The goal of this proposal is to elucidate the molecular bases of immunodeficiency in patients with adenosine deaminase (ADA) deficient severe combined immunodeficiency (SCID). Infants with this disorder have profound defects in T and B cell function which result in their dying of infections in the first few years of life. While HLA matched marrow transplantation has resulted in correction of the immunodeficiency in this condition, haploidentical stem cell transplants have often been unsuccessful. Because of this, efforts are now underway in several centers to correct the defects by gene insertion therapy. My Division has been very active in the care, transplantation, and characterization of a number of patients with this condition, and some of them will likely be candidates for gene therapy. I am proposing to characterize the molecular defects in these patients in order to correlate them with their immune defects and to provide a basis for studying the interaction in the future of a normal gene introduced into an environment in which a defective gene resides. The experiments are directed toward definition of the specific molecular defects in patients who have been well-characterized at the cellular immunologic level at Duke University Medical Center. For each patient, the size and quantity of ADA mRNA will first be determined by Northern analysis of poly (A) RNA purified from B lymphoblastoid cell lines I have established from affected individuals and obligate heterozygotes. Based on analysis of other defective genes, we can use the qualitative and quantitative features of the mRNA to predict the nature of the molecular defect. The specific defect will be further defined by S-1 nuclease analysis of RNA and by cDNA cloning and sequencing when appropriate. With these methods I will be able to correlate the patients' molecular defects with their immune phenotypes and responses to established therapies. This information could prove extremely useful in the analysis of patients who will potentially receive gene transfer therapy in the future.
| Markert, M L (1991) Purine nucleoside phosphorylase deficiency. Immunodefic Rev 3:45-81 |
