316. Successful Editing of the CD40LG Locus in Human Hematopoietic Stem Cells
Authors: Gabrielle Curinga, Sarah Leach, Swati Singh, Nick Hubbard, David Hagin, Karen Sommer, Iram Khan, Yumei Song, Troy Torgerson, Andy Scharenberg, David J. Rawlings
Journal: Molecular Therapy (2016)

X-Linked Hyper-IgM Syndrome (X-HIGM) is a genetic disorder caused by mutations in CD40LG that result in the loss of functional CD40L protein on the T cell surface. CD40L is required for T cells to provide "help" to B cells during an immune challenge; thus X-HIGM patients have impaired immunoglobulin class-switching and somatic hypermutation, and suffer recurrent infections. Murine gene transfer and X-HIGM patient studies suggest that safe and effective gene therapies for this disorder need to replicate cell surface expression patterns of WT endogenous CD40L, as well as knock-out expression of the mutant CD40L; requirements that are unlikely to be achievable using viral gene replacement. We recently reported a gene editing approach combining mRNA-delivered TALEN (targeting just upstream of the coding sequence of CD40LG) with an rAAV donor template for homology directed repair, targeting a promoter-less CD40L cDNA to the ATG start codon of the endogenous allele. This approach restored regulated cell surface expression of CD40L to X-HIGM T cells, disrupted expression of the mutant protein, and resulted in T cells that induced B cell class-switching in vitro, thus demonstrating it's potential as a T cell therapy for X-HIGM. Although the pathologies of X-HIGM are attributed mostly to the T cell defects, CD40L is expressed in most hematopoietic lineages. Besides providing a stable X-HIGM treatment, editing of autologous hematopoietic stem cells (HSC) would likely rescue regulated CD40L expression in all lineages. A selective advantage for gene corrected cells is not anticipated; however, patients with mixed donor chimerism post-transplantation have substantial improvements when as few as 10% of HSC have the WT allele. Here we report gene editing of CD40LG in adult human CD34+ PBSC at rates that are anticipated to provide such clinical benefit. Initial experiments using mRNA delivery of TALEN pairs targeting exon 1 of the CD40LG locus in human CD34+ cells demonstrated indel frequencies of >50%. To investigate the potential for HDR at the CD40LG locus in adult CD34+ cells, we combined delivery of the TALEN with an AAV6 donor template containing an MND promoter-GFP expression cassette flanked by 1 kb CD40LG homology arms. This donor template allowed us to track editing rates within the CD40LG locus (normally silent in HSCs) by flow cytometry. Using this co-delivery strategy, we consistently achieved editing rates of ~30% across multiple control human stem cell donors. Edited cells demonstrated minimal loss of viability, and expansion rates in culture equivalent to controls. Edited cells have been transplanted into immune deficient, NSG mice in order to track engraftment and differentiation potential. In parallel, we are currently evaluating editing rates using a more clinically relevant, promoter-less donor template encoding the CD40L cDNA. In summary, we have achieved clinically relevant rates of gene editing within the endogenous CD40LG locus in human HSC, setting the stage for additional work required to translate this approach into clinical application.