238. BTK-Promoter LV Vectors Utilizing Conserved Intron Element Mediate Functional Rescue in Murine XLA

X-linked agammaglobulinema (XLA) is a rare immunodeficiency disorder caused by a mutation in the gene coding Bruton's tryosine kinase (BTK). BTK is required for signal transduction downstream of the B cell antigen receptor (BCR) and is essential for normal B cell development, activation, and survival. Patients lacking BTK have very few mature B cells, markedly reduced antibody production, and suffer from recurrent and life-threatening infections. XLA is an ideal candidate for hematopoietic gene therapy, as B cells that express BTK are positively selected during development. We previously developed a SIN-lentiviral (LV) construct that used the human BTK promoter (BTKp) to drive BTK expression. We also incorporated a truncated, 0.7 kb, fragment derived from the HNRPA2B1/CBX3 ubiquitously acting chromatin opening element (UCOE) upstream of the BTKp to facilitate stable BTK expression over time. Bone marrow transplantation in a murine XLA model (BTK-/-Tec-/- mice) using LV transduced hematopoietic stem cells (HSCs) reconstituted myeloid and B cell numbers, BCR-dependent B cell proliferation and T-dependent antibody responses. The goal of the current study was to further optimize BTK expression in B and myeloid cells while reducing the viral copy number (VCN) required for efficient functional reconstitution. Using the ENCODE database, we identified several highly conserved upstream and intronic regions exhibiting histone modifications and transcription factor binding profiles in B cells consistent with enhancer regions. These conserved elements were introduced individually or in tandem into the LV construct between the UCOE and BTKp and tested for their ability to rescue BTK expression and B cell development in the murine XLA model. Addition of two key intronic regions led to consistent BTK expression and rescued the development and function of B cells, despite a >5-fold reduction in VCN per splenic B cell. Our findings illustrate the usefulness of this strategy for improving the efficacy of endogenous promoter-based genetic therapies. This new LV vector, UCOE. I4-5.BTKpro-hBTK, is currently being evaluated using mobilized CD34+ stem cells isolated from XLA patients and in additional pretoxicology studies designed to support its application in a future LV clinical trial for XLA.