siRNA-Induced Transient Silencing of PTEN Expression Enhances Human Hematopoietic Cell Engraftment in NOD/SCID/γ_c^null Mice and Increases Gene Transduction Efficiency.

PTEN (phosphatase and tensin homologue) has been implicated as a regulator of murine hematopoietic stem cell (HSC) self-renewal. In mice, PTEN deletion initially results in a transient expansion of HSC numbers, but the HSC pool subsequently becomes depleted. Therefore, constitutive down-modulation of PTEN would not be desirable for stem cell expansion, however transient depletion of PTEN activity could be beneficial for transplantation and gene therapy applications. Little is understood regarding the role of PTEN in human HSC regulation. We studied the impact of transient inhibition of PTEN expression in human CD34+ cells on their cell cycle profile, their susceptibility to retroviral transduction, their proliferative potential in vitro, and their ability to repopulate NOD/ SCID/γ_c^null mice. Human G-CSF-mobilized CD34+ cells were transfected with PTEN siRNA (PTEN1 or PTEN2) or control siRNA. Forty-eight hours later PTEN expression was inhibited by 73–100% (PTEN1) and 64–97% (PTEN2) compared to CD34+ cells transfected with control siRNA as measured by real-time RT-PCR. Reduced PTEN protein levels were confirmed by Western blot. Compared to control siRNA treated CD34+ cells, PTEN-silenced CD34+ cells showed a significant decrease in the proportion of cells in the G0 phase of the cell cycle (PTEN1: 10.9%; PTEN2: 13.0%; control: 19.5%; p < 0.05) and a concomitant increase in the proportion of cells in the S + G2/M phase of the cell cycle (PTEN1: 42.1%; PTEN2: 42.4%; control: 37.2%; p < 0.05). We hypothesized that the increased proportion of cycling CD34+ cells may enhance their susceptibility to retroviral transduction for gene therapy applications. Human CD34+ cells were nucleofected with PTEN or control siRNA, cultured for 2 days with cytokines and transduced for 48 hours with MND.MFG.YFP c10 retroviral vectors (MOI=0.3) on fibronectin-coated plates. Transduction efficiencies in the bulk CD34+ cells transfected with PTEN1 siRNA and PTEN2 siRNAs were significantly higher compared with CD34+ cells transfected with a control siRNA as determined by detection of YFP by flow cytometry. The average percentage of YFP+ cells from eight independent transductions were 41.7% (PTEN1, p=0.0001), 38.9% (PTEN2, p=0.003), compared with the control siRNA treated group (29.4%). We next tested whether transient inhibition of PTEN expression could result in improved engraftment of primitive human cells capable of repopulating nonobese diabetic/ severe combined immune-deficient-interleukin-2R^−/− (NOD/SCID/γ_c^null) mice. A total of 25 NOD/SCID/γ_c^null mice were transplanted with PTEN or control siRNA treated CD34+ cells immediately after nucleofection (PTEN1 n=10; PTEN2 n=5; control n=10) and 9 additional mice were transplanted with untreated human CD34+ cells. Mice transplanted with PTEN1 siRNA- or PTEN2 siRNA-treated CD34+ cells had increased percentages of human cell engraftment as determined by flow cytometry for human CD45 compared to mice transplanted with control siRNA-treated CD34+ cells or untreated CD34+ cells (PTEN1: 37.6%; PTEN2: 38.1%; control: 19.7%; untreated: 19.2%, p = 0.03). NOD/ SCID/γ_c^null mice transplanted with PTEN1 and PTEN2 siRNA-treated CD34+ cells showed no statistically significant differences in human lymphoid (CD3+, CD20+) or myeloid (CD15+) differentiation compared to control mice but a trend favoring an increased representation of myeloid (CD15+) cells was noted in PTEN1-treated mice (PTEN1: 41.7%; control: 18.8%, p = 0.06). None of these mice developed myeloproliferative disorders or leukemias. Overall, these data suggest that PTEN plays an important role in governing transitions between the quiescent and activated states of primitive human hematopoietic cells, and the transient inhibition of PTEN expression in these cells results in improved retroviral transduction efficiencies and increased engraftment. This approach may find clinical applications in gene therapy for inherited disorders and in adult cord blood stem cell transplantation where the number of HSC is limited.