299. Efficient Functional Oxidase Correction after CRISPR-Oligomer Gene Repair in X-CGD Patient Hematopoietic Stem Cells (HSC) Using Non-Viral, cGMP Compliant, Scalable, Closed System
Authors: Linhong Li, Suk see De Ravin, Cornell Allen, Pachai Natarajan, Harry L. Malech, Madhusudan V. Peshwa
Journal: Molecular Therapy (2015)

Gene therapy using integrating viral vectors in hematopoietic stem cells (HSC) has shown clinical benefit in genetic diseases. The safety concerns associated with random integration and the lack of gene expression regulation remain key challenges with use of viral vectors. Efficient and target-site specific correction of mutation(s) in patient HSC genomic DNA using non-viral methods may improve safety and gene expression regulation. We and others have previously reported that loading of Nucleases in the form of messenger RNA (mRNA) results in low toxicity and efficient functional gene editing when using MaxCyte's rapid, automated, cGMP and regulatory compliant, and closed cell loading platform. This platform has been cleared for use in human clinical trials for multiple applications of targeted gene editing in stem cells and immune cells. We report herein that transfecting CRISPR (Cas9+gRNA) mRNA along with donor oligomer specific to a hotspot mutation in the gp91phox gene (CYBB) located on exon 7 associated with the X-linked form of Chronic Granulomatous Disease (X-CGD), using MaxCyte GT System, results in efficient repair of the 676T (stop codon) to 676C (Arginine) mutation, resulting in gp91 protein expression and associated oxidase activity. Plasmids encoding Cas9 and gRNA were purchased from the Genomic Engineering Center at Washington University (St. Louis, MO). The mRNA encoding Cas9 and gRNA were synthesized at MaxCyte using mMESSAGE mMACHINE® T7 Ultra kit, (Ambion, Austin, TX). Patient EBV-transformed B cell line (B-LCL) and patient HSC were obtained under NIAID IRB approved protocol after patient written informed consent. The cells were cultured in RPMI-1640+10% FBS+2mM L-glutamine and StemSpan medium (Stem Cell Technologies, BC, Canada) supplemented with 100ng/ml SCF, Flit3L and TPO, respectively. We first optimized transfection conditions with the X-CGD patient B-LCL. It was found that B-LCL can be efficiently transfected with CRISPR and donor oligomer. Transfected B-LCL exhibit 80±6% viability, and maintained cell proliferation rate identical to control cells. Cel-1 assay shows 30-40% (N=8) efficiency of specific-site gene editing. Using donor oligomer containing a HindIII restriction site, it was determined that the DNA integration efficiency in Exon 7 was 35±8% (N=3). Using donor oligomer with the removal of HindIII restriction site, the expression of functional gp91 protein as determine by flow cytometry was 9±3% (N=3). These developed protocols were used to transfect HSC obtained from the same CGD patient (N=1). Following in vitro myeloid differentiation of gene mutation-repaired CGD HSC, 6.3% of the differentiating cells containing neutrophils and monocytes were oxidase positive in the dihydrorhodamine (DHR) flow cytometry oxidase function assay. These observations demonstrated restoration of phagocyte oxidase activity from the gene repair. Work is in progress to optimize and achieve clinical scale-up of the process which may have broad applicability to the treatment of monogenic diseases.