Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction

Mx, Li; Banerjee, D.; Sc, Zhao; Schweitzer, BI; Mineishi, Shin; Gilboa, Eli; Bertino, JR

doi:10.1182/blood.v83.11.3403.3403

Cited by 46 publications

(6 citation statements)

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“…DHFR was first tested for its ability to protect BM cells in vivo. HSCs transduced with DHFR variants become resistant to antifolate agents, such as methotrexate and trimetrexate (Li et al, 1994;Spencer et al, 1996), and can be positively selected both in vitro and in vivo. DHFR naturally functions to generate tetrahydrofolate, which is necessary for the de novo synthesis of pyrimidines and purines.…”

Section: Hscs In Vivo Selection Strategiesmentioning

confidence: 99%

“…This has been achieved by genes/factors that could confer on HSCs a growth advantage or would allow for their in vivo selection. A number of genes encoding drug-resistance molecules are currently under investigation, such as the human dehydrofolate reductase (DHFR), multi-drug resistance 1 (MDR1), and 53 methylguanine metyltransferase (MGMT) drug-resistance genes (Allay et al, 1998;Chinnasamy et al, 1998;Davis et al, 2000;Hickson et al, 1998;Li et al, 1994;Persons et al, 2003;Sawai et al, 2001;Spencer et al, 1996). A side effect to this strategy is the high toxicity of the selective drugs.…”

Section: Introductionmentioning

confidence: 99%

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Ex vivo expansion of hematopoietic stem cells for use in nonmyeloablative transplantation

Bakovic

Ohta

Imren

et al. 2008

Blood Cells, Molecules, and Diseases

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Hematopoietic stem cell transplantation (HSCT) is used to treat a wide range of hematologic and non-hematologic disorders. Recently, interest has grown in the potential of autologous HSCT coupled to gene therapy for the treatment of genetic blood disorders as a way of avoiding the severe immunologic reactions associated with allogeneic HSCT. However, the remaining risks in using myeloablative conditioning regimens to allow relatively small numbers of transplanted HSCs to be transplanted greatly limit the applicability of this approach. Nonmyeloablative regimens would be an appealing alternative but necessitate the generation of large numbers of genetically corrected HSCs to achieve therapeutic levels of chimerism. In this thesis I have explored the potential of forced overexpression of homeobox genes as a strategy to obtain the degree of HSC expansion required. In a first series of experiments, I found that HOXB4 and NUPHOX transduced and expanded HSCs maintain the ability of fresh HSCs to produce sustained, high level, polyclonal, lympho-myeloid chimerism when transplanted into mice given 2-2.5 Gy. I then tested the therapeutic efficacy of ex vivo expanded HSCs in nonmyeloablated mice with severe R-thalassemia caused by the homozygous deletion of the fj-maj or globin gene (P-MDD). The results of these experiments showed that this approach could produce a dramatic improvement in the hematocrit, hemoglobin and RBC morphology and ultimately the cure of the thalassemic phenotype that was not achievable in recipients of equivalent numbers of unmanipulated BM cells or of cells transplanted immediately after transduction. Again, the cured mice displayed a sustained, high level of polyclonal chimerism. Together these data provide "proof of principle" of the curative potential of ex vivo expanded ii HSCs in a preclinical model of ^-thalassemia treated with nonmyeloablative conditioning.

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Section: Hscs In Vivo Selection Strategiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Ex vivo expansion of hematopoietic stem cells for use in nonmyeloablative transplantation

Bakovic

Ohta

Imren

et al. 2008

Blood Cells, Molecules, and Diseases

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“…Likewise, vectors containing mutated DHFR genes have been engineered for transduction of hematopoietic progenitor cells [47][48][49]. Williams et al [50] and Cline et al [51] demonstrated protection of recipient animals from lethal doses of methotrexate.…”

Section: Animal Models For Transduction Of Chemoresistance Genes To Hmentioning

confidence: 99%

In Vivo Drug‐Selectable Genes: A New Concept in Gene Therapy

et al. 1997

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Chemoresistance genes, initially considered to be a major impediment to the successful treatment of cancer, may become useful tools for gene therapy of cancer and of genetically determined disorders. Various target cells are rendered resistant to anticancer drugs by transfer of chemoresistance genes encoding P-glycoprotein, the multidrug resistance-associated protein-transporter, dihydrofolate reductase, glutathione-S-transferase, O 6 -alkylguanine DNA alkyltransferase, or aldehyde reductase. These genes can be used for selection in vivo because of the pharmacology and pharmacokinetics of their substrates. In contrast, several other selectable marker genes conferring resistance to substrates like neomycin or hygromycin can only be utilized in tissue culture. Possible applications for chemoresistance genes include protection of bone marrow and other organs from adverse effects caused by the toxicity of chemotherapy. Strategies have also been developed to introduce and overexpress nonselectable genes in target cells by cotransduction with chemoresistance genes. Thereby expression of both transgenes can be increased following selection with drugs. Moreover, treatment with chemotherapeutic agents should restore transgene expression when or if expression levels decrease after several weeks or months. This approach may improve the efficacy of somatic gene therapy of hematopoietic disorders which is hampered by low or unstable gene expression in progenitor cells. In this article we review preclinical studies in tissue culture and animal models, and ongoing clinical trials on transfer of chemoresistance genes to hematopoietic precursor cells of cancer patients.

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“…The four-week delay allowed selection with a much larger dose of MTX. The delayed high dose selection with MTX resulted in deaths of all control animals within four weeks while more than 80% of experimental animals survived the treatment [48]. Presently we are investigating other clinically suitable protocols such as BMT without the need for prior whole body irradiation of the recipients so that the whole procedure can be made less hazardous.…”

Section: Transduction Of Murine Bone Marrow By Coculture Of Bone Marrmentioning

confidence: 99%

Gene therapy utilizing drug resistance genes: A review

Banerjee¹,

Zhao²,

Li³

et al. 1994

STEM CELLS

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The generation of drug resistant bone marrow may facilitate the development of aggressive chemotherapeutic regimens that might otherwise be lethal due to marrow toxicity. With the availability of technology that permits in vitro manipulation of human marrow and peripheral blood stem cells, it is now possible to introduce genes that confer drug resistance to these hematopoietic progenitors. Animal models and in vitro work with human progenitors using drug resistance genes are reviewed.

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Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction

Cited by 46 publications

References 0 publications

Ex vivo expansion of hematopoietic stem cells for use in nonmyeloablative transplantation

Ex vivo expansion of hematopoietic stem cells for use in nonmyeloablative transplantation

In Vivo Drug‐Selectable Genes: A New Concept in Gene Therapy

Gene therapy utilizing drug resistance genes: A review

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