Mitotically Stable Lentiviral Episomes for Stem Cell Gene Therapy

 描述(由申请人提供)：使用HIV-1来源的慢病毒载体对自体干细胞进行稳定的基因改造，为遗传性造血和免疫障碍患者提供了临床益处。基因传递到不同组织的广泛取向，前病毒整合成分稳定地传递给所有后代，以及规模化生产用于临床的能力，为将该载体系统用于干细胞基因治疗提供了强大的动机。然而，内源性基因插入激活的例子继续引起人们对整合慢载体在造血干细胞基因治疗中的使用的关注。目前的非整合载体提供了在有丝分裂后组织中的长期表达，但如果不能复制附体载体基因组，就会导致干细胞种群分裂的快速丧失。我们最近报道了一种新的、非整合的外体慢病毒载体的开发，它通过细胞分裂忠实地复制并表达感兴趣的基因--不需要选择。与类似载体不同的是，这种锚定的非整合慢病毒载体避免了异源病毒序列，依赖于人β-干扰素核支架/基质附着区(S/MAR)基因座序列。我们的主要假设是，aNILV将优化生物安全性，并为范可尼贫血小鼠模型的骨髓衰竭提供长期纠正。我们将开发第二代Episome载体设计(aNILV-II)，另外能够转化为无骨架的微环，以进一步增强生物安全性和长期转基因表达。这项建议是对PAS-13-006：血液学研究的新方向(SIRE-II)的独特回应。特异性目的1[A]在Fanconi贫血(FA)相关的骨髓衰竭小鼠模型中，展示了NILV介导的表型纠正、生物安全性和在生理和药物选择下的克隆多样性。特异性AIM-1[B]示踪在免疫缺陷小鼠体内连续移植nILV转导的人CD34祖细胞的分子持久性和多系表达。特定目的1[C]设计一种微环递送慢病毒载体(aNILV-II)，并系统地检测其在克隆性细胞系和体内的性能。拟议的平台承诺调和慢病毒载体传递的效率与表观染色体维持基因组固有的生物安全性。这种载体系统将对干细胞基因治疗产生强大的翻译影响，并可能显著节省成本，目前与患者的长期整合部位监测有关。

 DESCRIPTION (provided by applicant): Stable genetic modification of autologous stem cells using HIV-1 derived lentivirus vectors confers clinical benefit in patients with inherited disorder of hematopoiesis and immunity. Broad tropism for gene delivery to diverse tissues, stable passage of the proviral integrant to all progeny, and the ability to scale production for clinical use provide strong motivation for using this vector system in stem cell gene therapy. Nevertheless, instances of insertional activation of endogenous genes continue to raise concerns for the use of integrating lentivectors in hematopoietic stem cell gene therapy. Current non- integrating vectors provide long-term expression in postmitotic tissues, but failure to replicate the episomal vector genome leads to rapid loss in dividing stem cell populations. We recently reported the development of a novel, non-integrating episomal lentivector that faithfully replicates through cell division and expresses a gene of interest - without selection. In distinctin to similar vectors, this anchored Non-Integrating LentiVector (aNILV) avoids heterologous viral sequences and relies on human β-interferon nuclear scaffold/matrix attachment region (S/MAR) locus sequences. Our principal hypothesis is that aNILV will optimize biosafety and provide long-term correction of bone marrow failure in a murine model of Fanconi Anemia. We will develop a second-generation episome vector design (aNILV-II) additionally capable of conversion to backbone-free minicircles to further enhance biosafety and long-term transgene expression. This proposal is uniquely responsive to PAS-13-006: New Directions in Hematology Research (SHINE-II). SPECIFIC AIM 1 [A] Demonstrate aNILV-mediated phenotype correction, biosafety and clonal diversity under physiologic and pharmacological selection in a murine model of Fanconi Anemia (FA) associated bone marrow failure. SPECIFIC AIM 1 [B] Track molecular persistence and multilineage expression during serial transplantation of aNILV transduced human CD34 progenitor cells in immunodeficient mice. SPECIFIC AIM 1 [C] Design a minicircle-delivering lentivector (aNILV-II) and systematically determine its performance in clonally-derived cell lines and in vivo. The proposed platform promises to reconcile the efficiency of lentivector delivery with the inherent biosafety of episomally maintained genomes. This vector system will have a strong translational impact on stem cell gene therapy and may allow for significant cost savings, currently associated with long- term integration site surveillance in patients.