Severe combined immunodeficiency (SCID) comprises a group of heterogeneous genetic disorders that are fatal, unless treated by hematopoietic cell transplantation (HCT). Mutations of RAG1 and RAG2 genes are the most common cause of T-B-NK+ SCID in humans. Hypomorphic defects in the same gene may cause leaky SCID or Omenn syndrome (OS), a severe immunodeficiency associated with multiorgan damage due to infiltrating and oligoclonal T cells. Long-term survival rate after HCT for RAG deficiency is only 50%, and is even worse in leaky SCID and OS. Recently, virus-mediated gene transfer into hematopoietic CD34+ progenitor cells has been used to treat some forms of SCID, when HLA-matched donors are lacking. However, this approach has been associated with insertional mutagenesis. Therefore, the pursue of novel and safer technologies for gene correction is of outmost importance. Homing endonucleases (HE) recognize large (>12 bp) DNA target sequences in a specific manner, and could be used to attempt gene correction. Our collaborator Frederic Paques has engineered a RAG1-specific HE. One of the major limitations of the preclinical studies that aim at exploring the efficacy of gene transfer in humans with immunodeficiency is the limited availability of patient-derived target cells. However, fibroblasts can be reprogrammed in vitro into induced pluripotent stem cells (iPSCs) through virus-mediated transduction of a combination of transcription factors. These iPSCs can be targeted multilineage differentiation (including T lymphocytes) in vitro. We have generated a repository of fibroblast cell lines from patients with SCID or OS, carrying different RAG1 mutations. We now intend to generate iPSCs from RAG1-mutated patients, and to characterize their stemness and pluripotency profile, chromosomal integrity and patient-specific derivation. In collaboration with Dr. Zuniga-Pflucker, we will investigate the ability of these patient-derived iPSCs to proceed along T-cell differentiation in vitro. We will also explore the ability of the RAG1-specific HE to correct the mutant RAG1 locus in patient-derived iPSCs, and to support V(D)J recombination and T cell differentiation in vitro. We will compare the ability of gene-corrected corrected and uncorrected patient-derived iPSCs to support T-cell development in vitro. This study will permit to define the specific ability of various RAG1 mutations to support T-lymphocyte differentiation, and may thus help explain the basis of the phenotypic diversity of RAG defects in humans. Furthermore, this project will also explore the efficacy and safety of a novel approach to gene therapy of SCID, based on gene-specific endonuclease-mediated homologous recombination.

Public Health Relevance

Defects of the RAG1 and RAG2 genes in humans cause a spectrum of severe immunodeficiencies that range from complete absence of lymphocytes (SCID) to a condition characterized by immunodeficiency and autoimmunity (Omenn syndrome). The basis for this diversity remains poorly defined. To address this issue, we will induce skin cells (fibroblasts) from patients with RAG1 gene defects, to become pluripotent stem cells (iPSCs), and we will then study their ability to differentiate into lymphocytes in a test tube. Moreover, we will use a novel protein to repair the RAG1 gene defect in patient-derived stem cells. This may pave the way to novel and safer approaches to correct genetic diseases by gene repair.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-IMM-K (52))
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Johnson, David R
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Children's Hospital Boston
United States
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Weinacht, Katja G; Brauer, Patrick M; Felgentreff, Kerstin et al. (2012) The role of induced pluripotent stem cells in research and therapy of primary immunodeficiencies. Curr Opin Immunol 24:617-24
Pessach, Itai M; Notarangelo, Luigi D (2011) Gene therapy for primary immunodeficiencies: looking ahead, toward gene correction. J Allergy Clin Immunol 127:1344-50
Pessach, Itai M; Ordovas-Montanes, Jose; Zhang, Shen-Ying et al. (2011) Induced pluripotent stem cells: a novel frontier in the study of human primary immunodeficiencies. J Allergy Clin Immunol 127:1400-7.e4