title: Flow cytometry analysis of adenosine deaminase (ADA) expression: a simple and reliable tool for the assessment of ADA-deficient patients before and after gene therapy.
abstract: Clinical gene therapy trials for adenosine deaminase (ADA) deficiency have shown limited success of corrective gene transfer into autologous T lymphocytes and CD34(+) cells. In these trials, the levels of gene transduction and expression in hematopoietic cells have been assessed by DNA- or RNA-based assays and measurement of ADA enzyme activity. Although informative, these methods are rarely applied to clonal analysis. The results of these assays therefore provide best estimates of transduction efficiency and gene expression in bulk populations based on the assumption that gene transfer and expression are uniformly distributed among transduced cells. As a useful additional tool for evaluation of ADA gene expression, we have developed a flow cytometry (fluorescence-activated cell sorting, FACS) assay capable of estimating the levels of intracellular ADA on a single-cell basis. We validated this technique with T cell lines and peripheral blood mononuclear cells (PBMCs) from ADA-deficient patients that showed severely reduced levels of ADA expression (ADA-dull) by FACS and Western blot analyses. After retrovirus-mediated ADA gene transfer, these cells showed clearly distinguishable populations exhibiting ADA expression (ADA-bright), thus allowing estimation of transduction efficiency. By mixing ADA-deficient and normal cells and using enzymatic amplification, we determined that our staining procedure could detect as little as 5% ADA-bright cells. This technique, therefore, will be useful to quickly assess the expression of ADA in hematopoietic cells of severe combined immunodeficient patients and represents an important tool for the follow-up of patients treated in clinical gene transfer protocols.
authors.affiliation: Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Makoto
authors.initials: M
authors.lastname: Otsu
authors.forename: Michael S
authors.initials: MS
authors.lastname: Hershfield
authors.forename: Laura M
authors.initials: LM
authors.lastname: Tuschong
authors.forename: Linda M
authors.initials: LM
authors.lastname: Muul
authors.forename: Masafumi
authors.initials: M
authors.lastname: Onodera
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ariga
authors.forename: Yukio
authors.initials: Y
authors.lastname: Sakiyama
authors.forename: Fabio
authors.initials: F
authors.lastname: Candotti
mesh_terms: D000243:Adenosine Deaminase / Q000032:analysis* / Q000235:genetics* / Q000627:therapeutic use; D001681:Biological Assay; D002460:Cell Line; D002986:Clinical Trials as Topic; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling; D015316:Genetic Therapy; D015368:Human T-lymphotropic virus 1; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D012680:Sensitivity and Specificity; D016511:Severe Combined Immunodeficiency / Q000175:diagnosis* / Q000235:genetics / Q000628:therapy*; D013601:T-Lymphocytes / Q000201:enzymology*
chemical_list: D000243:Adenosine Deaminase