132. Targeting the BTK Locus in Primary Human Hematopoietic Cells with TALENs and AAV Donor Template

Abstract

X-linked agammaglobulinemia (XLA), a rare immunodeficiency, is caused by mutations in the Bruton's Tyrosine Kinase (BTK) gene. BTK is required for B-cell maturation, activation and BCR-mediated signaling. Patients lacking functional BTK have predominantly immature B-cells with near absence of mature B cells, minimal antibody production and are prone to recurrent and life-threatening bacterial infections. Available treatments are limited to intravenous immunoglobulin therapy, which lessens the severity of these infections and judicious use of antibiotic therapy. BTK is an ideal target for gene editing as BTK-expressing B cells exhibit a marked selective advantage in vivo, and even a small number of corrected HSC or B cell progenitors rescues B cell development and function in murine models and is expected to rescue the B cell compartment in XLA patients. Here we outline a strategy to edit the endogenous BTK locus in primary human cells, targeting a codon-optimized BTK cDNA into the first coding exon of BTK using BTK specific DNA nucleases and donor template. Four pairs of transcription activator-like effector nucleases (TALENs) cleaving within introns 1 or 2 of BTK were constructed and their cleavage efficiency in primary human T cells evaluated. Using the T7 Endonuclease assay, we determined that disruption (indel) rates of ~75% were obtained using the TALENs. Next, utilizing co-delivery of TALEN mRNA and an AAV donor template (consisting of an MND promoter-GFP cDNA expression cassette flanked by 1.0 kb BTK homology arms), we achieved up to 25% homology directed repair (HDR) in primary T cells. The translation of this gene editing strategy to human mobilized peripheral CD34+ cells is currently being optimized. If successful, this targeting approach will be used to test expression levels of the targeted cDNA as regulated by the endogenous BTK promoter.