title: Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions.
abstract: Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.
authors.affiliation: Department of Biology and Biotechnology, Istituto Scientifico San Raffaele, Milano, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Ferrari
authors.forename: S
authors.initials: S
authors.lastname: Rossini
authors.forename: N
authors.initials: N
authors.lastname: Nobili
authors.forename: D
authors.initials: D
authors.lastname: Maggioni
authors.forename: A
authors.initials: A
authors.lastname: Garofalo
authors.forename: R
authors.initials: R
authors.lastname: Giavazzi
authors.forename: F
authors.initials: F
authors.lastname: Mavilio
authors.forename: C
authors.initials: C
authors.lastname: Bordignon
mesh_terms: D000243:Adenosine Deaminase / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D002675:Child, Preschool; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D013601:T-Lymphocytes / Q000201:enzymology*; D014162:Transfection
chemical_list: D000243:Adenosine Deaminase