HSC transduction in situ by cellular delivery of integrating viral vectors

描述(由申请人提供)：涉及干细胞基因改造的疗法在治疗血液和免疫系统疾病方面具有很大的前景。它们还面临令人望而生畏的障碍，限制了它们的可行性和效率，部分原因与目标细胞获取困难和干细胞在培养中的存活受到影响有关。我们最近做了一系列新的观察，证明了造血细胞“载体”延长了HIV来源的慢病毒载体颗粒在细胞内的滞留时间，并证明了它们对二次转导事件的能力。在小鼠移植研究中，完整颗粒的释放导致了颗粒优先扩散到宿主的造血器官。这些发现揭示了一种独特的治疗机会，即结合颗粒捕获和活性载体细胞归巢，将慢病毒载体输送到骨髓微环境。我们假设，当与已建立的归巢机制相结合时，可以利用和优化由造血细胞“载体”细胞捕获和释放的慢载体颗粒来进行骨髓干细胞的原位遗传修饰。我们将在三个具体目标上系统地检验这一假设。在干细胞缺陷的小鼠移植模型中，我们将通过原位颗粒传递来确定载体细胞纠正长期存活干细胞的表型能力，评估发生这种情况的机制，并确定扩散到其他器官的能力。我们将测试基于载体细胞归巢和宿主条件调节的策略，以改善载体细胞进入微环境的能力和颗粒输送的效率。最后，结合生化分析和活细胞成像，我们将研究载体细胞内的颗粒运输，以此作为理解和操纵负责载体颗粒滞留和释放的机制的一种手段。建议的策略是基于我们实验室的观察，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输送。成功地将慢病毒载体颗粒通过自体细胞载体直接输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。如果不能更好地接触到组织和靶细胞，基因治疗的治疗潜力在很大程度上仍然没有得到发挥。因此，建议开发一种新的细胞方法来传递基因有效载荷，这不仅对于预期纠正血液和免疫系统的遗传疾病至关重要，而且作为再生医学中损伤后修复的工具也是至关重要的。

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.