Non-viral and viral hematopoietic progenitor cell gene therapy

Unrestricted 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/yC(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 HSB-transposon 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.

无限制：造血干细胞（hematopoietic stem cells, HSCs）的多能性特性使其成为基因治疗的优质候选细胞。常用病毒基因转导系统（viral gene transfer system）存在的安全缺陷，使得该领域对非病毒或嵌合型基因转导系统等替代方案的探索愈发受到关注。睡美人转座子（Sleeping Beauty, SB）介导的基因转导系统便是其中之一。通过逐步优化睡美人转座子系统的各组成元件，并采用非病毒方式递送SB质粒，本研究将稳定基因转导效率提升至既往报道水平的最高20倍。体内实验结果显示，经SB修饰的人CD34阳性（CD34+）细胞可在NOD/SCID/γc敲除（NOD/SCID/yC(null), NSG）小鼠体内定植，并分化为多谱系细胞类型，同时维持转基因的稳定表达。二次移植的NSG小鼠体内仍可检测到持续的转基因表达，表明HSB转座子系统可实现长期稳定的基因整合。研究同时探索了非整合型慢病毒（Non-integrating lentiviral, NIL）载体作为SB质粒递送工具的可行性。将SB转座子系统的稳定整合特性与NIL载体的高效递送能力相结合，可构建一种被命名为“NILting beauty”的嵌合载体系统，该系统兼具病毒与非病毒载体系统的优势，可为造血干细胞的基因修饰提供更高效、更安全的策略。研究采用K562细胞与人造血干细胞评估了NILting beauty系统实现稳定转基因整合的可行性与应用潜力。结果显示，经NILting beauty载体转导后，K562细胞的稳定转基因表达率可达7%，人CD34阳性细胞的表达率约为1%。在临床造血干细胞基因治疗中，另一种可提升长期转基因表达且不良反应相对轻微的策略是采用非清髓性预处理方案（non-myeloablative conditioning regimen）。为实现在造血干细胞基因治疗中的长期稳定基因标记，研究探索了将白消安（busulfan）与氟达拉滨（fludarabine）联合使用作为替代预处理方案的可行性，该方案或具备更优的治疗效果。本研究提出假说：作为免疫抑制性化疗药物的氟达拉滨，可通过降低宿主对异体转基因产物的免疫应答，从而促进造血干细胞的定植与长期转基因表达。为验证该假说，研究采用了符合临床场景的幼年恒河猴骨髓移植（bone marrow transplant, BMT）模型。初步实验数据显示，白消安的给药剂量与其血药浓度曲线下面积（area-under-the curve, AUC）存在显著相关性。实验中观察到一过性中性粒细胞减少，但未出现淋巴细胞减少的情况。未给予氟达拉滨的实验组猴中，基因标记水平较高的个体可检测到抗增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）抗体；而给予氟达拉滨的实验组猴则未出现针对eGFP的体液免疫应答。上述结果表明，针对转基因产物的免疫应答可能对造血干细胞基因治疗的成功与否起到关键作用，而氟达拉滨可对这类免疫应答起到调节作用。由于氟达拉滨治疗并未实现显著的淋巴细胞清除，后续需评估更高剂量的氟达拉滨对造血干细胞定植与长期转基因表达的影响。尽管NILting beauty嵌合载体系统与宿主预处理方案仍需进一步改进与优化，但本论文所述研究证实，优化后的HSB转座子系统在基于造血干细胞的睡美人转座子基因治疗领域具有广阔的应用前景。