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Activation of proto-oncogenes by retroviral insertion is an important issue delaying clinical development of gene therapy. We have reported the nonrandom persistence of hematopoietic clones with vector insertions within the MDS1/EVI1 locus following transplantation of rhesus macaques. We now ask whether prolonged culture of transduced CD34+ cells before transplantation selects for clones with insertions in the MDS1/EVI11 or other proto-oncogene loci. CD34+ cells were transduced with standard retroviral vectors for 4 days and then continued in culture for an additional 6 days before transplantation. A 15% of insertions identified in granulocytes 6 months post-transplant were in MDS1/EVI11, significantly increased compared to the frequency in animals transplanted with cells immediately following transduction. MDS1/EVI1 clones became more dominant over time post-transplantation in one animal that was followed long term, accompanied by an increased overall copy number of vector-containing granulocytes, with one MDS1/EVI1 clone eventually accounting for 100% of transduced granulocytes and marrow colony-forming unit (CFU). This vector insertion increased the expression of Evi1 mRNA. There was no overrepresentation of MDS1/EVI1 insertions contributing to lymphoid lineages. Strategies involving prolonged ex vivo expansion of transduced cells may increase the risk of genotoxicity. [ABSTRACT FROM AUTHOR]

The ability to expand hematopoietic stem and progenitor cells (HSPCs) in vitro will enhance the success of a wide range of transplant-related therapies. PTEN (phosphatase and tensin homologue deleted on chromosome 10) has been implicated as a regulator of murine HSPC self-renewal, but little is understood about the role of PTEN in human HSPC regulation. We tested the impact of transient small interfering RNA (siRNA)–induced inhibition of PTEN expression in human CD34+ cells on their cell cycle profile, their susceptibility to retroviral transduction, and their ability to self-renew and repopulate nonobese diabetic/severe combined immunodeficiency disease with interleukin-2 receptor γ-chain deficiency mice. Reduced PTEN messenger RNA and protein levels were confirmed in PTEN siRNA-treated CD34+ cells compared with control siRNA-treated CD34+ cells. Transient silencing of PTEN in CD34+ cells promoted their entry into cell cycle, and increased their expansion in vitro compared with control siRNA-treated CD34+ cells. When these cells were transduced with retroviral vectors, transduction efficiencies in the bulk CD34+ cells transfected with PTEN siRNA were significantly higher compared with CD34+ cells transfected with a control siRNA. Transient PTEN suppression in CD34+ cells also increased their proliferation and engraftment potential in nonobese diabetic/severe combined immunodeficiency disease with interleukin-2 receptor γ-chain deficiency mice, and maintained their multilineage differentiation capacity in vivo. No mice developed myeloproliferative disorders or leukemias. Similar to findings with murine HSPC, PTEN may also promote quiescence of human HSPC. With optimization of technologies for transfer of siRNA in primary CD34+ cells, this approach may facilitate investigations into the mechanisms underlying HSPC self-renewal, and could find clinical applications in gene therapy protocols. [Copyright &y& Elsevier]

Compared to other integrating viral vectors, foamy virus (FV) vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34 + cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy. However, infectious titers of FV-human(h)CD18 were low and not suitable for manufacturing of clinical-grade product. Herein, we developed a scalable production and purification process that resulted in 60-fold higher FV-hCD18 titers from ~1.7 × 10 4 to 1.0 × 10 6 infectious units (IU)/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimized gag , heparin affinity chromatography, tangential flow filtration, and ultracentrifugation, which reproducibly resulted in 5,000-fold concentrated and purified virus, an overall yield of 19 ± 3%, and final titers of 1-2 × 10 9 IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO) concentration, thereby avoiding DMSO-induced toxicity to CD34 + cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production.

Induced pluripotent stem cell (iPSC) therapies offer a promising path for patient-specific regenerative medicine. However, tumor formation from residual undifferentiated iPSC or transformation of iPSC or their derivatives is a risk. Inclusion of a suicide gene is one approach to risk mitigation. We introduced a dimerizable-"inducible caspase-9" (iCasp9) suicide gene into mouse iPSC (miPSC) and rhesus iPSC (RhiPSC) via a lentivirus, driving expression from either a cytomegalovirus (CMV), elongation factor-1 α (EF1α) or pluripotency-specific EOS-C(3+) promoter. Exposure of the iPSC to the synthetic chemical dimerizer, AP1903, in vitro induced effective apoptosis in EF1α-iCasp9-expressing (EF1α)-iPSC, with less effective killing of EOS-C(3+)-iPSC and CMV-iPSC, proportional to transgene expression in these cells. AP1903 treatment of EF1α-iCasp9 miPSC in vitro delayed or prevented teratomas. AP1903 administration following subcutaneous or intravenous delivery of EF1α-iPSC resulted in delayed teratoma progression but did not ablate tumors. EF1α-iCasp9 expression was downregulated during in vitro and in vivo differentiation due to DNA methylation at CpG islands within the promoter, and methylation, and thus decreased expression, could be reversed by 5-azacytidine treatment. The level and stability of suicide gene expression will be important for the development of suicide gene strategies in iPSC regenerative medicine.

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4(+) human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas β2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

Hematopoietic stem cells (HSCs) are ideal targets for genetic manipulation in the treatment of several congenital and acquired disorders affecting the hematopoietic compartment. Although G-CSF-mobilized peripheral blood CD34(+) cells are the favored source of hematopoietic stem cells in clinical transplantation, this source of stem cells does not provide meaningful engraftment levels of genetically modified cells compared with G-CSF + stem cell factor (SCF)-mobilized cells in nonhuman primates. Furthermore, the use of G-CSF mobilization can have disastrous consequences in patients with sickle cell disease, a long-held target disorder for HSC-based gene therapy approaches. We therefore conducted a study to compare the levels of genetically modified cells attainable after retroviral transduction of CD34(+) cells collected from a bone marrow (BM) harvest with CD34(+) cells collected from a leukapheresis product after mobilization with G-CSF (n = 3) or G-CSF in combination with SCF (n = 3) in the rhesus macaque autologous transplantation model. Transductions were performed using retroviral vector supernatant on fibronectin-coated plates for 96 hours in the presence of stimulatory cytokines. BM was equal to or better than G-CSF-mobilized peripheral blood as a source of HSCs for retroviral transduction. Although the highest marking observed was derived from G-SCF + SCF-mobilized peripheral blood in two animals, marking in the third originated only from the BM fraction. These results demonstrate that steady-state BM is at least equivalent to G-CSF-mobilized peripheral blood as a source of HSCs for retroviral gene transfer and the only currently available source for patients with sickle cell disease.