This competing renewal application seeks funding for studies aimed at elucidating how and why mutations in the recombinase activating gene 1 (RAG1) or RAG2 cause a spectrum of immune phenotypes, including severe combined immune deficiency (SCID), Omenn syndrome (OS), leaky SCID (LS), and delayed onset combined immunodeficiency with granuloma and/or autoimmunity (CID-G/A). We propose to test the overall hypothesis that hypomorphic RAG mutations associated with distinct clinical phenotypes differentially shape composition of the T and B cell antigen receptor repertoire, and perturb NK cell phenotype and function. We will continue studies of the currently funded project and use gene editing to model faulty T cell differentiation and to develop an in vitro pre-clinical model o correction of human RAG1 deficiency using human induced pluripotent stem cells (iPSCs).
In Aim 1, we will test the disease-causing role of naturally occurring human RAG1 and RAG2 mutations, and analyze mechanisms of immune dysregulation in this disease. We will use next generation sequencing (NGS) to study the diversity and composition of the TCR and BCR repertoire in patients with diverse clinical phenotypes. We will characterize the spectrum and avidity of the autoantibodies produced, and we will use single cell cloning to study the frequency and specificity of circulating autoreactive B cells. To test the hypothesis that RAG mutations affect cellular fitness of NK cell progenitors, we will perform an extensive phenotypic and functional characterization of NK lymphocytes.
In Aim 2, in order to gain mechanistic insights into the immune dysregulation of RAG deficiency, we propose to characterize a new mouse model that we have generated, and that is homozygous for the Rag1 F971L mutation. The equivalent mutation in humans is associated with CID-G/A. We will analyze T, B and NK cell development and function, and mechanisms of immune dysregulation in this model, and results will be compared to those observed in Rag1S723C/S723C and Rag2R229Q/R229Q mice, which are models of LS and OS, respectively.
In Aim 3, we propose to model and correct RAG1 deficiency with iPSCs. We will use CRISPR/Cas9 to generate isogenic iPSCs that harbor different RAG1 mutations, and we will investigate the ability of these mutations to support in vitro T cell differentiation and generation of a diversified T cell receptor repertoire. We will alo investigate the ability of CRISPR/Cas9 gene editing approach to correct RAG1 mutations in patient-derived iPSCs and restore T cell differentiation in vitro. Overall, these studies will provide novel insights into the pathophysiology of human RAG deficiency and may provide the basis for future development of innovative forms of treatment based on gene editing.
This project aims at investigating the mechanisms underlying the diverse clinical presentation associated with genetic defects responsible for a severe congenital form of immunodeficiency. To this purpose, we will take advantage of our large repository of patient samples and a newly generated mouse model of the disease. We will use state-of-the art techniques to introduce or repair the genetic defect in human stem cells, and perform studies of disease modeling and correction. These studies may pave the way to novel and safer approaches to gene therapy.
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