Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: References located at the end of each chapter.

Wiskott-Aldrich Syndrome (WAS) is a rare X-linked primary immunodeficiency affecting approximately 1 in 100,000 live XY births in North America and is caused by a mutation to the WAS gene which is expressed across hematopoietic lineages. The WAS protein (WASp) plays a role in regulating actin polymerization. On a cellular level, there are a variety of effects of a lack of WASp or expression of a dysfunctional WASp protein for patients including issues with migration, adhesion, chemotactic response, phagocytosis, activation, and proliferation across different cell types in addition to reduced platelet size and output. This can lead to several systematic effects for the patients however because mutations to the WAS gene are not limited to one location or type there is a great amount of variability between patient symptoms making it challenging to diagnose. Major symptoms include frequent and recurrent infections, uncontrolled bleeding episodes, issues associated with autoimmunity, and malignancy, the most common form being lymphoma. Without treatment, the life expectancy of an individual diagnosed with WAS is 14 years of age, and the only curative treatment strategy available is hematopoietic stem and progenitor cell transfer (HSPCT). If not performed with an HLA-identical donor, which is available to less than 10% of patients, and within the first two years of life, the risk of graft versus host disease (GvHD) increases drastically for the patient. A gene therapy using autologous and genetically corrected CD34+ cells would be advantageous to the patients due to a reduction in preparative conditioning, immunosuppressive aftercare, and the risk of GvHD. CSL Behring is currently in the development of a lentiviral gene therapy to fulfill this gap in care, however, to develop the assays required to assess and characterize the drug substance usually an uncorrected patient sample is compared with a gene-edited sample. The limitation here is that due to the risk of infection and bleeding patient sample is very limited and therefore the development of a mock patient sample is necessary for early development. The goal of the project is to develop a WAS-KO protocol utilizing CRISPR/Cas9 and its characterization.

Thèse de doctorat : Bioingénierie : Nancy 1 : 2007. / Titre provenant de l'écran-titre.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 122-140).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 214-243).

Stem cells, with the ability to self-renew and differentiate into intended cell types, are a valuable tool for disease modeling and mechanistic study. CRISPR-Cas9 has been widely used for genome editing due to its high efficiency and convenience. However, CRISPR-Cas9 has large-deletion safety issues that dramatically restrict its applications. Wiskott-Aldrich syndrome (WAS) is an inborn immunological disorder caused by WASP deficiency. WASP functions in the nucleus, which may help to understand WAS pathology, are poorly defined. Pannexin 1 (PANX1) forms large plasma membrane pores to exchange intracellular small molecules with the extracellular environment and functions in inflammatory processes. The regulatory mechanisms of the PANX1 channel remain obscure. In this dissertation, I focused on mechanistic studies of CRISPR-Cas9 genome editing, and two immune disease relevant genes, WASP and PANX1 using stem cell-derived immune cells. We first found that CRISPR-induced large deletions (LDs) are predominantly mediated by the MMEJ repair pathway through statistical studies. Further, we found POLQ and RPA play vital roles in CRISPR-induced LDs. Modulation of POLQ and RPA can decrease CRISPR-induced LDs and increase HDR efficiency. Using three isogenic WAS iPSC models generated via gene editing, we successfully recapitulated WAS phenotypes, and for the first time, revealed that WASP regulates RNA splicing via epigenetically controlling the transcription of splicing factors and directly participating in the splicing machinery through a liquid-liquid phase separation process. We established a full-length human PANX1 (hPANX1) channel model via cryo-electron microscopy experiments and molecular dynamics simulation study, and found that hPANX1 channel is a homo-heptamer with both the N- and C-termini stretching deeply into the pore funnel. Functional studies of three selected residues support the new hPANX1 channel model and suggest the potential regulatory role of hPANX1 in pyroptosis upon immune responses. Overall, the mechanistic studies of WASP, PANX1 and CRISPR genome editing revealed new roles of WASP in regulating RNA splicing, new functional insights of PANX1 in pyroptosis, and uncovered two critical players POLQ and RPA in CRISPR-induced LDs.

The cytoskeleton is an intrinsic aspect of all cells, and is essential for many cellular events including cell motility, endocytosis, cell division and wound healing. Remodeling of the cytoskeleton in response to these cellular activities leads to significant alterations in the morphology of the cell. One such alteration is the formation of an actomyosin contractile array required for cytokinesis, wound healing and embryonic development. / Cellular structure and shape depends upon tensional prestress brought about by the organization of cytoskeletal components. Using the Xenopus laevis oocyte wound healing model, it is first described how diminished cellular tension affects the balance of the Rho family of GTPases, and subsequently prevents the formation of actomyosin contractile arrays. This suggests that cellular tension in the cell is not created at the level of the cytoskeletal elements but rather via the upstream signaling molecules: RhoA and Cdc42. / The role of N-WASP (Neural-Wiscott Aldrich Syndrome Protein), a mediator of Arp2/3 based actin polymerization, is next examined for its putative role in cellular wound healing. Xenopus laevis oocytes injected with mutant N-WASP constructs reveals in vivo evidence that functional N-WASP is required for appropriate contractile array formation and wound closure. / Lastly, it is revealed that the cellular structures involved with single cell wound healing in other model systems are also important for the initial repair of severed muscle cells. Actin, non-muscle myosin-II, microtubules, sarcomeric myosin and Cdc42 are all recruited and reorganized at the edge of damaged C2C12 myotubes. This data promotes the possibility that an actomyosin array may be established in injured muscle cells as well.

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En fonction du contexte génétique d’un individu et de son environnement, les mutations responsables de certaines maladies génétiques peuvent avoir des effets différents. Ces effets créent donc un besoin pour des traitements médicaux personnalisés. Ces traitements, pour être développés, demandent une meilleure compréhension de l’implication du contexte génétique au niveau moléculaire, incluant le mode d’action des gènes modificateurs. Pour mieux comprendre leur mode d’action, dans le cadre de mon projet, j’utilise un modèle du Syndrome de Wiskott-Aldrich chez la levure Saccharomyces cerevisiae impliquant le gène LAS17. Grâce à des expériences d’évolution expérimentale, des gènes ciblés par des mutations et, par la suite, une molécule ont été identifiés permettant de corriger le phénotype malade. Dans certains cas, cette correction est spécifique au contexte génétique et la majorité des gènes ciblés codent pour des partenaires d’interaction physique de Las17p. Une exception est la sous-unité Cnb1p de la calcineurine, la protéine ciblée par la cyclosporine A, qui ne fait pas partie de ces partenaires. La réponse à cette molécule est pourtant spécifique à un contexte génétique. J’ai utilisé différentes approches, dont des analyses QTL, pour identifier les gènes modificateurs sous-jacents à cette spécificité. Plusieurs locus ont été identifiés et la plupart incluaient des gènes au niveau du réseau d’interaction protéique ou génétique de Las17p et des sous-unités de la calcineurine. Le gène candidat principal, END3, un interactant physique de Las17p, semble présenter des effets de dosage au niveau protéique. Les liens avec la littérature suggèrent une importance dans la balance protéique de réseaux d’interaction à la fois du gène causant la maladie et celui ciblé par le traitement. L’ensemble des résultats du modèle soutiennent l’importance de tenir compte du contexte génétique pour élaborer de nouveaux traitements et suggèrent que les partenaires d’interactions devraient être les principales cibles de ces interventions. / Depending on the genetic background of an individual and their environment, the mutations responsible for some genetic diseases may have different effects. These effects create a need for personalized medical treatments. To be developed, these treatments require a better understanding of the implication of the genetic background at the molecular level, including the mode of action of modifier genes. To better understand their mode of action, as part of my project, I used a model of the Wiskott-Aldrich Syndrome in the yeast Saccharomyces cerevisiae involving the LAS17 gene. Through experimental evolution experiments, several genes targeted by mutations and, subsequently, a molecule have been identified to rescue the diseased phenotype in yeast. In some cases, this phenotypic rescue was specific to the genetic context and the majority of the targeted genes coded for Las17p physical interaction partners. An exception was the Cnb1p subunit of calcineurin, the protein targeted by cyclosporin A, which was not one of these partners. The response to this molecule was nevertheless specific to a genetic background. I used different approaches, including QTL analysis, to identify modifier genes underlying this specificity. Several loci were identified and most of them included genes in the protein or genetic interaction networks of Las17p and of calcineurin subunits. The main candidate gene, END3, a physical interactant of Las17p, appeared to exhibit protein-level effects. The literature suggests an importance in the protein balance of the interaction networks of both the gene causing the disease and the one targeted by the treatment. The overall model results support the importance of considering the genetic background for developing new treatments and suggest that interaction partners should be primary targets for these interventions.