X-linked severe combined immune deficiency (X-SCID) is the most common form of SCID caused by mutations in the gene for the common gammac cytokine receptor chain. Human and canine X-SCID are marked by a block in thymopoiesis, peripheral T and NK lymphopenia, and presence of phenotypic B cells with defective antibody responses. Murine knockouts for gammac differ from humans and dogs in that the affected mice have both T and B lymphopenia. Bone marrow transplant (BMT), by introducing normal hematopoietic stem cells (HSC), can be used to cure patients with X-SCID. Because of the immunoincompetence of the recipients and the selective advantage to the normal HSC, histocompatible BMT can generally be performed without prior cytoablative chemo- or radiotherapy. Most patients do not have histocompatible donors and receive alternate therapy with histoincompatible BMT, which is less successful. 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 histoincompatible BMT. The present techniques for transducing HSC with retroviral vectors are inefficient and result in low numbers of transduced HSC for transplantation. Our clinical trial of gene therapy for ADA deficient SCID has 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. Gene therapy of canine X- SCID is an ideal large animal pre-clinical model, in that canine X-SCID is phenotypically identical to that of humans, and it is likely that the same vectors and reagents to be used in a clinical trial could be tested in the dog model. 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 analyze transduction, engraftment, and functional correction of the immune system in X-SCID dogs receiving autologous CD34+ marrow cells from X- SCID dogs after transduction with retroviral vectors containing the human gammac cDNA. The studies will use a combination of biochemical and immunologic analyses, in vitro culture of transduced hematopoietic progenitors, and in vivo transplantation of transduced HSC into X-SCID dogs to test gene therapy. Modifications of the vector LTR will be tested for the ability to give activation-independent expression and to prevent vector silencing. We will test whether the administration of the thymopoietic cytokine IL-7 after gene therapy will accelerate the usually slow development of functional immunity from HSC.
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