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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.

<p>  </p> <p>Cancer immunotherapies have revolutionized anticancer treatment, saving lives by utilizing patients’ immune systems. Immunogenic cell death inducing chemotherapies have recently gained interest as they can not only inhibit the growth of the tumor but also allows the patient to develop a long-lasting immune response to the tumor. However, due to the poor retention of chemotherapies in the tumor and immunosuppressive tumor microenvironment, the activity of immunogenic cell death inducing chemotherapy is limited. To overcome the limitations, I have developed immunofunctional poly(lactic-co-glycolic acid) nanoparticles to enhance the retention of immunogenic cell death inducers at the tumor and increase the recruitment of antigen-presenting cells to the tumor.</p> <p><br></p> <p>In our study, paclitaxel and carfilzomib were determined as immunogenic cell death inducers, supported by in vitro screening of damage-associated molecular patterns and in vivo vaccination study. Both drugs were identified as immunogenic cell death inducing chemotherapy agents. To deliver immunogenic cell death inducers, immunofunctional poly(lactic-co-glycolic acid) nanoparticles were developed by modifying the surface with adenosine triphosphate. The coating of adenosine triphosphate attracted dendritic cells in a concentration gradient manner and improved the stability of adenosine triphosphate against its degrading enzyme. Both paclitaxel and carfilzomib were successfully encapsulated into the developed nanoparticle formulation. Paclitaxel encapsulated nanoparticles were chosen as a lead candidate due to the inherent immunotoxicity of carfilzomib.</p> <p>Paclitaxel encapsulated nanoparticles coated with ATP effectively suppressed tumor growth in CT26 murine carcinoma and B16F10 murine melanoma. The formulation also increased the immune cell infiltration into the tumor, which may explain the enhanced efficacy of the nanoparticle formulation. Combinational therapy of nanoparticles with anti-PD-1 antibodies significantly increased the complete regression rate in tumor-bearing mice by invigorating the immunosuppressive environment. </p> <p><br></p> <p>In summary, paclitaxel (an immunogenic cell death inducer) encapsulated in adenosine triphosphate-coated poly(lactic-co-glycolic acid) nanoparticles attracted dendritic cells in a concentration gradient manner and effectively suppressed tumor. Additional anti-PD-1 antibodies further improved the antitumor effect, inducing complete tumor regression in 75% of CT26-bearing mice, by inhibiting the interactions between T cells and immunosuppressive cells (regulatory T cells and myeloid-derived suppressor cells). </p> <p><br></p> <p>Chapter 1 discusses the current understanding of immunotherapy and delivery systems to enhance immunotherapy. Chapter 2 describes the determination of immunogenic cell death inducers and the development of immunofunctional nanocarrier. The in vivo antitumor efficacy of the nanocarrier was tested in Chapter 3. </p>

Glioblastoma (GBM) remains the most aggressive and prevalent malignant primary brain tumor in adults. Unchanged since 2005, standard of care (SoC) consists of surgical resection, followed by radiation therapy (RT) with concurrent and adjuvant chemotherapy with temozolomide (TMZ). Despite these therapeutic efforts, patients succumb to recurrent disease with a median overall survival of 14.6 months and a five-year survival rate of 5.5-6.8%. Therapeutic failure is largely explained by ITH and the presence of treatment-resistant GBM stem-like cells (GSCs). Given the lack of understanding of recurrent GBM and absence of second line therapies patients, I hypothesize that genome-scale functional genetic interrogation will unravel recurrent GBM-specific tumor biology and inform development of novel therapeutics. First, I compared primary and recurrent GBM at the genetic, transcriptomic, proteomic and functional genetic levels. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 repress axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and exploit a genetic dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. Given the genetic dependency on ROBO signaling and enrichment of ROBO1 expression in GBM tissues, I undertook a campaign to evaluate ROBO1 as a therapeutic target in recurrent GBM and develop anti-ROBO1 chimeric antigen receptor T (CAR-T) cells using camelid single-domain antibodies targeting human ROBO1. I optimized the design of anti-ROBO1 CAR-T cells and tested the anti-tumor activity of these modalities in in vitro using patient-derived recurrent GBM lines and orthotopic patient-derived xenograft models. I present data to expand the repertoire of GBM-enriched antigens suitable for effective CAR-T cell therapy. Given that resistance to SoC and disease relapse are inevitable for GBM patients, pre-clinical and clinical advancement of immunotherapeutic modalities, combined with recent insights into the tumor immune microenvironment, are poised to improve clinical outcomes for this patient population. / Thesis / Doctor of Philosophy (PhD) / Glioblastoma remains the most lethal and prevalent primary brain tumor in adults. Standard of care for patients remains unchanged since 2005, consisting of surgery to remove visible tumor at diagnosis (primary tumor), followed by radiation therapy and chemotherapy to treat remaining tumor cells. Despite these therapeutic efforts, tumor relapse (recurrent tumor) is inevitable with no standardized second-line therapy. Patients succumb to recurrent disease with a median overall survival of 14.6 months and only 5.5-6.8% of patients survive five years post diagnosis. Therapy failure and tumor relapse are explained by immense diversity among tumor cells at the DNA and protein levels, giving rise to a subset of tumor cells with abilities to resist therapy and seed the recurrent tumor. Previous studies have presented evolution of tumor cells through therapy, with recurrent tumor cells harboring novel changes at the DNA and protein levels. However, the impact of these changes on tumor cell function has not been evaluated. In this thesis, we developed and applied a genetic screening technique to determine the functional role of thousands of genes in primary and recurrent tumor cells from the same patient. This analysis revealed numerous genes that exhibit differential effects on survival of primary and recurrent tumor cells, including genes that drive recurrent tumor cell growth but are dispensable in primary tumor cells. Functional remodeling of these genes and pathways revealed a new functional role of multiple proteins belonging to a process called axonal guidance in recurrent tumor cells. To evaluate the therapeutic potential of these findings, we deeply interrogated the mechanism by which axonal guidance drives recurrent tumor cells and targeted crucial molecular players using chemical and immunological therapies. Using models that predict clinical effectiveness, we engineered and tested a novel therapy that redirects immune cells to target recurrent tumor cells driven by dysfunctional axonal guidance activity. The goal of this thesis was to discover the functional differences between primary and recurrent tumor cells, thereby leveraging this information to engineer candidate therapies for treatment of recurrent glioblastoma.