Non-viral and viral hematopoietic progenitor cell gene therapy

The pluripotent characteristic of hematopoietic stem cells (HSCs) makes them a good candidate for gene therapy. The safety drawbacks of the commonly used viral gene transfer system have made the search for alternative gene transfer methods such as non-viral or hybrid gene transfer systems became increasingly appealing in the field. One such system is the Sleeping Beauty (SB) transposon-mediated gene transfer system. Using a non-viral approach to delivery SB plasmids we were able to significantly increase the efficiency of stable gene up to 20-fold higher than previously published data by incrementally optimizing each element of the SB transposon system. In vivo studies demonstrated that SB-modified human CD34+ cells were engrafted in NOD/SCID/gammaC(null) (NSG) mice and differentiated into multi-lineage cell types with stable transgene expression. Transgene expression remained persistent in the secondary transplanted NSG mice indicating a long-term stable integration achieved by HSBtransposon system. Non-integrating lentiviral (NIL) vectors were also investigated as another method for SB plasmid delivery. Combining the stable integration of the SB transposon system with the delivery efficiency of NIL, termed NILting beauty, could produce a hybrid vector system that synergizes the advantages of both viral and non-viral vector systems and provide a more effective and safer approach to genetically modify HSCs. The feasibility and potential of utilizing NILting beauty to achieve stable transgene integration was evaluated using K562 and human HSCs. Up to 7% stable transgene expression was achieved in K562 cells and around 1% for human CD34+ cells when transduced with NILting beauty vectors. The other approach to increase long-term transgene expression with relatively minimal adverse effects in clinical HSC gene therapy is using non-myeloablative conditioning regimen. The feasibility of combining busulfan with fludarabine as an alternative and potentially more effective conditioning regimen was explored to achieve long-term stable gene marking in HSC gene therapy. We hypothesized that the addition of the immunosuppressive chemotherapeutic agent fludarabine may contribute to better HSC engraftment and long term transgene expression by reducing host immunological responses to the foreign transgene product. To evaluate this hypothesis, a clinically relevant infant rhesus monkey bone marrow transplant (BMT) model was used. Preliminary data showed a strong correlation between the busulfan dose and the busulfan area-under-the curve (AUC). Transient neutropenia was noted whereas lymphopenia was not observed. While monkeys with high levels of eGFP gene marking also showed detectable levels of anti-eGFP antibodies when no fludarabine was given, they lacked humoral immune responses to eGFP if they received fludarabine. These data suggest that the immune responses against the transgene may play a significant role in the successful outcome of HSC gene therapy and that fludarabine may be able to modulate these responses. Since significant lymphodepletion was not achieved by the fludarabine treatment, higher doses of fludarabine may need to be evaluated for an effect on engraftment and long-term transgene expression. Although further improvements and optimization are required for the NILting beauty hybrid system and the host conditioning regimen, studies described in this thesis demonstrated that the application of the optimized HSB transposon system holds great promise for further advancement of SB-transposon based gene therapy using hematopoietic stem cells.