Lentiviral Gene Therapy and Genome Editing for Wiskott-Aldrich Syndrome

项目摘要--项目2 大卫·罗林斯博士将指导这个项目的整体活动，包括在西雅图完成的所有工作 儿童研究所(SCRI)和协调我们的合作项目站点所开展的工作。我们 将实施并参与一项基于慢病毒(LV)的新型临床基因治疗试验，用于Wiskott患者- 奥尔德里奇综合征(WASS)。本试验将对CL20-i650-MND-huwas LV载体进行检测。将产生临床LV 由圣犹大儿童研究医院(圣犹大)使用稳定的生产者克隆。将使用GMP LV库存 两次法转导G-CSF/PLERIXA动员的IS患者外周血CD34细胞 协议。转导的细胞将在使用氟达拉滨和 有针对性的白花丹。我们将在我们的三个研究地点招募最多15名患者：西雅图儿童医院， NIH临床中心和圣犹大。总体而言，这项试验将提供有关使用 要治疗的LV是，以及其他需要在多个谱系中高水平表达治疗性基因的疾病。 在这项试验的同时，我们将在西雅图进行基因编辑研究。我们将利用我们广泛的专业知识 在原代细胞中进行核酸酶工程和基因编辑，以开发新一代临床前工具 基因打靶。供体模板与编码新型归巢内切酶TALEN或 CRISPR试剂将被用来编辑内源性基因座或候选避风港。跟随 CD34-HSC体外控制的优化，我们将评估NSG受体植入后的体内功能 老鼠。最后，我们将使用来自HAS受试者的HSC进行临床前研究。

PROJECT SUMMARY – PROJECT 2 Dr. David Rawlings, PI, will direct overall activities in this project, including all work performed at Seattle Children’s Research Institute (SCRI) and coordination of work performed by our collaborating program sites. We will implement and participate in a novel lentiviral (LV)-based clinical gene therapy trial for patients with Wiskott- Aldrich Syndrome (WAS). This trial will test the CL20-i650-MND-huWAS LV vector. Clinical LV will be generated by St. Jude Children’s Research Hospital (St. Jude) using a stable producer clone. GMP LV stocks will be used to transduce G-CSF/plerixafor mobilized peripheral blood CD34+ cells from patients with WAS using a two-hit protocol. Transduced cells will be re-infused into the patient after subablative conditioning using fludarabine and targeted busulfan. We will enroll up to 15 total patients at our three study sites: Seattle Children’s Hospital, the NIH Clinical Center, and St. Jude. Overall, this trial will provide important new information regarding the use of LV to treat WAS, as well as other disorders requiring high-level therapeutic gene expression in multiple lineages. In parallel with this trial, we will perform WAS gene editing studies at Seattle. We will leverage our broad expertise in nuclease engineering and gene editing in primary cells to develop next-generation pre-clinical tools for WAS gene targeting. Co-delivery of donor template and mRNA encoding novel homing endonuclease, TALEN or CRISPR reagents will be used to edit the endogenous WAS locus or candidate safe-harbor sites. Following optimization in control CD34+ HSC in vitro, we will assess function in vivo following engraftment in NSG recipient mice. Finally, we will perform pre-clinical studies using HSC from WAS subjects.