Gene Therapy in Hematopoietic Cells

该项目的总体目标是制定战略，这将使高效和安全的 通过造血干细胞（HSC）基因疗法治疗造血系统疾病。不幸的是， 体外研究和小鼠模型研究都不能预测基因转移的安全性和有效性 在大型动物或人类中，因此我们将使用狗模型，这也将使我们能够评估改进的 直接在疾病模型中的策略。虽然在狗模型和其他动物模型中基因转移到HSC中的效率 在过去的几年里，大型动物的白血病发生率有了显着提高， 法国X连锁严重联合免疫缺陷（XSCID）基因治疗试验中的患者发生了变化， 从强调有效性到强调安全性。因此，我们将采用犬模型研究3种常用药物的安全性 使用整合载体系统：γ逆转录病毒、慢病毒和泡沫病毒载体。利用这些矢量 系统，我们已经能够实现犬长期再生细胞的有效转导， 在相当数量的狗中稳定的基因转移水平>5%。因此，我们现在有一个独特的资源， 可用于研究特异性目的1中HSC基因转移的安全性。在具体目标2中， 将进一步优化泡沫和慢病毒载体的转导条件，以最大限度地减少插入风险。 诱变我们将集中于慢病毒和泡沫病毒载体，因为与γ逆转录病毒相比， 载体，这些载体系统允许使用短转导培养物进行有效转导。这是 对于非清髓性移植环境中的干细胞基因治疗尤其重要， 干细胞的维持对于与存活的内源性干细胞竞争的能力至关重要。因此在 具体目标3：我们将在非清髓性环境中使用这些载体系统，并探索是否在 体内选择策略可以提高非清髓性条件处理后的基因转移水平。最后在 具体目标4我们将在犬遗传病模型中测试改进的基因转移方案。的 临床相关的大型动物模型的可用性应该使我们能够快速将我们的发现转化为 临床HSC基因治疗研究。

The overall objective of this project is to develop strategies, which will allow the efficient and safe treatment of hematopoietic diseases by hematopoietic stem cell (HSC) gene therapy. Unfortunately neither in vitro studies nor studies in the mouse model have been predictive for safety and efficacy of gene transfer in large animals or humans, thus we will use the dog model which will also allow us to evaluate improved strategies directly in a disease model. While gene transfer efficiencies into HSCs in the dog model and other large animals have improved significantly over the past several years, the development of leukemia in 3 patients in the French X-linked severe combined immunodeficiency (XSCID) gene therapy trial has shifted the emphasis from efficacy to safety. Thus, we will use the dog model to study the safety of 3 commonly used integrating vector systems: gammaretrovirus, lentivirus, and foamy virus vectors. Using these vector systems, we have been able to achieve efficient transduction of canine long-term repopulating cells with stable gene transfer levels >5% in a significant number of dogs. Thus, we now have a unique resource available to study in Specific Aim 1 the safety of HSC gene transfer with these vectors. In Specific Aim 2, we will further optimize transduction conditions for foamy and lentiviral vectors to minimize risks from insertional mutagenesis. We will focus on lentivirus and foamy virus vectors since, in contrast to gammaretroviral vectors, these vector systems allow for efficient transduction using short transduction cultures. This is particularly important for stem cell gene therapy in a nonmyeloablative transplant setting where maintenance of stem cells is crucial for the ability to compete with surviving endogenous stem cells. Thus, in Specific Aim 3 we will use these vector systems in a nonmyeloablative setting and also explore whether in vivo selection strategies can improve gene transfer levels after nonmyeloablative conditioning. Finally, in Specific Aim 4 we will test improved gene transfer protocols in a canine genetic disease model. The availability of a clinically relevant large animal model should allow us to quickly translate our findings to clinical HSC gene therapy studies.