| X-linked agammaglobulinemia (XLA) is a human primary immunodeficiency characterized by a profound lack of mature B-lineage cells and a marked reduction in serum levels of IgM, IgG, and IgA. These features are secondary to a severe block in early B-cell development and result in recurrent bacterial infections. XLA is caused by mutations in Bruton's tyrosine kinase (Btk), a non-receptor tyrosine kinase that is critical for signaling through the pre-B-cell receptor (preBCR) and the B-cell receptor (BCR), both of which regulate key checkpoints in the B-cell developmental pathway.; Patients with XLA are currently treated with intravenous immunoglobulin (IVIG) and antibiotics. However, even with comprehensive care, morbidity rates remain high. Transplantation of autologous hematopoietic stem cells that have been corrected by gene transfer of wildtype Btk would theoretically provide a superior alternative to current supportive XLA therapies. Considerable evidence exists to suggest that even small numbers of corrected stem cells would undergo a significant selective advantage in vivo, and that even a partial recovery of B-lineage cells would result in clinical benefit.; The data presented here represent a series of studies on retroviral vector mediated Btk gene therapy in three animal models of XLA: X-linked immunodeficiency (Xid) mice; Btk-/- knockout mice; and Btk-/Tec- double knockout mice. Briefly, bone marrow populations from Btk deficient mice were transduced with Moloney murine leukemia virus (MMLV) based Btk vectors and transplanted into recipient mice that were then monitored for transgenic Btk expression and rescue of B-cell development and function.; Xid committed B-lineage progenitors were utilized as transduction targets in the first set of experiments, where we observed high levels of initial transduction followed by rapid transgene silencing in vivo. In the second set of experiments, transplantation with corrected Xid or Btk-/- hematopoietic stem cell enriched bone marrow populations resulted in long-term transgene expression and partial restoration of B-cell development and serum immunoglobulin levels. In the final set of experiments, transduction and transplantation of Btk-/Tec- hematopoietic stem cell enriched bone marrow populations led to complete rescue of the Btk deficient phenotype. Together, these results constitute the first demonstration of successful Btk gene therapy in an animal model. |
