? 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.
? PROJECT 2 WAS represents an ideal candidate disorder to apply therapeutic gene replacement and/or gene editing. We will perform a clinical trial testing the efficacy and safety of lentiviral-based WAS gene therapy. In parallel, we will apply novel methods to promote gene editing in hematopoietic cells with the goal of identifying approaches amenable to future clinical application.
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