Knockdown of HPRT Enables Selection of Genetically Modified Human Hematopoietic Progenitor Cells.

Abstract 3772 Genetic modification of autologous hematopoietic stem cells (HSC) has the potential for effective treatment of a wide variety of inherited blood disorders. However, HSC gene therapy has shown limited clinical efficacy (with notable exceptions), in part because of the small proportion of engrafted genetically corrected HSCs. The use of drug-resistance genes to enable selection for transduced HSCs has been explored, but with limited success. Previous studies from our laboratory have indicated that murine HSC can be selected with 6-Thioguanine (6TG), a relatively non-toxic drug used in the treatment of leukemias, after knocking down the expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT), an enzyme that metabolizes 6TG to its active state. We sought to determine if these findings can be translated to human hematopoietic cells. In the present study, we transduced human myeloid (Molm13, MV4-11) and lymphoid cell lines (Reh) with lentiviral vectors expressing shRNA constructs targeting HPRT or a non-targeted control sequence (Ctrl). Two of the most promising constructs directed against HPRT (491 and 50) were studied in more detail to determine which is most effective. Cells were selected in puromycin and cell lysates analyzed for HPRT gene expression. Reverse-transcription, real-time PCR (RT qPCR) and western blotting demonstrated that construct 491 was most efficient in knocking down HPRT in human hematopoietic cell lines compared to construct 50 (and Ctrl). To determine whether knockdown of HPRT provided resistance to 6TG, cells were cultured in the absence or presence of different doses of 6TG and live cell concentrations were determined. While Ctrl transduced cells decreased in a dose dependent manner after 72h of 6TG treatment, cells transduced with constructs 491 and 50 were relatively resistant to 6TG. IC50 values for construct 491 were significantly higher (114μM for Molm13 and 46μM for Reh cell lines) than construct 50 (1μM for Molm13 and 10μM for Reh) in comparison to control transduced cells (0.4μM for Molm13 and 3.5μM for Reh). We assessed cell death in human hematopoietic cell lines by annexin V staining after exposure to 6TG at 48 and 72h. As expected, control transduced cells died of apoptosis upon 6TG treatment, while 491 and 50 transduced cells were resistant. Furthermore, 491 transduced cells were more resistant to apoptosis than 50 transduced cells. Based on these results, construct 491 was used to transduce human CD34+ progenitor cells isolated from umbilical cord blood along with control shRNA. Transduction efficiency varied from 25–35% as determined by %GFP expression by flow cytometry. Sorted GFP+ cells showed reduced expression of HPRT in 491 transduced cells compared to controls, as measured by RT qPCR. Similar to the effects in cell lines, in vitro proliferation of control transduced CD34+ cells diminished in response to increasing 6TG concentrations. There was an increase in the percentage of GFP+ cells in 6TG treated 491 transduced cells compared to untreated controls in a dose dependent fashion, indicating a selective advantage conferred to 491 transduced cells in the presence of 6TG. Importantly, 491 transduced cells continued to proliferate despite treatment with 6TG. Like 6TG, cisplatin requires mismatch repair (MMR) for cytotoxicity. To determine if HPRT knockdown had off-target effects impairing MMR, transduced cells were also treated with cisplatin. Both control and 491 transduced cells stopped proliferating in the presence of cisplatin indicating that MMR remained intact. These data indicate that human hematopoietic progenitor cells can be selected in vitro by knock-down of HPRT and treatment with 6TG. Xenografts of Ctrl and 491 transduced human CD34+ cord blood cells have been generated and are being treated with 6TG to determine if human cells can be selected with 6TG in vivo. Disclosures: Off Label Use: Off label use of 6-thioguanine will be suggested.