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. We have developed and tested a self-inactivating lentiviral vector (LV) containing a B lineage-specific promoter/enhancer driving human Btk expression in murine HSC derived from Btk/Tec-/- mice. Using this approach, we have achieved full rescue of bone marrow and peripheral B-cell development, and correction of Btk-dependent B cell functional responses. These data demonstrate that LV Btk gene transfer can rescue immune function in an animal model of XLA, and strongly support the further pursuit of this therapeutic approach. Recent clinical trials for primary immunodeficiency disorders highlight the great potential for genetic correction as well as an unanticipated risk for gamma-retroviral associated malignancies. While conceptually simple, implementation of definitive genetic therapy in XLA requires a concerted program of basic and applied research. Several key issues, in particular, remain to be addressed before this approach can be utilized in patients including: a safety and efficacy comparison of our validated B-cell-specific LV vs. LV that utilizes the endogenous Btk promoter;a direct test of safety and efficacy in HSC derived from XLA patients in vivo;and direct testing of our planned clinical gene delivery protocol in a non-human primate model. Using a series of integrated experimental approaches we will test the hypotheses that: (1) LV utilizing the Btk endogenous promoter will mediate sustained, temporally appropriate Btk gene expression; and rescue of function in murine and human cells in vivo and in vitro;(2) these LV will exhibit a low risk of vector-mediated mutagenesis in vitro and in vivo, and (3) our anticipated clinical gene transfer protocol will lead to sustained Btk marking, without evidence for toxicity, in a relevant non-human primate model. Together, these data will set the stage for an LV clinical trial in XLA;and may also provide insight into gene delivery methods for other congenital immune disorders that lack a similar selective advantage.

Public Health Relevance

Development of genetic therapy for X-linked agammaglobulinemia is an important next step in the evolution of gene therapy for primary immunodeficiency disorders. This application is designed to address most of the key questions required to move forward to a clinical trial. Our work will identify an optimal lentiviral vector capable of rescuing Btk function in mouse and human cells. In addition, we will test this vector for safety and efficiency using a relevant non-human primate model. Because B cell function impacts a broad array of human diseases, studies in this rare disorder may also benefit future therapies for more common immune disorders.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
High Priority, Short Term Project Award (R56)
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Special Emphasis Panel (ZRG1-IMM-K (52))
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Johnson, David R
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Seattle Children's Hospital
United States
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