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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.

Publication ,  Journal Article
Otsu, M; Hershfield, MS; Tuschong, LM; Muul, LM; Onodera, M; Ariga, T; Sakiyama, Y; Candotti, F
Published in: Hum Gene Ther
February 10, 2002

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.

Duke Scholars

Published In

Hum Gene Ther

DOI

ISSN

1043-0342

Publication Date

February 10, 2002

Volume

13

Issue

3

Start / End Page

425 / 432

Location

United States

Related Subject Headings

  • T-Lymphocytes
  • Severe Combined Immunodeficiency
  • Sensitivity and Specificity
  • Leukocytes, Mononuclear
  • Humans
  • Human T-lymphotropic virus 1
  • Genetic Therapy
  • Gene Expression Profiling
  • Flow Cytometry
  • Clinical Trials as Topic
 

Citation

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Otsu, M., Hershfield, M. S., Tuschong, L. M., Muul, L. M., Onodera, M., Ariga, T., … Candotti, F. (2002). 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. Hum Gene Ther, 13(3), 425–432. https://doi.org/10.1089/10430340252792558
Otsu, Makoto, Michael S. Hershfield, Laura M. Tuschong, Linda M. Muul, Masafumi Onodera, Tadashi Ariga, Yukio Sakiyama, and Fabio Candotti. “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.Hum Gene Ther 13, no. 3 (February 10, 2002): 425–32. https://doi.org/10.1089/10430340252792558.
Otsu M, Hershfield MS, Tuschong LM, Muul LM, Onodera M, Ariga T, et al. 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. Hum Gene Ther. 2002 Feb 10;13(3):425–32.
Otsu, Makoto, et al. “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.Hum Gene Ther, vol. 13, no. 3, Feb. 2002, pp. 425–32. Pubmed, doi:10.1089/10430340252792558.
Otsu M, Hershfield MS, Tuschong LM, Muul LM, Onodera M, Ariga T, Sakiyama Y, Candotti F. 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. Hum Gene Ther. 2002 Feb 10;13(3):425–432.
Journal cover image

Published In

Hum Gene Ther

DOI

ISSN

1043-0342

Publication Date

February 10, 2002

Volume

13

Issue

3

Start / End Page

425 / 432

Location

United States

Related Subject Headings

  • T-Lymphocytes
  • Severe Combined Immunodeficiency
  • Sensitivity and Specificity
  • Leukocytes, Mononuclear
  • Humans
  • Human T-lymphotropic virus 1
  • Genetic Therapy
  • Gene Expression Profiling
  • Flow Cytometry
  • Clinical Trials as Topic