Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1999-07-06
2002-11-26
Crouch, Deborah (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093100, C424S093200, C435S325000, C514S04400A
Reexamination Certificate
active
06485722
ABSTRACT:
BACKGROUND OF THE INVENTION
As many hereditary diseases are a result of defects in single genes, there are many potential applications of gene therapy in the treatment of single gene disorders. Moreover, gene therapy can also be useful in the treatment of acquired diseases, e.g., cancer and infectious diseases. In particular, many of these diseases can potentially be treated or prevented by the introduction of a therapeutic gene(s) into hematopoietic stem cells (HSC), since the entire hematopoietic system can theoretically be regenerated from a single stem cell.
With a few exceptions (e.g., hormones), most anticancer drugs used in the clinic today cause moderate to severe bone marrow toxicity (e.g., vinblastine, cisplatin, methotrexate, alkylating agents, and anthracyclines). The introduction of a gene that confers resistance to a chemotherapeutic drug (termed a drug resistance gene) into HSC can convert these bone marrow progenitor cells to a drug resistant state, thus allowing larger than conventional doses of chemotherapeutic agents to be administered safely to patients, without toxicity to bone marrow, the gastrointestinal tract, and other normal proliferating tissue.
The first eukaryotic drug resistance gene to be transferred to reconstituting bone marrow cells was a methotrexate (MTX) resistant rodent dihydrofolate reductase (mDHFR) gene. Mice transplanted with cells transduced with a mDHFR containing retroviral vector were protected from methotrexate induced myelosuppression (Corey et al.,
Blood,
75, 337 (1990); Williams et al.,
J. Exp. Med.
166, 210 (1987)). Subsequent experiments have suggested that methotrexate can also be used to select for murine hemopoietic cells expressing transferred MDHFR genes (Vinh and McIvor,
J. Pharmacol. Exp. Ther.,
267, 989 (1993)).
Transfer of the human mdr1 gene to hemopoietic cells has also been described. P-glycoprotein, the product of the mdr1 gene, functions as a drug efflux pump and confers resistance to a wide variety of naturally occurring chemotherapeutic agents. Mice transplanted with mdr1 transduced cells showed attenuation of taxol induced myelosuppression (Sorrentino et al.,
Science,
257, 99 (1992); Hanania et al.,
Blood,
82, 1260 (1990)). In taxol treated animals, the proportion of circulating leukocytes transduced with the mdr1 virus increased with drug treatment, suggesting that cells expressing the transferred mdr1 gene can be dominantly selected in vivo with taxol (Sorrentino et al., supra; Podda et al.,
Proc. Nat'l Acad. Sci. USA,
89, 9676 (1992)). More recent work has suggested that the mdr1 gene can be used to select for the presence of other therapeutic genes when the therapeutic genes are linked to the mdr1 cDNA in bicistronic retroviral vectors. Thus, drug resistance genes can be used to attenuate drug induced myelosuppression and can act as dominant selectable markers for genetically altered hemopoietic cells.
While HSC are tempting targets for gene transfer, these cells can be transduced with only limited efficiency, since generally less than 0.01% of the cells in bone marrow are HSC. This limits the implementation of clinical protocols based on gene modified HSC. Moreover, even if a therapeutic gene can be stably integrated into HSC, once transferred to a recipient, the transgenic HSC have no selective growth advantage relative to their nontransgenic counterparts. Without such an advantage, the engraftment of HSC containing the therapeutic gene is uncertain and, thus, the curative effect of the expression of the therapeutic gene in HSC and their progeny is unlikely.
One way to increase the representation of successfully transduced HSC would be to mediate selective engraftment by expression of a drug resistance gene. Although selective expansion of hematopoietic cells derived from transduced stem cells has been demonstrated for stem cells transduced with drug resistance genes, the ability of these drug-resistance genes to confer selective engraftment of HSC has not been established by long-term reconstitution studies.
Thus, what is needed is an improved method to select for engraftment of transplanted hematopoietic cells.
SUMMARY OF THE INVENTION
The present invention provides a method for selective engrafinent of hematopoietic stem cell in vivo. The method comprises administering to a mammal a population of stem cells comprising transgenic stem cells, the genome of which has been augmented by a first preselected DNA segment which is operably linked to a promoter functional in stem cells. The first preselected DNA segment encodes resistance to an agent which is normally toxic to stem cells. A preferred embodiment of the invention includes a first preselected DNA segment which encodes resistance to a chemotherapeutic agent, such as an antineoplastic or cytotoxic agent. The genome of the transgenic stem cells can also be augmented by a second preselected DNA segment which encodes a therapeutic agent. The expression of the first preselected DNA segment in the transgenic stem cells is effective to impart resistance or tolerance to said transgenic cells to an amount of the agent which is toxic to the corresponding nontransgenic stem cells. The agent is administered to said mammal in an amount, and for a time, so as to increase the engraftment and proliferation of transgenic stem cells relative to the engraftment and proliferation of nontransgenic stem cells. A preferred embodiment of the invention includes daily administration of the agent.
As used herein, the term “hematopoietic stem cells (HSC)” means a population of primitive progenitor cells which can provide long term reconstitution of both myeloid and lymphoid cell lineages in a host.
As used herein, a cell which is “resistant or tolerant” to an agent means a cell which has been genetically modified so that the cell proliferates in the presence of an amount of an agent that inhibits or prevents proliferation of a cell without the modification.
As used herein, a preselected DNA segment that encodes “resistance” to an agent, such as a preselected DNA segment that encodes resistance to a chemotherapeutic agent, e.g. methotrexate, means that the expression of the preselected DNA segment in a cell permits that cell to proliferate in the presence of the agent to a greater extent than the proliferation of a corresponding cell without the preselected DNA segment. A preselected DNA segment of the invention can encode resistance to methotrexate, vinblastine, cisplatin, alkylating agents, or anthracyclines, their analogs or derivatives, and the like.
As used herein with respect to an agent, the term “therapeutically effective amount” means an amount of the agent that inhibits or prevents proliferation of untransformed cells in a mammalian host.
As used herein, the term “a preselected DNA segment encoding a therapeutic agent” is defined as a preselected DNA segment encoding any polypeptide, peptide, protein, or sense or antisense RNA that imparts a desired effect to the mammal when it is produced by the cells of said mammal, including, but not limited to, an enzyme (e.g, adenosine deaminase, thymidine kinase, glucose cerebrosidase), a hormone (e.g., human growth hormone, insulin), a cytokine, clotting factors, a hormonal regulator (e.g., amylin, erythropoietin), antisense oligonucleotides (e.g., antisense to the mRNA encoding P210
BCR/ABL
) and the like.
As used herein, the term “engraftment” with respect to HSC means that HSC which are introduced into a recipient are localized in the bone marrow of the recipient and can provide long term reconstitution of both myeloid and lymphoid cell lineages in that recipient.
REFERENCES:
patent: 5436151 (1995-07-01), McGlave et al.
patent: 5460964 (1995-10-01), McGlave
patent: 0 341 966 (1989-11-01), None
Li et al., Blood 83(11):3403-3408, Jun. 1994.*
Flasshove et al., Leukemia 9:534-537, Oct. 1995.*
May et al., Blood 86(6):2439-2448, Oct. 1995.*
Sorrentino et al., Science 257:99-103, Jul. 1992.*
Morris et al., Biochemical Pharmacology 47(7):1207-1220, Oct. 1994.*
Corey et al., Blood 75(2):337-343, Jan. 1990.*
Bodi
James Rohaizah I.
May Chad
McIvor R. Scott
Crouch Deborah
Regents of the University of Minnesota
Schwegman Lundberg Woessner & Kluth P.A.
Woitach Joseph
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