In vivo selection of primitive hematopoietic cells

Details In vivo selection of primitive hematopoietic cells In vivo selection of primitive hematopoietic cells

1999-05-04

2002-12-31

Reynolds, Deborah J. (Department: 1632)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C435S320100, C435S325000, C435S456000, C514S04400A, C530S385000

Reexamination Certificate

active

06500421

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
2. Background Art
Retroviral-mediated gene transfer is a potential therapeutic strategy for a number of diseases that affect the hematopoietic system. (Karisson S. “Treatment of genetic defects in hematopoietic cell function by gene transfer.” Blood 78:2481 (1991)). 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. Although retroviral vectors provide one of the best methods for HSC transduction, recent clinical trials have shown that current protocols result in very low levels of HSC gene transfer. (Dunbar et al. “Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraft ment after autologous transplantation.” Blood 85:3048 (1995) and Brenner et al. “Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients.” Lancet 342:1134 (1993)) Because the overall proportion of modified cells ranges from 10 to 0.01% after hematopoietic reconstitution, there are insufficient numbers of modified cells to be therapeutically effective. Thus, gene transfer is currently not a feasible treatment option for many diseases such as hemoglobinopathies, AIDS, chronic granulomatous disease, and cancer.
Because of the problem of low numbers of modified cells after hematopoietic reconstitution, selection of primitive HSCs is required for significant enrichment of modified cells. Selection of more differentiated cells would allow only a transient enrichment of modified cells due to the limited self-renewal capacity of these more mature cells.
One means of selecting modified cells that has been the focus of intense investigation involves dihydrofolate reductase. 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. 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. Anti. Acad. Sci. U S A 80:2495 (1983)) raised the possibility that mutant DHFRs could potentially be used as drug resistance genes.
Mutant DHFR genes were the first drug resistance genes to be transferred to primary hematopoietic cells. Despite the theoretical advantages for using DHFR as an in vivo selectable marker, and the potential protection of hematopoiesis conferred by DHPR variants, evidence for in vivo selection has been equivocal using this experimental system. In 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. “Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells.” Blood 75:337 (1990)) However, when Southern blot analysis was done to confirm an enrichment of vector-modified cells in myeloid tissue, no such enrichment could be documented. The authors suggested that more prolonged MTX exposure may be required for in vivo selection of DHFR-modified immature hematopoietic cells, thus implying that cell cycle status may play a significant role in antifolate resistance. In analogous experiments done using a human DHFR variant, only a 2-fold increase in vector-expressing myeloid progenitor cells was seen following MTX treatment. (Zhao et al. “Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase.” Cancer Gene Therapy 1:27 (1994)) Hence, the authors conclude from this study that a modest level of in vivo selection of DHFR-modified progenitors can be accomplished utilizing MTX alone. However, several important controls were omitted from these experiments, such as the percentage of MTX resistant progenitors from DHFR mice that were not treated with MTX, so that even this modest level of selection remains unconvincing.
Another study reports that human myeloid progenitor cells transduced with a vector expressing a human DHFR variant were selected and expanded in vitro in MTX-containing cultures. (Flasshove et al. “Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer.” Blood 85:566 (1995)) Vector-expressing progenitor cells were amplified two-fold using this approach. However, this approach was in vitro. Further, it is not clear if transduced hematopoietic stem cells could be amplified using this method.
Consistent with these disappointing results, recent findings demonstrate that hematopoietic stem cells and progenitors are highly resistant to antifolates. (Blau et al. “Cytokine prestimulation as a strategy for in vivo selection: resistance of hemopoietic progenitors to folate analogs.” Stem Cell Gene Therapy: Biology and Techniques. Sep. 28-Oct. 1 (1995)) These findings lead the authors to conclude that antifolates are poorly suited for the in vivo selection of transduced hematopoietic progenitor and stem cells. (Blau 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. To accomplish this result, 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.
“The use of nucleoside transport inhibitors has 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. However, this use has produced perplexing results. It has been well demonstrated in cultured cell lines that exogenously added nucleosides can reverse the toxicity of these drugs and that nucleoside transport inhibitors such as NBMPR and dipyridamole can restore toxicity. (Marina et al. “Effect of nucleoside transport inhibitors on thymidine salvage and the toxicity of nucleoside analogs in mouse bone marrow granulocyte-macrophage progenitor cells.” Cancer Communications 3:367 (1991)) However, in vivo studies with mice have shown either no antitumor effect of the transport inhibitor, no potentiation of the antitumor effect, or an increased somatic toxicity with little change in the therapeutic index. In addition, clinical phase I and II studies combining dipyridamole with acivicin, methotrexate, 5-fluorouracil, or PALA have revealed only limited responses.”
Thus, 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 util

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