CML is malignant disease of the hematopoietic stem cell (HSC) characterized by the BCR/ABL gene rearrangement. The only curative treatment for most patients with CML is transplantation of HSC from an allogeneic donor. Although transplantation of autologous, unpurged or purged bone marrow or blood progenitors can restore Ph-hematopoiesis in some patients, most patients relapse within a year after transplant due in part to contamination of the graft with Ph+ cells and in part due to disease persisting in the host after the preparative regimen. Therefore, new approaches to eliminate residual disease are required. One possible post transplant therapy is the administration of chemotherapy, such as methotrexate (MTX). However, MTX will not only affect Ph+ HSC but also NL HSC. Transduction of NL HSC with mutant dihydrofolate reductase (DHFR) genes, such as the tyr22-DHFR, may allow use of MTX post-transplant to eliminate residual leukemia but selectively spare the genetically modified transplanted HSC. Since CML progenitors are at least as sensitive to MTX as NL progenitors, transduction of the autograft with a MTXr gene may allow selective elimination of the Ph+ clone persisting in the host after transplant with MTX. The applicant has developed methods to select CD34+HLA-DR-cells from chronic phase CML BM which are highly enriched in Ph- primitive progenitors in quantities sufficient for transplantation. Since small numbers of Ph+ progenitors may persist in the FACS selected graft, transduction of the graft with MTXr genes will also render Ph+ HSC MTX resistant. The malignant phenotype of CML progenitors can be attributed to the presence of the BCR/ABL encoded p210BCR/ABL tyrosine kinase. Since elimination of the BCR/ABL mRNA with breakpoint specific anti-sense oligonucleotides results in normalization of the phenotypic characteristics of Ph+ progenitors, transduction of anti-BCR/ABL antisense sequences (AS) may result in phenotypically normal Ph+ CML progenitors. Based on the applicant's preliminary studies, she proposes to generate retroviral vectors that will confer MTX resistance to the infused HSC population to allow administration of MTX post transplant to eliminate residual disease persisting in the host. Since the graft may contain Ph+ cells, she will couple the MTXr gene with anti-BCR/ABL AS sequences to eliminate the malignant phenotype in Ph+ HSC that contaminate the graft. In SA1, she will construct retroviral vectors containing both the tyr22-DHFR gene and AS sequences to identify the construct that allows equal expression of the MTXr component and the AS sequence. The expression level of the DHFR and AS sequences as well as the capacity and specificity with which a retroviral vector eliminates the BCR/ABL mRNA and oncoprotein and its associated malignant phenotype will be quantified using the 32DBCR/ABL cell line. The specificity and activity of the AS sequence will then be studied in vitro in primary CML progenitors (SA2) and in vivo in a murine model of CML (SA3). Experiments in SA2 and SA3 will also examine the transduction efficiency into primary CML and NL 34+ cells, transfer and persistent expression of drug-resistance in vitro and in vivo. Once a vector(s) has been identified which fulfills criteria set forth in the application, future clinical trials in which CML autografts are transduced and patients receive MTX post transplant will be initiated to confirm the clinical efficiency of this approach.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Experimental Therapeutics Subcommittee 1 (ET)
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Wolpert, Mary K
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University of Minnesota Twin Cities
Internal Medicine/Medicine
Schools of Medicine
United States
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