HSC transduction in situ by cellular delivery of integrating viral vectors

描述（由申请人提供）：涉及干细胞遗传修饰的疗法对血液和免疫系统疾病的治疗有很大的希望。它们还面临着限制其可行性和效率的令人生畏的障碍，部分原因是靶细胞获取困难和干细胞在培养中的存活率受损。我们最近进行了一系列新的观察，证明了造血“载体”细胞对HIV衍生的慢病毒载体颗粒的细胞内滞留时间延长及其次级转导事件的能力。在小鼠移植研究中，完整颗粒的释放导致优先颗粒扩散到宿主造血器官。这些发现揭示了将联合收割机颗粒捕获和主动载体细胞归巢结合用于将慢病毒载体递送至骨髓微环境的独特治疗机会。我们假设，慢病毒载体颗粒的捕获和释放造血“载体”细胞，当与建立归巢机制相结合，可以利用和优化骨髓干细胞原位遗传修饰。我们将在三个具体目标中系统地检验这一假设。在具有干细胞缺陷的鼠移植模型中，我们将确定载体细胞通过原位颗粒递送来表型校正长寿命干细胞的能力，评估这种情况发生的机制并确定向其他器官的传播。我们将测试基于载体细胞归巢和宿主调节的策略，以改善载体细胞进入微环境和颗粒递送效率。最后，我们将结合生物化学检测和活细胞成像，研究载体细胞内的颗粒运输，作为理解和操纵载体颗粒保留和释放机制的一种手段。所提出的策略是基于我们实验室的观察结果，并适应了已建立的癌症基因治疗范例，即使用细胞载体靶向肿瘤组织进行病毒颗粒递送。通过自体细胞载体将慢病毒载体颗粒成功地全身递送到骨髓干细胞将有助于克服目前造血和免疫疾病基因治疗的障碍。如果不改善对组织和靶细胞的获取，基因治疗的治疗潜力在很大程度上仍未实现。因此，提出的开发一种新的细胞方法来递送遗传有效载荷不仅对于预期的血液和免疫系统遗传疾病的校正至关重要，而且作为再生医学中损伤后修复的工具也至关重要。

DESCRIPTION (provided by applicant): Therapies involving the genetic modification of stem cells hold substantial promise for the treatment of blood- and immunesystem disorders. They also face daunting obstacles limiting their feasibility and efficiency, related in part to poor target cell access and compromised stem cell survival in culture. We have recently made a series of novel observations that demonstrate the prolonged intracellular retention of HIV-derived lentivector particles by hematopoietic "carrier" cells and their capacity for secondary transduction events. The release of intact particles in murine transplantation studies led to preferential particle dissemination to host hematopoietic organs. These findings reveal a unique therapeutic opportunity to combine particle capture and active carrier cell homing for the delivery of lentivector to the marrow microenvironment. We hypothesize that lentivector particle capture and release by hematopoietic "carrier" cells, when combined with established homing mechanisms, can be exploited and optimized for the genetic modification of bone marrow stem cells in situ. We will systematically test this hypothesis in three specific aims. In a murine transplantation model with a stem cell defect we will ascertain the ability of carrier cells to phenotypically correct long-lived stem cells by in situ particle delivery, evaluate the mechanism by which this occurs and determine the dissemination to other organs. We will test strategies based on carrier cell homing and host conditioning to improve carrier cell access to the microenvironment and the efficiency of particle delivery. Finally, using a combination of biochemical assays and live-cell imaging we will investigate particle trafficking within carrier cells as a means of understanding and manipulating the mechanisms responsible for vector particle retention and release. The proposed strategy is based on our laboratory's observations, and adapts the established cancer gene therapy paradigm of using cellular carriers to target tumor tissue for virus particle delivery. Successful systemic delivery of lentivector particles by autologous cellular carriers directly to bone marrow stem cells will help overcome current impediments to gene therapy for hematopoietic and immune disorders. Without improved access to tissues and target cells, the therapeutic potential of gene therapy remains largely unfulfilled. The proposed development of a novel cellular approach to the delivery of genetic payload is therefore critically important not only for the intended correction of genetic disorders of the blood and immune system, but also as a tool for post injury repair in regenerative medicine.