Severe combined immune deficiency (SCID) is a syndrome in which T and B lymphocyte function is absent. The most common forms of SCID are due to mutations in the genes for the common gc cytokine receptor chain (X-linked SCID), adenosine deaminase (ADA), the JAK3 tyrosine kinase, the ZAP-70 tyrosine kinase, and the CIITA and RF-X proteins required for MHC proteins required for MHC expression. Bone marrow transplant (BMT), by introducing normal hematopoietic stem cells (HSC), can be used to cure patients with SCID. Because of the immuno-incompetence of the recipients and the selective advantage to the normal HSC, histocompatible BMT can generally be performed without prior cytoablative chemo- or radiotherapy. Unfortunately, most patients do not have histocompatible donors and receive alternate therapy with non-histocompatible BMT, which has a high rate of serious immunologic complications. Transplantation of autologous HSC genetically modified to express the defective gene (stem cell gene therapy) is an alternative approach which may ultimately prove to be superior to histocompatible BMT. The present techniques for transducing HSC with retroviral vectors are inefficient and result in low numbers of transduced HSC for transplantation. In a clinical trial of stem cell gene therapy for adenosine deaminase deficiency, we have demonstrated that retrovirally transduced HSC can engraft without cytoablation, generating transduced T, B, and myeloid cells expressing normal levels of ADA. The frequency of vector-positive cells T cells has risen because of the selective advantage conferred to the transduced cells by the expression of ADA. In the present proposal, gene therapy of X-SCID and ZAP-70- SCID will be investigated. As in ADA deficiency, there is an expected selective advantage to T cell progenitors expressing the normal gene product, which may allow restoration of immune function in spite of the relatively low levels of HSC transduction. The studies will use a combination of biochemical and immunologic analysis, in vitro culture of transduced hematopoietic progenitors, and in vivo transplantation of transduced HSC into bnx-Hu mice to test gene therapy for SCID. Cell lines from patients with X-SCID or ZAP-70-SCID will be transduced with a normal gc or ZAP-70 gene to determine which retroviral vector is optimal for correction of cytokine receptor function or T cell receptor (TCR) mediated signaling, respectively. Transduced HSC from patients with X-SCID and ZAP-70- SCID patients will be transplanted into immunodeficient mice, and the correction of T lymphopoiesis measured. Dominant-negative mutations and IL-4 receptor defects in X-SCID will be studied. The role of syk in the immune dysfunction of ZAP-70- SCID will be determined. The results are expected to lead to important clinical gene therapy trials for both diseases within the next 5 years.
