Lentiviral Gene Therapy and Genome Editing for Wiskott-Aldrich Syndrome

项目概要-项目2 博士PI大卫·罗林斯（David Rawlings）将指导该项目的整体活动，包括在西雅图执行的所有工作 儿童研究所（SCRI）和协调我们的合作计划网站执行的工作。我们 将实施并参与一项针对Wiskott患者的新型慢病毒（LV）临床基因治疗试验， 奥尔德里奇综合征（WAS）。本试验将测试CL 20-i650-MND-huWAS LV载体。将生成临床LV 由圣裘德儿童研究医院（圣裘德）使用稳定的生产者克隆。将使用GMP LV储备液 使用二次打击法，从WAS患者中动员G-CSF/plerixafor动员的外周血CD 34+细胞 议定书转导的细胞将在使用氟达拉滨的亚消融预处理后重新输注到患者体内， 目标是白消安。我们将在我们的三个研究中心招募多达15名患者：西雅图儿童医院， 美国国立卫生研究院临床中心和圣裘德。总体而言，本试验将提供关于使用 LV治疗WAS，以及其他需要高水平治疗基因表达的疾病。 与此同时，我们将在西雅图进行WAS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，为WAS开发下一代临床前工具 基因靶向供体模板和编码新型归巢核酸内切酶TALEN或TALEN的mRNA的共递送 CRISPR试剂将用于编辑内源性WAS基因座或候选安全港位点。以下 为了在体外优化对照CD 34 + HSC，我们将评估NSG受体植入后的体内功能 小鼠最后，我们将使用WAS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.