Gene transfer of cytidine deaminase protects myelopoiesis from cytidine analogs in an in vivo murine transplant model

Abstract

Hematopoietic stem cell gene transfer of the drug-resistance gene cytidine deaminase (CDD) protecting cells from the cytotoxic cytidine analogs cytarabine and gemcitabine was investigated in a murine transplant model. Following transplantation of CDD-transduced cells and cytarabine application (500 mg/kg; days 1-4; intraperitoneally) significant myeloprotection was demonstrated with nadir counts of peripheral blood granulocytes and thrombocytes of 2.9 ؎ 0.6/nL versus 0.7 ؎ 0.1/nL (P < .001) and 509 ؎ 147/nL versus 80 ؎ 9/nL (P ‫؍‬ .008), respectively (CDD versus control). Protection also was observed from otherwise lethal gemcitabine treatment (250 mg/kg; days 1-3). Stable levels of gene-marked cells in primary and secondary recipients were demonstrated for up to 9 months, and whereas CDD overexpression clearly reduced Band T-lymphocyte numbers, no major toxicity was observed in the myeloid compartment. Despite the profound myeloprotective properties, however, CDD overexpression did not allow for pharmacologic enrichment of transduced hematopoiesis in our model. Thus, in summary, our data establish CDD as a drug-resistance gene highly suitable for myeloprotective purposes, which, given the lack of selection observed in our hands, might best be used in combination with selectable drugresistance genes such as MGMT (

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

“…CDD represents a clinically highly relevant CTX-R gene, as overexpression of this enzyme protects lymphohematopoietic cells from deoxycytidine analogs such as cytosine-arabinoside (1-b-D-arabinofuranosylcytosine, Ara-C), the most effective single-agent in the treatment of acute leukemias. Myeloprotection as well as chemoselection by hCDD gene transfer has been established in murine and human clonogenic progenitor cells [21][22][23] as well as in murine in vivo bone marrow transplant models [24][25][26]. However, in leukemias, HSC-based myeloprotective gene therapy approaches always carry the risk of inadvertent transduction of malignant cells.…”

Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues

“…74 Finally, in vivo myeloprotection has been observed in mice treated with araC or gemcitabine after transplantation of CDAtransduced murine bone marrow cells. 75 Nadir values of granulocytes and thrombocytes in araC-treated CDA overexpressing mice were fourfold and sixfold higher than in control mice whereas threefold differences were observed with gemcitabine.…”

MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy

“…23,24 Thus, in the context of HSC gene therapy, miRNAs represent an attractive approach to detarget gene expression from unwanted cells, thereby preventing transgene toxicity. Based on our previous experience with transgenic expression of the drug resistance gene cytidine deaminase in lymphohematopoietic cells, 17 we have specifically investigated transgene detargeting in the lymphoid compartment. We here demonstrate effective shut-down of transgene expression in differentiated B and T cells by a complementary, tetrameric binding cassette for miRNA-150, while expression in primitive stem and progenitor cells or in the myeloid lineage remains unaltered.…”

“…Substantial transgene-related lymphotoxicity has been encountered when gene transfer of the drug resistance gene cytidine deaminase as a mean to protect the hematopoietic system from the toxicity of deoxycytidine-analog type cytotoxic drugs such as cytosine arabinoside (Ara-C) or gemcitabine was investigated. 17 Therefore, we here investigated targeting of miRNA-150 as a strategy to suppress transgene expression in the lymphatic compartment. MiRNA-150 is highly expressed in mature, resting T and B cells and miRNA-150 expression has been reported to be upregulated during the terminal maturation of B or T cells at the intrasplenic T1 to T2/3 or at the intrathymic double-negative to single-positive stage transition, respectively.…”