This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The central hypothesis of this proposal is: Retroviral-mediated transfer of a normal human ADA cDNA into CD34+ hematopoietic stem cells of infants and children with ADA-deficient SCID, who have undergone marrow cytoreduction and are not on PEG-ADA replacement, can be performed safely and will result in the production of mature T lymphocytes, expressing ADA enzyme activity and restoring immune function. The study objectives are: 1. Examine the safety of isolating CD34+ cells from autologous bone marrow of infants or children with ADA-deficient SCID, taken off PEG-ADA, performing ex vivo gene transduction with the GCsap-M-ADA and MND-ADA retroviral vectors, performing marrow cytoreduction with busulfan, and re-infusing the cells into the donors. Busulfan pharmacokinetics will be performed following the first intravenous dose. Long-term follow-up (15 years) for replication-competent retrovirus and for leukemia monitoring (resulting from serious adverse events occurring in an X-SCID gene transfer trial in France; Hacein-Bey-Abina S, 2003) will be provided (first two years under this protocol, then after informed consent under protocol CCI #03-220). 2. Assess the efficacy of stem cell transduction/engraftment by serial examination of peripheral blood lymphocytes and hematopoietic cells to quantitate the percentages of cells containing the ADA cDNA by semi-quantitative DNA-PCR. 3. Assess vector expression by ADA enzymatic activity, and possibly by RT-linked PCR of peripheral blood leukocytes. 4. Compare the numbers of cells which accumulate containing the GCsap-M-ADA vector or the MND-ADA vector, to determine if either vector confers a greater selective advantage. 5. Examine the effects of ADA gene expression on the immune function by performing a controlled trial of PEG-ADA withdrawal in patients who have been enrolled while on PEG-ADA treatment, if milestones for engraftment and expression of the transduced gene are met. In all, these clinical studies will determine the safety and effectiveness of current retroviral-mediated gene transfer techniques to introduce genes into human hematopoietic stem cells from the bone marrow. Two different retroviral vectors, GCsap-M-ADA and MND-ADA, will be compared for their abilities to express the human ADA cDNA and confer a selective survival advantage upon T lymphocytes. Marrow cytoreduction in the absence of PEG-ADA will be tested, to assess the safety of the approach and its effect on engraftment and selective survival of gene-corrected cells. Additionally, the specific benefits for reconstitution of immunity in ADA-deficient SCID patients will be assessed.
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