Lentiviral Gene Therapy of X-Linked Agammaglobulinemia

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DESCRIPTION (provided by applicant): X-linked agammaglobulinemia (XLA) results from deficient function of Bruton's tyrosine kinase (Btk) and is characterized by a severe block in early B-cell development. B cells that express wildtype Btk exhibit a strong selective advantage in vivo; suggesting that introduction of a normal Btk cDNA into autologous hematopoietic stem cells (HSC) may lead to long-term immunologic reconstitution in XLA. In previous work, we demonstrated that both gamma-retroviral and B lineage-specific, lentiviral (LV) Btk gene therapy can lead to rescue of Btk-dependent, B lineage development and function in vivo. Notably, in addition to its role in B cells, Btk is activated via multiple receptors expressed on myeloid cells implying a broader role for Btk in host immune responses. To optimize the expression profile of the Btk transgene in both B and myeloid lineage cells, we developed a novel LV platform using the endogenous Btk promoter (Btkp) in association with two alternative elements [the IgH chain enhancer Em or a ubiquitous chromatin opening element (UCOE) element]. Using these LV to express codon-optimized human Btk, we observed rescue of Btk-dependent, B-lineage development and function; and restoration of Toll-like receptor (TLR) signaling in myeloid cells. Together, these data strongly support our goal of using a Btkp-based LV in a future gene therapy trial for patients with XLA. Several key issues; however, remain to be addressed before this approach can be moved forward into patients. First, we need a comprehensive in vivo comparison of the safety and efficacy of EmBtkp vs. UCOE.Btkp LV in both murine XLA and primary stem cells derived from XLA patients. Second, we need a detailed assessment of potential genotoxicity of these candidate LV. Accordingly, this proposal is designed to test the hypotheses that: (1) LV utilizing the endogenous Btk promoter will mediate sustained, temporally appropriate levels of Btk gene expression and rescue of B and myeloid function in murine and human cells; (2) Efficient Btk rescue will also be achieved using a non-myeloablative marrow conditioning regimen that closely mimics a future clinical approach in XLA; and (3)These LVs will exhibit little or no risk of vector- mediated mutagenesis.