? The treatment of genetic and acquired immunodeficiencies by gene transfer is one of the most advanced field of gene therapy. Using the ZAP-70 protein tyrosine kinase deficiency as a paradigm, we previously demonstrated the feasibility of oncoretroviral-mediated hematopotetic stem cell (HSC) gene correction for ZAP-70-deficiency in a knock out mouse model (Steinberg/Otsu et al., Blood, 2002). While this approach has been successfully used in SCID patients, we hypothesized that some of the associated drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. In vivo intrathymic transfer of a gene providing a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny allowing long-term immune reconstitution. Indeed, following intrathymic injection of a T cell-specific ZAP-70-expressing lentiviral vector into thymi of ZAP-70-/- mice, we observed long-term (>12 month) differentiation of mature TCRap thymocytes in a subset of mice, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70-expressing T cells were generated in these mice, demonstrated a partially diversified receptor repertoire and were responsive to allo-antigens (Adjali et al., J Clin Invest, 2005). We now propose to optimize the efficacy and safety of this technology. We will improve delivery of virions to the thymus; notably, by injecting higher amounts of virions, performing repeat injections, and stimulating thymocyte proliferation. Additionally, we will improve the ZAP-70-expressing lentiviral vector (i) to enhance gene expression and specificity from the CD4 promoter, and (ii) to include safety measures such as HS4 insulator sequences and the HSV1-TK suicide gene. The immune reconstitution and function following intrathymic injections with these new methods and vectors will be compared with that obtained following ex vivo lentiviral-mediated correction of HSC. Ultimately, we will perform experiments using improved methods and vectors to assess T cell differentiation of human CD34+ derived cells from ZAP-70-deficient infants in NOD/Rag-/- mice. Altogether, the data emerging from these experiments should promote the development of an optimal and safe gene therapy strategy for patients with ZAP-70-deficiency as well as other forms of SCID. ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI059349-02
Application #
7483117
Study Section
Special Emphasis Panel (ZRG1-GTIE-A (01))
Program Officer
Gondre-Lewis, Timothy A
Project Start
2007-08-15
Project End
2011-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
2
Fiscal Year
2008
Total Cost
$196,200
Indirect Cost
Name
Cnrs Delegation Languedoc-Roussillon
Department
Type
DUNS #
735293318
City
Montpellier
State
Country
France
Zip Code
34000
de Barros, Stephanie C; Vicente, Rita; Chebli, Karim et al. (2013) Intrathymic progenitor cell transplantation across histocompatibility barriers results in the persistence of early thymic progenitors and T-cell differentiation. Blood 121:2144-53
De Barros, Stéphanie C; Zimmermann, Valérie S; Taylor, Naomi (2013) Concise review: hematopoietic stem cell transplantation: targeting the thymus. Stem Cells 31:1245-51
Loisel-Meyer, Severine; Swainson, Louise; Craveiro, Marco et al. (2012) Glut1-mediated glucose transport regulates HIV infection. Proc Natl Acad Sci U S A 109:2549-54
Vicente, Rita; Swainson, Louise; Marty-Gres, Sophie et al. (2010) Molecular and cellular basis of T cell lineage commitment. Semin Immunol 22:270-5
Vicente, Rita; Adjali, Oumeya; Jacquet, Chantal et al. (2010) Intrathymic transplantation of bone marrow-derived progenitors provides long-term thymopoiesis. Blood 115:1913-20
Mondino, Anna; Dardalhon, Valérie; Hess Michelini, Rodrigo et al. (2010) Redirecting the immune response: role of adoptive T cell therapy. Hum Gene Ther 21:533-41
Moreau, Aurélie; Vicente, Rita; Dubreil, Laurence et al. (2009) Efficient intrathymic gene transfer following in situ administration of a rAAV serotype 8 vector in mice and nonhuman primates. Mol Ther 17:472-9
Adjali, Oumeya; Montel-Hagen, Amélie; Swainson, Louise et al. (2009) In vivo and ex vivo gene transfer in thymocytes and thymocyte precursors. Methods Mol Biol 506:171-90