Date of Graduation

5-2014

Document Type

Dissertation (PhD)

Program Affiliation

Human and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Brian R. Davis, PhD

Committee Member

Phillip Carpenter, PhD

Committee Member

Laurence J. N. Cooper, MD, PhD

Committee Member

Gilbert Cote, PhD

Committee Member

Patrick Zweidler-McKay, MD, PhD

Abstract

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease characterized by thrombocytopenia, recurrent infections and increased autoimmunity. This disease is caused by mutations in the WAS gene (WAS) which encodes for the WAS protein (WASp), exclusively expressed in hematopoietic cells and required for proper platelet production and lymphoid cell function. Approximately 11% of patients with WAS exhibit a phenomenon called Somatic Revertant Mosaicism which is characterized by the presence of lymphocytes which naturally revert back to normal phenotype by restoring WASp expression. To date, the mechanisms of this naturally-occurring gene therapy remains poorly understood, and the full extent of the repertoire of revertant genotypes has not yet been elucidated. The remaining 89% of WAS patients require treatment early in life, or the disease leads to premature death. At present, cure for WAS can only be attained through allogeneic stem cell transplantation or lentiviral hematopoietic stem cell gene therapy.

In this work, we focused on both groups of WAS patients. In order to gain insight into the extent of the revertant repertoire in WAS revertant patients, we used next generation sequencing technology to analyze DNA from WASp+ T-cells isolated from two revertant patients carrying the same germline mutation. We identified over a hundred different revertant genotypes. In this report we describe those which directly reverted the original germline mutation, as well as those which are believed to provide a compensatory effect by inducing alternative splicing. Our findings represent, to our knowledge, the first report of ultra-deep analysis of somatic reversions in WAS patients.

In our study of non-revertant WAS patients, we investigated restoration of T- and NK-cell functionality following an in vitro virus-free zinc-finger nuclease (ZFN)-mediated genome editing strategy for correction of WAS muations. We generated induced pluripotent stem cells (iPSC) from skin fibroblasts of a WAS patient carrying an insertional frame-shift mutation. Subsequently, a WAS-2A-eGFP transgene was targeted at the endogenous chromosomal location by homology-directed repair using ZFN, thereby correcting the gene defect and creating a GFP reporter for WASp expression. Hematopoietic progenitor cells were generated from WAS iPSC and gene-corrected iPSC (cWAS) in vitro via spin embryoid bodies. Human embryonic stem cell lines WA01 and WA09 were used as control. GFP expression was pronounced in all CD43+ hematopoietic lineages including myeloid, monocytic, lymphoid, erythroid and megakaryocytic lineages. Hematopoietic precursors were further cultured on OP9-DL1 to generate NK cells. NK cells were readily obtained from cWAS and WA01/WA09 progenitors, but to a far more limited extent from WAS progenitors. WAS-derived NK cells were unable to generate interferon-γ or tumor necrosis factor-α upon stimulation with K562. Cytokine production was restored in cWAS-derived NK cells.

Taken together these results indicate that targeted endogenous integration of the WAS gene in WAS-iPSC results in restoration of the lymphoid defect observed in WAS-iPSC. Transplantation of gene-corrected iPSC-derived hematopoietic precursors may offer an alternative to lentiviral gene therapy which carries an inherent risk for insertional oncogenesis.

Keywords

Wiskott-Aldrich Syndrome, somatic reversion, genome editing, iPSC, hematopoietic progenitors, targeted endogenous integration, next-generation sequencing

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