antifolate. This invention, therefore, solves the problems identified by Blau et al. using a completely different approach.
  • nucleoside transport inhibitors have previously been proposed to potentiate the sensitivity of tumors to a variety of antifolates, including PALA, methotrexate, 5-fluorouracil, and acivicin, and potentially make these more effective by blocking the salvage of exogenous nucleosides.
  • PALA methotrexate
  • 5-fluorouracil 5-fluorouracil
  • acivicin acivicin
  • this invention utilizes for the first time nucleoside transport inhibitors to select against unmodified hematopoietic progenitor and stem cells to provide an effective means to utilize gene therapy to treat many diseases of hematopoietic cells.
  • this invention in one aspect, provides a method of in vivo selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells in a subject comprising genetically modifying hematopoietic progenitor cells by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which, when expressed, can confer antifolate resistance, administering to the subject the genetically modified hematopoietic progenitor cells, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect
  • the invention further provides a method of in vivo selection for hematopoietic progenitor cells genetically modified to contain and express a nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase from nonmodified hematopoietic cells in a subject comprising administering to the subject the genetically modified hematopoietic progenitor cells, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo for the genetically modified hematopo
  • the invention provides a method of in vitro selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells comprising genetically modifying hematopoietic progenitor cells by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to hematopoietic cells comprising the genetically modified hematopoietic progenitor cells and nonmodified hematopoietic cells an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset by nucleoside salvage, and administering to the hematopoietic cells a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hema
  • the invention provides a method of in vitro selecting for genetically modified hematopoietic progenitor cells containing and expressing a nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase from nonmodified hematopoietic cells comprising administering to hematopoietic cells comprising genetically modified hematopoietic progenitor cells and nonmodified hematopoietic cells an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset by nucleoside salvage, and administering to the hematopoietic cells a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects
  • the invention provides a method of in vivo selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells in a subject comprising, genetically modifying hematopoietic progenitor cells in the subject by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo
  • nucleic acid comprising a sequence encoding a dihydrofolate reductase which is relatively resistant to an antifolate can further comprise a heterologous gene.
  • the methods of the present invention can be used in gene therapy procedures.
  • the present invention further provides nucleic acids encoding mutant dihydrofolate reductases and cells expressing those nucleic acids.
  • Fig. 1 shows TTIs effectively block nucleoside salvage and potentiate TMTX toxicity at physiological concentrations of thymidine.
  • Unseparated bone marrow from normal C57B1/6 or WBBF, +/+ mice were cultured in DMEM plus dialyzed FBS with or without 150 nM TMTX, 100 mM hypoxanthine and 0.1, 1.0 or 10 ⁇ M thymidine.
  • either A) NBMPR or B) draflazine at 0.1 or 1.0 ⁇ M were added to the indicated suspension cultures.
  • Progenitors were assayed by a semisolid methylcellulose- based myeloid progenitor assay after four days of suspension culture.
  • Fig. 2 shows L22Y-DHFR transduced progenitors are protected from TMTX + NBMPR toxicity in vitro.
  • Unseparated bone marrow from C57B1/6 or WBBF j +/+ mice was prestimulated for 48 hrs in medium supplemented with IL-3, IL-6 and SCF, and then layered on 100% confluent, irradiated L22Y-DHFR or control MDR1 ecotropic retroviral producers for an additional 48 hrs.
  • Nonadherent marrow cells were then harvested, washed extensively in PBS and placed in a suspension culture system in DMEM supplemented with optimal concentrations of IL-3, IL-6 and SCF, and either undialyzed or dialyzed fetal bovine serum (FBS), with or without 1.0 ⁇ M NBMPR. Progenitor content was then assayed after four days of suspension culture as described in the legend to figure 1. Progenitor survival was scored relative to control cultures without TMTX addition.
  • FBS dialyzed fetal bovine serum
  • Fig. 3 shows thymidine transport inhibitors sensitize murine progenitors to TMTX in vivo.
  • Normal C57B1/6, WBBF, +/+ or B6.C-Hl 6 /By (HW80) mice were administered TMTX (130 mg kg), NBMPR-P (20 mg/kg) or draflazine (20 mg/kg) alone, or the combination of TMTX + NBMPR-P or TMTX + draflazine ip for five consecutive days. 24 hrs following the last treatment, mice were sacrificed and bone marrow cellularity and myeloid progenitor content per femur were analyzed relative to untreated mice.
  • Figure 4 shows in vivo protection of L22Y-DHFR modified bone marrow cells (4A) and progenitors (4B) in mice treated with TMTX + NBMPR-P.
  • bone marrow was harvested from C57B1/6 mice and prestimulated and transduced with L22Y-DHFR or MDR1 retroviral vectors.
  • 2 x 10 6 transduced marrow cells were then transplanted into genetically anemic W/W v mice.
  • Transplanted normal donor cells competitively reconstitute host hematopoiesis completely due to the defective c-kit receptor expressed on host hematopoietic cells.
  • mice were administered TMTX + NBMPR-P for five consecutive days. 24 hrs following the final treatment, bone marrow cellularity (4A) and hind limb progenitor (4B) content were analyzed relative to untreated transplanted mice.
  • Figure 5 shows evidence for in vivo selection of L22Y-DHFR transduced myeloid progenitors in mice treated with TMTX + NBMPR-P 6 months after transplant.
  • Individual progenitor-derived colonies isolated from methylcellulose plates from untreated or TMTX + NBMPR-P treated mice were examined by PCR for presence of integrated provirus (L22Y-DHFR vector).
  • Primers specific for the endogenous mouse ⁇ -globin gene were used as an internal control (globin control).
  • FIG. 6 shows TMTX and NBMPR-P kill hematopoietic stem cells in normal mice.
  • C57B1/6 donor mice were administered either 100 mg/kg TMTX, 100 mg/kg TMTX + 20 mg/kg NBMPR-P, or 130 mg/kg TMTX + 20 mg/kg NBMPR-P ip for five consecutive days.
  • 24 hrs following the last treatment bone marrow was harvested and transplanted into genetically anemic W/W v mice. The hemoglobin phenotype of transplanted mice was determined after 8 weeks. Bone marrow from either untreated mice or mice that received treatments that did not kill stem cells completely repopulated recipients, as evidenced by the absence of the host hemoglobin phenotype. Bone marrow from donor mice that received treatments that were cytotoxic to stem cells failed to reconstitute host hematopoiesis, as evidenced by the maintenance of the host hemoglobin phenotype.
  • Figure 7 shows draflazine potentiates TMTX toxicity in human myeloid progenitors.
  • CD34-enriched, mobilized peripheral blood progenitors were assayed in the defined suspension culture system, as described for murine cells in the legend to figure 1. Two separate human samples were examined, and progenitor survival is represented relative to progenitors cultured without TMTX.
  • Figure 8 shows L22Y-DHFR-transduced human myeloid progenitors have increased TMTX resistance in vitro.
  • Peripheral blood cells collected following chemotherapy and growth factor mobilization were prestimulated for 48 hrs with IL-3, IL-6 and SCF, followed by coculture on irradiated amphotropic retroviral producers (L22Y-DHFR or neo) for an additional 48 hrs.
  • Nonadherent hematopoietic cells were then assayed for drug resistant progenitors by addition of increasing concentrations of TMTX to the semisolid methylcellulose cultures.
  • the invention provides a method of in vivo selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells in a subject comprising genetically modifying hematopoietic progenitor cells by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to the subject the genetically modified hematopoietic progenitor cells, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in
  • the present invention provides a method of in vivo selection for genetically modified hematopoietic stem cells from nonmodified hematopoietic stem cells in a subject comprising genetically modifying hematopoietic stem cells by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to the subject the genetically modified hematopoietic stem cells, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic stem cells, wherein the inhibition of the nonmodified hematopoietic stem cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic stem cells, whereby the combination of the antifolate
  • Hematopoietic progenitor cells and hematopoietic stem cells are well known in the art.
  • Hematopoietic progenitor cells represent those cells which are derived early in hematopoiesis from hematopoietic stem cells and are committed to a specific differentiation path for each type of mature blood cell.
  • Techniques are widely known in the art which are used to identify hematopoietic progenitor cells, such as their ability to form myeloid colonies in semisolid media (Inscove et al. Proc. Soc. Exp. Biol. Med. 134:33 (1970)), the ability to form large IL-1 responsive colonies in culture (Bertoncello et al. Exp. Hematol.
  • selection is a term familiar to one of ordinary skill in the art and is used herein to describe supporting or sustaining the growth of a particular cell versus a different cell. For example, by genetically modifying hematopoietic progenitor cells and hematopoietic stem cells so that these cells are relatively resistant to the inhibitory effects of an antifolate drug, these modified cells can be "selected” for in a mixed population of cells comprising modified cells and nonmodified cells when the population is treated with an antifolate.
  • the growth of the nonmodified cells in the population can be inhibited by the antifolate whereas the growth of the modified cells is either not inhibited, or is inhibited to a lesser degree than the nonmodified cells.
  • the inhibition of the nonmodified cells is not necessarily complete and the resistance of the modified cells to the inhibitory effects of the antifolate is not necessarily total, but when these two types of cells are treated with comparable amounts of an antifolate, the genetically modified cells are less affected by the drug than the nonmodified cells, i.e.
  • the genetically modified cells are able to outcompete and/or outgrow the nonmodified cells, which can result in the predominance of the modified cells from a mixed population of cells comprising genetically modified cells and nonmodified cells.
  • genetically modified is another term familiar to one of ordinary skill in the art and is used herein to describe either introducing into cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which has been mutated versus the wild-type dihydrofolate reductase such that the mutant dihydrofolate reductase is relatively resistant to the inhibitory effects of an antifolate versus the wild- type dihydrofolate reductase.
  • antifolates such as aminopterin, methotrexate (amethopterin), trimetrexate, edatrexate, pyritrexim, trimethoprim, pyrimethamine, 5, 10 dideazatetrahydrofolate, 10-ethyl-lO-deaza- aminopterin, 10-propargyl-5,8 dideazafolate, and 2,4-diamino 5(3', 4' dichlorophenyl) 6 methylpyrimidine (DDMP) block the reduction of dihydrofolate to tetrahydrofolate and thereby inhibit de novo synthesis of nucleotides which ultimately leads to an inhibition of cell growth in certain cells dependent upon de novo nucleotide synthesis by inhibiting dihydrofolate reductase.
  • DDMP 2,4-diamino 5(3', 4' dichlorophenyl) 6 methylpyrimidine
  • Certain mutants of dihydrofolate reductase are relatively resistant to the inhibitory effects of antifolate drugs and the presence and expression of genes encoding these mutant dihydrofolate reductases in a certain cell can render these cells relatively resistant to the inhibitory effects of the antifolates.
  • Other mutations to the nucleic acid encoding the dihydrofolate reductase are possible, but as used herein, the term "genetically modified" refers to the specific alterations to the nucleic acid, and therefore changes in the polypeptide, which render the dihydrofolate reductase relatively resistant to the inhibitory effects of an antifolate.
  • nonmodified is not limited to a nucleic acid encoding a dihydrofolate reductase or the dihydrofolate reductase itself being totally homologous to the wild-type nucleic acid or polypeptide, but this specifically refers to the absence of modifications which can render the polypeptide relatively resistant to the inhibitory effects of an antifolate.
  • nucleoside transport inhibitors to effectively render nonmodified hematopoietic cells more susceptible to the inhibitory effects of an antifolate in combination with a mutant dihydrofolate reductase which is relatively resistant to the inhibitory effects of an antifolate.
  • the nucleoside transport inhibitor blocks the salvage of nucleosides which, in primitive hematopoietic cells such as hematopoietic progenitor cells and hematopoietic stem cells, can bypass the de novo nucleotide synthesis block induced by the antifolate.
  • the de novo synthesis of inosine monophosphate and therefore the de novo synthesis of d ATP and dGTP is blocked by the inhibition of DHFR.
  • This blockage can be bypassed by a salvage pathway utilizing hypoxanthine.
  • the de novo synthesis of dTMP from dUMP is blocked by the inhibition of DHFR, but this blockage can be bypassed by the salvage pathway utilizing thymidine. Therefore nonmodified hematopoietic progenitor cells and nonmodified hematopoietic stem cells, when treated with a suitable nucleoside transport inhibitor and an antifolate, are unable to grow and replicate since DNA synthesis is inhibited.
  • nucleoside transport inhibitors which specifically block the salvage of thymidine include, but are not limited to dilazep, draflazine, a 5' monophosphate derivative of nitrobenzylmercaptopurine riboside (NBMPR or NBMPR-P), dipyridamole, mioflazine, soluflazine, and R57974.
  • NBMPR nitrobenzylmercaptopurine riboside
  • hematopoietic progenitor cells or the hematopoietic stem cells can be genetically modified.
  • retroviral vector systems which can package a recombinant retroviral genome containing a nucleic acid encoding a modified dihydrofolate reductase.
  • nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which, when expressed, can confer antifolate resistance to hematopoietic progenitor cells and/or hematopoietic stem cells
  • nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase which, when expressed, can confer antifolate resistance to hematopoietic progenitor cells and/or hematopoietic stem cells
  • This additional sequence is not limited to the entire sequence comprising a "gene" but can comprise the sequence encoding the gene product.
  • the coding region of the gene can be operatively linked to an additional sequence, such as a promoter in a vector, whereby the nucleic acid is expressed and the gene product is produced.
  • the heterologous gene can be any gene encoding a gene product having clinical usefulness, for example, any gene or gene product that directly or indirectly enhances the therapeutic effects of the cells.
  • the heterologous gene can be any gene or gene product that allows the cells to exert a therapeutic effect that the cells would not otherwise exert.
  • heterologous genes which can be used for genetic therapy, are for example, those that encode cytokines such as tumor necrosis factor (TNF), interleukins (for example, interleukins 1-12), interferons (alpha, beta, and gamma-interferons), T-cell receptor proteins and the Fc receptors for antigen-binding domains of antibodies, such as immunoglobulins.
  • TNF tumor necrosis factor
  • interleukins for example, interleukins 1-12
  • interferons alpha, beta, and gamma-interferons
  • T-cell receptor proteins T-cell receptor proteins
  • Fc receptors for antigen-binding domains of antibodies, such as immunoglobulins.
  • Other examples of gene therapy are the supplementation of genes of the host, that for some reason do not produce a functional gene product or insufficient gene product, with a wild-type gene or a modified gene, which when expressed by the host, can produce a gene product which alleviates the condition of the host which is
  • hemoglobinopathies ⁇ -globin
  • HIV infection ⁇ -globin
  • HPRT Lesch-Nyhan syndrome
  • ADA severe combined immunodeficiency
  • GC GC
  • the hematopoietic progenitor cells or hematopoietic stem cells can be removed from a subject, modified outside the subject, and administered to the same subject after modification (e.g. autologous cells).
  • the hematopoietic progenitor cells or hematopoietic stem cells can be from a different subject than the subject to whom they are to be administered as long as they are compatable so that the modified cells are not rejected by the subjects immune system.
  • the methods for facilitating engraftment are well established in the art. See, for example, Torok-Storb et al. "Role of marrow microenvironment in engraftment and maintenance of allogeneic hematopoietic stem cells.
  • the particular hematopoietic cells which are to be genetically modified or those that are already genetically modified can be allogeneic with respect to the subject that is to receive the genetically modified cells. Additionally, the particular hematopoietic cells which are to be genetically modified or those that are already genetically modified can be xenogeneic with respect to the subject that is to receive the genetically modified cells. In either situation, it is contemplated that the genetically modified cells are or will become compatable to the host, either genetically or through immune suppression, so that the modified cells are not rejected by the subject's immune system.
  • these cells can also be genetically modified by techniques such as site-specific recombination or mutagenesis whereby an altered nucleic acid is exchanged for the wild-type nucleic acid within the cell or the wild-type nucleic acid is altered within the cell.
  • genetically modifying cells by introducing into cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase is not limited to simply adding a modified nucleic acid to the cells, but also includes any technique whereby a modified nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase is expressed in a cell that previously expressed a nucleic acid encoding a nonmodified dihydrofolate reductase.
  • genetically modifying these cells includes altering the expression of the nucleic acids such that the nucleic acid encoding the modified dihydrofolate reductase is favored to the extent that the cell is relatively resistant to the inhibitory effects of an antifolate.
  • the methods described herein are not limited to specific methods in which each manipulation can be performed.
  • the present invention describes the discovery of and claims the manipulation of cells whereby specific cells in a population, such as hematopoietic progenitor cells or hematopoietic stem cells, can be selected.
  • hematopoietic progenitor cells or hematopoietic stem cells after being genetically modified by introducing into these cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which is relatively resistant to an antifolate, are administered to a subject.
  • This subject can be a human or any other mammal, such as canine, equine, feline, porcine, bovine, or non-human primates.
  • the techniques used for administering the genetically modified hematopoietic progenitor cells or the genetically modified hematopoietic stem cells will be apparent to one of ordinary skill in the art.
  • these modified cells can be administered by IN. to a patient in an amount sufficient to repopulate the patient's hematopoietic and immune system.
  • the genetically modified cells can be directly introduced into a subject as a bone marrow administration. Precise, effective quantities can be readily determined by those skilled in the art and will depend, of course, upon the exact condition being treated, the severity of the condition, the size of the subject, etc.
  • the subject that is being administered the genetically modified hematopoietic progenitor cells or hematopoietic stem cells is also administered an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells in the presence of a suitable nucleoside transport inhibitor.
  • the effective amount of the antifolate can readily be determined by one skilled in the art and can typically range from 5 to 500 nM if the cells are selected in culture with trimetrexate or from 5 to 11 mg/square meter of body surface area given daily for five consecutive days where the cells are selected in vivo with trimetrexate. These dosages are exemplary only and are not meant to be limiting.
  • the specific dosage will of course vary, and may depend upon such variables such as the nucleoside transport inhibitor, the mass of the subject, the condition of the subject, the type and severity of the condition the subject is experiencing which requires therapy, the expression levels of nucleic acid encoding the nonmodified dihydrofolate reductase, etc.
  • the therapeutically effective amount can readily be determined by routine optimization procedures.
  • the administration of the antifolate can have the additional advantage in certain subjects of inhibiting the growth of not only the nonmodified hematopoietic cells, but of other rapidly growing cells as well, such as cancer cells and especially certain types of leukemias.
  • This type of selection therefore, has the particular advantage of being able to select for modified hematopoietic cells that have resistance to an antifolate in a subject that is being administered an antifolate as an anticancer chemotherapeutic.
  • the modified hematopoietic cells can therefore divide and differentiate within a chemotherapy patient so that the patient's hematopoietic system can become reestablished with these genetically modified hematopoietic cells which will also be resistant to further chemotherapy treatments.
  • the nucleoside transport inhibitor blocks the salvage pathway of nucleotide synthesis, specifically thymidylic acid synthesis; therefore cells exposed to a nucleoside transport inhibitor are dependent upon de novo thymidylic acid synthesis.
  • This de novo synthesis can be blocked in cells by an antifolate which inhibits dihydrofolate reductase.
  • the inhibitory effects of the antifolate can be offset or rescued in nonmodified cells by the nucleotide salvage pathway.
  • This offset or rescue from inhibitory effects of the antifolate can be either a complete elimination of the inhibitory effects or less than a complete offset or rescue where the inhibitory effects are reduced to the extent that the nonmodified hematopoietic cells are able to survive and grow despite some dependence on the de novo synthesis of thymidylic acid, but these cells cannot be selected against versus genetically modified hematopoietic progenitor cells or the genetically modified hematopoietic stem cells in the presence of an antifolate without a nucleoside transport inhibitor.
  • the nucleoside transport inhibitor prevents this offset or rescue from the inhibitory effects of the antifolate such that genetically modified hematopoietic progenitor cells or genetically modified hematopoietic stem cells expressing a mutant nucleic acid encoding a dihydrofolate reductase which is resistant to an antifolate are able to grow and replicate in the presence of the antifolate and the nucleoside transport inhibitor whereas nonmodified hematopoietic cells cannot grow or replicate, or can only grow or replicate to a lesser degree.
  • the nucleoside transport inhibitor which is suitable for use in the methods described herein are those which can inhibit nucleoside transport in vivo and/or in vitro, depending upon which method is utilized.
  • the combination of the nucleic acid encoding a mutant dihydrofolate reductase which is resistant to an antifolate with the antifolate and the suitable nucleoside transport inhibitor therefore, allows the efficient selection of the genetically modified cells from a mixed population of cells comprising genetically modified cells and nonmodified cells.
  • a typical dosage of NBMPR-P can range from 60 to 100 mg disodium NBMPR-P dissolved in 250 ml saline over 2 hours via an intravenous infusion where the cells are selected in vivo in human subjects. These dosages are also exemplary only and are not meant to be limiting. This amount will of course vary, and will depend among variables such as the dosage of the antifolate, the concentration of nucleosides in the subject or culture, the mass of the subject, the condition of the subject, the schedule of administration of the antifolate and/or the nucleoside transport inhibitor, etc.
  • the therapeutically effective amount can readily be determined by routine optimization procedures.
  • the nucleic acid encoding dihydrofolate reductase is well known to one of ordinary skill in the art. This enzyme is well known to be essential for the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate which is required for the biosynthesis of thymidylate and purine nucleotides. Narious mutations in the dihydrofolate reductase gene yield mutants with varying degrees of resistance to antifolate drugs.
  • mutants include Gly (G) to Trp (W) at codon 15, Leu (L) to Arg (R) or Leu (L) to Phe (F) or Leu (L) to Tyr (Y) at codon 22, and Phe (F) to Ser (S) or Phe (F) to Trp (W) or Phe (F) to Gly (G) at codon 31.
  • the mutant dihydrofolate reductase used in any of the selection methods described herein can comprise the dihydrofolate reductase containing a tyrosine at codon 22.
  • This mutant represents a mutant with approximately 100-fold higher resistance to an antifolate than the wild-type dihydrofolate reductase.
  • the use of this mutant as part of the selection method therefore, can better enable one to select genetically modified hematopoietic progenitor cells or genetically modified hematopoietic stem cells.
  • the present invention also provides that the amino acid substitution of Arginine for Phenylalanine at codon 31 of dihydrofolate reductase can result in a mutant which is especially resistant to antifolates. Therefore in another aspect of the present invention, the mutant dihydrofolate reductase used in any of the selection methods described herein can comprise the dihydrofolate reductase containing an arginine at codon 31.
  • the mutant dihydrofolate reductase used in any of the selection methods described herein can comprise the dihydrofolate reductase containing a double mutation.
  • a presently preferred double mutation includes a tyrosine at codon 22 and an arginine at codon 31.
  • Various other combinations including multiple mutations or later discovered mutations can also be utilized.
  • the present invention also provides purified mutant dihydrofolate reductases that are relatively resistant to the inhibitory effects of an antifolate.
  • the mutant dihydrofolate reductase has an arginine at codon 31.
  • the mutant dihydrofolate reductase has a tyrosine at codon 22 and an arginine at codon 31.
  • the mutant dihydrofolate reductases provided for by the present invention may be obtained in any number of ways.
  • a DNA molecule encoding a dihydrofolate reductase can be isolated from the organism in which it is normally found.
  • a genomic DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA).
  • the nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps.
  • a dihydrofolate reductase from any organism is contemplated.
  • the example contained herein discloses the use of a mutant dihydrofolate reductase from humans.
  • the methods, compounds, and compositions of the present invention are not limited to this particular isolate as it is disclosed only as an exemplary model and any dihydrofolate reductase that either is modified such that it is relatively resistant to the inhibitory effects of an antifolate, or one that can be modified to be relatively resistant to the inhibitory effects of an antifolate, or one that is naturally relatively resistant to the inhibitory effects of an antifolate is contemplated.
  • other species mutant dihydrofolate reductases can be utilized.
  • any specific amino acid can be altered, if desired, at any particular amino acid position by techniques well known in the art.
  • PCR primers can be designed which span the amino acid position or positions and which can substitute a basic amino acid for a non-basic amino acid.
  • a nucleic acid can be amplified and inserted into the wild-type dihydrofolate reductase coding sequence in order to obtain any of a number of possible combinations of basic amino acids at any position of the dihydrofolate reductase.
  • one skilled in the art can introduce specific mutations at any point in a particular nucleic acid sequence through techniques for point mutagenesis.
  • Another example of a method of obtaining a DNA molecule encoding a specific dihydrofolate reductase is to synthesize a recombinant DNA molecule which encodes the dihydrofolate reductase.
  • oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis.
  • a nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand.
  • One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector.
  • Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together.
  • Cunningham, et al. "Receptor and Antibody Epitopes in Human Growth Hormone Identified by Homolog- Scanning Mutagenesis," Science, 243: 1330-1336 (1989), have constructed a synthetic gene encoding the human growth hormone gene by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together. See also, Ferretti, et al, Proc. Nat. Acad. Sci.
  • nucleic acid encoding a particular dihydrofolate reductase of interest, or a region of that nucleic acid is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of that wild-type and/or modified dihydrofolate reductase.
  • the vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted gene, or hybrid gene.
  • These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region which may serve to facilitate the expression of the inserted gene or hybrid gene. (See generally, Sambrook et al.).
  • This invention also provides a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase containing an arginine at codon 31.
  • the invention provides a hematopoietic progenitor cell or a hematopoietic stem cell expressing a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase containing an arginine at codon 31.
  • This invention further provides a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase containing a tyrosine at codon 22 and an arginine at codon 31.
  • the invention provides a hematopoietic progenitor cell or a hematopoietic stem cell expressing a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase containing a tyrosine at codon 22 and an arginine at codon 31.
  • the mutant dihydrofolate reductase contains a tyrosine at codon 22 and the antifolate is trimetrexate.
  • the mutant dihydrofolate reductase contains a tyrosine at codon 22 and an arginine at codon 31 and the antifolate is trimetrexate.
  • the present invention also provides a method of in vivo selection for hematopoietic progenitor cells genetically modified to contain and express a nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase from nonmodified hematopoietic cells in a subject comprising administering to the subject the genetically modified hematopoietic progenitor cells, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo for the genetically modified hematop
  • the present invention further provides a method of in vivo selection for hematopoietic stem cells genetically modified to contain and express a nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase from nonmodified hematopoietic stem cells in a subject comprising administering to the subject the genetically modified hematopoietic stem cells, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic stem cells, wherein the inhibition of the nonmodified hematopoietic stem cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic stem cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo for the genetically modified hematopoi
  • the genetically modified hematopoietic progenitor cells or the genetically modified hematopoietic stem cells have previously been genetically modified such that they contain and are capable of or can express a mutant dihydrofolate reductase which is resistant to an antifolate.
  • various mutant dihydrofolate reductases are known in the art and one of ordinary skill in the art will readily appreciate there are numerous commercial entities which can genetically modify hematopoietic progenitor cells and/or hematopoietic stem cells such that these cells contain and express a mutant dihydrofolate reductase for any number of purposes.
  • dihydrofolate reductase is commonly used as a gene targeted for inhibition by antifolates in the study of gene amplification.
  • These genetically modified hematopoietic progenitor cells and genetically modified hematopoietic stem cells can be the subject of this type of study as well. It is not necessary, therefore, that the genetically modified hematopoietic progenitor cells or genetically modified hematopoietic stem cells be genetically modified for use specifically as a component of one of any of the methods described herein, but their use in any of these methods is contemplated.
  • the present invention also provides a method o ⁇ in vitro selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells comprising genetically modifying hematopoietic progenitor cells by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to hematopoietic cells comprising the genetically modified hematopoietic progenitor cells and nonmodified hematopoietic cells an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset by nucleoside salvage, and administering to the hematopoietic cells a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hem
  • in vitro is a term familiar to one of ordinary skill in the art and is used herein to describe selecting genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells outside the subject, i.e.
  • the genetically modified cells whether modified as part of a selection method or previously modified and later used in a selection method, can be selected for from a mixed population of cells comprising genetically modified hematopoietic progenitor cells and nonmodified hematopoietic cells
  • This selection in vitro has the advantage of allowing one to selectively propagate the genetically modified hematopoietic progenitor cells in vitro so that where these genetically modified cells are subsequently administered to a subject, the subject receives a higher percent of genetically modified cells versus nonmodified hematopoietic cells which can allow the subject, for example, to recover from a myeloablative procedure more rapidly than where the selection for the genetically modified hematopoietic progenitor cells is performed in vivo
  • Another advantage is that the subject is spared the toxic effects of drug selection Antifolates not only cause myeolsuppression, they can also cause mucositis, nausea, vomiting, chemical hepatitis, dermatiti
  • the invention also provides a method of in vitro selection for genetically modified hematopoietic stem cells from nonmodified hematopoietic stem cells comprising genetically modifying hematopoietic stem cells by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to hematopoietic stem cells comprising the genetically modified hematopoietic stem cells and nonmodified hematopoietic stem cells an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic stem cells, wherein the inhibition of the nonmodified hematopoietic stem cells by the antifolate can be offset by nucleoside salvage, and administering to the hematopoietic stem cells a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of
  • This method allows one to select for genetically modified hematopoietic stem cells in vitro such that nonmodified cells or cells not expressing the mutant dihydrofolate reductase can be eliminated prior to administering the cells to the subject. Having the mutant dihydrofolate reductase allows one to use the same selection technique both in vivo and in vitro such that the genetically modified hematopoietic stem cells can, but need not be expanded prior to in vivo selection.
  • the present invention also provides a method of in vitro selecting for genetically modified hematopoietic progenitor cells containing and expressing a nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase from nonmodified hematopoietic cells comprising administering to hematopoietic cells comprising genetically modified hematopoietic progenitor cells and nonmodified hematopoietic cells an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset by nucleoside salvage, and administering to the hematopoietic cells a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in
  • the present invention also provides a method of in vitro selecting for genetically modified hematopoietic stem cells containing and expressing a nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase from nonmodified hematopoietic stem cells comprising administering to hematopoietic stem cells comprising genetically modified hematopoietic stem cells and nonmodified hematopoietic stem cells an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic stem cells, wherein the inhibition of the nonmodified hematopoietic stem cells by the antifolate can be offset by nucleoside salvage, and administering to the hematopoietic stem cells a suitable nucleoside transport inhibitor in
  • the genetic modification of the hematopoietic progenitor cells as well as the selection of the genetically modified hematopoietic progenitor cells can occur in the subject itself.
  • gene therapy vehicles that are capable of targeting a particular cell type and can therefore deliver a foreign nucleic acid specifically to those cells.
  • intravenous injection of DNA/liposome complexes to mice results in gene transfer and expression in numerous organs, including the bone marrow and the spleen (Zhu et al. "Systemic gene expression after intravenous DNA delivery into adult mice. " Science 261 :209-211 (1993)).
  • This procedure has the advantage, among others, of exposing the subject to fewer procedures such extraction of that subjects own hematopoietic progenitor cells and replacement of those cells after they have been genetically modified comprising introducing into those cells a nucleic acid encoding a dihydrofolate reductase which is relatively resistant to an antifolate.
  • An additional major advantage of the method described here is that the in vivo modification would bypass the need for the toxic myeloablative conditioning that is necessary for engraftment in the autologous transplant setting.
  • the present invention therefore provides a method of in vivo selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells in a subject comprising genetically modifying hematopoietic progenitor cells in the subject by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo for the
  • This same method of genetically modifying hematopoietic cells within a subject and then selecting for these genetically modified cells using an antifolate in conjunction with a suitable nucleoside transport inhibitor can be performed by genetically modifying hematopoietic stem cells.
  • the present invention therefore also provides a method of in vivo selection for genetically modified hematopoietic stem cells from nonmodified hematopoietic cells in a subject comprising genetically modifying hematopoietic stem cells in the subject by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo for the genetically modified hem
  • the most common method for the genetic alteration of cells of the hematopoietic system involves harvesting autologous stem cells either from the blood or from the bone marrow These cells are then transduced with genetic vectors which may contain a therapeutic gene using a variety of in vitro culture conditions After some period of time, the genetically altered cells are returned to the patient, typically after the patient undergoes myeloablative conditioning therapy After patients recover from the conditioning therapy, the number of genetically-modified cells in hematopoietic compartments is very low and drops with time
  • the DHFR selection system can be applied at this time Serial treatment courses with TMTX plus either NBMPR-P or draflazine can be given to enrich for primitive hematopoietic cells that express the DHFR-mutant Ultimately, repeated rounds of selection can be used to eliminate most if not all cells which remained unmodified so that the genetically-modified cells comprise most if not all of the hematopoietic compartment
  • the selection system can be applied to a number of genetic and acquired diseases by generating vectors that have a therapeutic gene linked to the DHFR-mutant gene Post-transplant selection can then be used to enrich for cells that express the linked therapeutic gene
  • This approach involves the generation of selectable bicistronic vectors where one of the two genes is the DHFR-mutant gene
  • a single retroviral promoter can direct the expression of the DHFR-mutant and a second gene, such as a therapeutic gene (e g MDRl), or a reporter gene (e g CD24)
  • the vector may comprise an additional promoter which can direct the expression of a second gene, and the promoter or promoters can be designed to express only under certain conditions
  • Sickle cell anemia is a disease amenable to treatment with this approach
  • This first step is to generate a vector that expresses both a therapeutic gene for sickle cell anemia and a drug resistant mutant of DHFR such as L22Y Therapeutic genes for the hemoglobino-pathies such as a normal beta-like globin gene, or activator of gamma- globin transcription, can be incorporated in vectors with a drug resistant DHFR
  • a clinically certified, high titer retroviral producer clone can then be isolated for this vector.
  • Patients with sickle cell anemia can then have hematopoietic cells collected, either from bone marrow aspirates or from mobilized peripheral blood samples.
  • the CD34+ cells can then be immunopurified for stem cells expressing the CD34 antigen using any one of several commercially available methods.
  • the CD34+ cells can then be transduced with retroviral supernatant using the best available transduction protocol. These protocols typically involve culturing bone marrow cells with retroviral supernatant in the presence of hematopoietic cytokines such as IL3, IL6, SCF, and FLT3 ligand. Some groups have used plates coated with fibronectin, while others have transduced cells in the presence of autologous stromal cells. Typically these transductions are done for 24-96 hours in the presence of a polycation such as protamine sulfate.
  • a polycation such as protamine sulfate.
  • the patient Before reinfusion of the transduced cells, the patient will be treated with a conditioning regimen that is intended to reduce endogenous hematopoiesis and allow efficient engraftment of modified cells.
  • Cancer patients typically receive high doses of cyclophosphamide and radiation although a variety of different conditioning regimens have been reported.
  • the modified hematopoietic cells can be reinfused intravenously. Patients are supported while awaiting engraftment with hematopoietic cytokines such as G-CSF, intravenous antibiotics, transfusion products such as packed red blood cells, and other supportive means. After recovering from the myelosuppressive effects of the conditioning regimen, the number of genetically-corrected erythrocytes can be measured.
  • the proportion of cells which express the transferred therapeutic gene will likely be much lower than that required to exert a significant clinical benefit, being estimated to be between 1 and O.OO /o of all red blood cells.
  • patients can then be treated with intravenous doses of TMTX and either NBMPR-P or draflazine. This selective therapy will amplify the genetically-corrected population of erythroid cells.
  • patients may require serial rounds of selective treatment. This can be accomplished by allowing patients to recover from drug selection and then administering repeated doses of TMTX and transport inhibitors. This strategy will allow serial selection of corrected erythrocytes so that eventually the majority of erythrocytes will express the anti-sickling gene.
  • our system for in vivo selection can be used not only for treatment of hemoglobinopathies, but for any genetic disease of myeloid cells.
  • Candidates for this therapy include chronic granulomatous disease, Fanconi's anemia, red cell enzymopathies such as pyruvate kinase deficiency, and leukocyte adhesion deficiency.
  • lysosomal storage diseases arise from defects in macrophages, diseases such as Gaucher's disease, mucopolysaccharidosis, Niemann-Pick type B, and metachromatic leukodystrophy can also be potential applications for the DHFR in vivo selection system.
  • any non-malignant genetic disease which is correctable by hematopoietic transplantation can be treated with selectable vectors which incorporate a linked therapuetic gene specific for the disease.
  • DHFR mutants can also be used as a novel cancer therapy.
  • the DHFR vector can serve as a means to protect hematopoiesis from antifolate drugs used in the treatment of malignancy.
  • antifolates such as TMTX and transport inhibitors can increase the sensitivity of tumor cells to TMTX while providing selection of drug resistant cells within the bone marrow.
  • the simultaneous chemosensitization of the tumor through the use of the transport inhibitor, and the protection of hematopoiesis conferred by the DHFR mutant such as L22Y-DHFR leads to a widened therapeutic index and improved clinical response.
  • lymphoid cells are also progeny of hematopoietic stem cells, diseases of the immune system are also candidates for our in vivo selection system.
  • Genetic correction of congenital immunodeficiency caused by adenosine deaminase deficiency (ADA), purine nucleoside phosphorylase deficiency, and JAK3 deficiency have all been potential applications for genetic therapy.
  • patients with ADA deficiency who have been treated with genetically modified lymphocytes have been shown to have relatively low numbers of corrected cells in vivo.
  • Application of the described selection methods of the present invention in any of these situations can enhance the immune correction by providing an increased number of functionally intact lymphoid cells.
  • an anti-HIV vector is targeted either to lymphocytes or to hematopoietic stem cells with intent to generate cells that are resistant to further infection with HIV.
  • One strategy can be applied by developing a selectable anti-HIV vector by inserting the L22Y-DHFR cDNA into the construct. In vivo selection can then be used to enrich and expand normally functioning lymphoid cells that are resistant to HIV infection. The need to increase the number of HIV-resistant lymphocytes after gene therapy is highlighted by recent studies of HIV gene therapy.
  • Another application is the selection of cells used for adoptive immunotherapy.
  • a number of investigators have been able to isolate and expand lymphocytes that react against proteins present on diseased cells. These cells are reinfused into the patient and contribute to elimination of the diseased cells.
  • This approach has been used to treat disease in bone marrow transplant patients caused by Cytomegalovirus and Epstein Barr virus and is also being considered in a number of malignancies such as Hodgkins disease.
  • the viral antigen specific lymphocytes can be transduced with DHFR selection vector. This strategy would allow selective amplification of the alloreactive lymphocytes in vivo and thereby allow modulation of the anti-disease response.
  • TMTX antifolate trimetrexate
  • differentiated murine hematopoietic cells are highly sensitive to TMTX in vivo, immature myeloid progenitors resist it's cytotoxic effects.
  • TMTX addition 150 nM allowed survival of at least 50% of the myeloid progenitors in cultures containing undialyzed serum compared to 5% progenitor survival in cultures containing dialyzed serum.
  • Addition of high concentrations of thymidine (1.0 ⁇ M) alone did not restore TMTX resistance to progenitors in dialyzed serum.
  • addition of hypoxanthine (100 ⁇ M) alone to cultures containing dialyzed serum treated with thymidine phosphorylase did not rescue myeloid progenitors from TMTX toxicity.
  • Thymidine transport inhibitors were added to our defined liquid culture system. As shown in figures 1A and IB, the addition of either nitrobenzylmercaptopurine riboside (NBMPR) (at 1.0 ⁇ M or 0.1 ⁇ M) or draflazine (at 1.0 ⁇ M or 0.1 ⁇ M) to cultures containing 0.1 ⁇ M or 1 ⁇ M thymidine (approximate physiological serum concentrations in humans and mice, respectively) effectively sensitized progenitor cells to TMTX (150 nM). However, supraphysiological thymidine levels (10 ⁇ M) were able to overcome the TTI-specific blockage of thymidine import, restoring full progenitor survival.
  • NBMPR nitrobenzylmercaptopurine riboside
  • draflazine at 1.0 ⁇ M or 0.1 ⁇ M
  • DHFR-modified progenitor cells grown in dialyzed serum supplemented with 1.0 ⁇ M thymidine, 100 ⁇ M hypoxanthine, and 150 nM trimetrexate were completely resistant to TMTX and NBMPR (1.0 ⁇ M), whereas 85%> of progenitor cells modified with a control virus were killed.
  • murine myeloid progenitor cells can be sensitized to TMTX through the utilization of thymidine transport inhibitors and this toxicity can be completely reversed in vitro by expression of the L22Y-DHFR vector.
  • mice were intraperitoneally administered TMTX (130 mg/kg) together with either NBMPR-5' monophosphate (NBMPR-P) (20 mg/kg) or draflazine (20 mg/kg) for five consecutive days. 24 hrs after the last treatment, the combination of TMTX and either NBMPR-P or draflazine reduced marrow cellularity to a greater degree than TMTX alone, while neither TTI alone had any measurable effect on marrow cellularity (Fig. 3A). The myeloid progenitor content in the marrow was also measured following these various treatments.
  • NBMPR-5' monophosphate NBMPR-5' monophosphate
  • draflazine 20 mg/kg
  • TMTX alone actually resulted in a modest increase in the number of myeloid progenitors.
  • Treatment with either of NBMPR-P or draflazine alone had no effect on progenitor survival, however the combination of TMTX and either TTI severely reduced the number of myeloid progenitors relative to untreated control mice.
  • either draflazine or NBMPR-P can be used to sensitize progenitors to TMTX thereby allowing elimination of unmodified cells in vivo.
  • mice transplanted with L22 Y-DHFR or control (MDRl) transduced bone marrow were intraperitoneally administered TMTX (130 mg/kg) and NBMPR-P (20 mg/kg) for five consecutive days.
  • TMTX 130 mg/kg
  • NBMPR-P 20 mg/kg
  • Repopulation can be monitored by hemoglobin electrophoresis, which can distinguish the pattern obtained from C57B1/6J erthryocytes versus the W/W v background.
  • Figure 6 shows five daily doses of TMTX at 100 mg/kg/day failed to adversely effect the hematopoietic repopulating ability of donor marrow.
  • mice were treated for five consecutive days with the combination of 130 mg/kg TMTX and 20 mg/kg NBMPR-P, repopulating capacity of donor marrow was significantly reduced, as indicated by the recipient hemoglobin pattern after transplant.
  • Marrow derived from donor mice treated with 100 mg/kg TMTX and 20 mg/kg NBMPR-P had intermediate repopulating ability.
  • TMTX and NBMPR-P eliminated cells capable of repopulating both lymphoid and myeloid lineages.
  • TMTX alone does not appear to be sufficient for in vivo selection of hematopoietic stem or progenitor cells
  • the combination of TMTX and NBMPR-P effectively depletes both nonmodified progenitor cells and nonmodified repopulating stem cells.
  • CD34 selection was done using a Ceprate cell column (CellPro) according to the manufacturers instructions. Similar to the results observed for murine myeloid progenitors described above, draflazine effectively potentiated TMTX toxicity in the presence of varying concentrations of thymidine. Thus, similar to their murine counterparts, these experiments demonstrate that draflazine can potentiate TMTX toxicity in nonmodified human myeloid progenitors at physiologic thymidine concentrations.
  • Human progenitor cells were transduced as follows. Mononuclear cells were isolated by passing peripheral blood cells over a Ficoll gradient. These cells were cultured at 5 X 10 5 cells/ml in the presence of human IL-3 at 20 ng/ml, human IL-6 at 50 ng/ml, and human SCF at 50 ng/ml and 15% v/v fetal calf serum.
  • Figure 8 shows that following coculture with amphotropic L22 Y-DHFR producer cells in medium that has been supplemented with human interleukin-3, human interleukin-6, human stem cell factor and protamine sulphate, mobilized human peripheral blood progenitor cells had increased TMTX-resistance relative to control (neo) transduced progenitor cells
  • L22Y-DHFR effectively transduces and expresses within human myeloid progenitor cells
  • in vivo selection of human hematopoietic cells can occur in a manner analogous to that demonstrated in the murine system

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Abstract

Cette invention concerne un procédé de sélection in vivo de cellules mères hématopoïétiques génétiquement modifiées qu'on sélectionne parmi des cellules hématopoïétiques non modifiées chez un individu. Pour effectuer cette sélection on introduit des gènes mutants de dihydrofolate réductase dans les cellules mères hématopoïétiques puis on administre à un individu abritant les cellules transformées résultantes un antifolique et un inhibiteur de vecteur nucléosidique. Cette invention concerne également des acides nucléiques codant des gènes mutants de dihydrofolate réductase et des cellules hématopoïétiques transformées avec ces gènes mutants.
PCT/US1996/017660 1996-11-04 1996-11-04 Procede de selection in vivo de cellules hematopoietiques primitives WO1998019540A1 (fr)

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US10709739B2 (en) 2011-04-20 2020-07-14 The Regents Of The University Of California Method for chemoselection
EP3799876A1 (fr) 2011-04-20 2021-04-07 The Regents of the University of California Procédé pour le conditionnement et la chimiosélection combinés dans un seul cycle
US11607427B2 (en) 2011-04-20 2023-03-21 The Regents Of The University Of California Method for chemoselection
US11377659B2 (en) 2016-02-19 2022-07-05 The Regents Of The University Oe California Short hairpin RNA (shRNA734) and use of same to positively select and eliminate genetically modified cells
US10426798B2 (en) 2017-07-18 2019-10-01 Calimmune, Inc. Modulatable switch for selection of donor modified cells

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