Colin Ross

Anthracyclines (e.g., doxorubicin) are effective chemotherapeutics used to treat a broad-spectrum of childhood and adult cancers. However, the clinical utility of anthracyclines is considerably limited by anthracycline-induced cardiotoxicity (ACT). ACT remains a significant burden in the clinic, where >50% of patients receiving anthracyclines experience some degree of ACT, initially presenting as left ventricular dysfunction, and up to 16% of patients develop heart failure. Advancements in patient genetics show substantial promise as a predictive measure of ACT. The gene variants RARG-rs2229774, UGT1A6-rs17863783, and SLC28A3-rs7853758 have the strongest evidence for association with ACT in anthracycline-treated patients. First, this thesis aimed to understand the scope of genetics, focusing on the proposed roles of genes RARG, UGT1A6, and SLC28A3 in the development and prevention of ACT. Second, I investigated the roles of UGT1A6 and RARG in the development of ACT using cellular models. The research replicated the ACT susceptibility of the UGT1A6 locus using a cellular model (HEK293) of doxorubicin-induced cytotoxicity. Further, I identified that ligand-mediated activation of RARG, using all-trans retinoic acid (ATRA) and CD1530, significantly increases the cell viability of H9c2 cardiomyoblasts. Lastly, I evaluated the efficacy of ATRA in preventing the development of cardiotoxicity using a human cardiomyocyte and a mouse model of ACT. In human cardiomyocytes, ATRA enhanced cell survival during doxorubicin exposure. The protective effect of ATRA was also observed in a mouse model (B6C3F1/J) of ACT, in which ATRA treatment improved heart function of doxorubicin-treated mice. Histological analyses also indicated that ATRA treatment reduced the pathology associated with ACT. Further, in human cardiomyocytes, ATRA induces the gene expression of retinoic acid receptors (RARG, RARB), while repressing topoisomerase-II enzyme genes (TOP2A, TOP2B), which encode for the molecular targets of anthracyclines, and repressed downstream ACT response genes. This mechanism partially explains the protective effect of ATRA and why patients presenting with dysregulation of this pathway may be susceptible to ACT. Overall, the work performed in this thesis shows that gene variants that modify ACT risk belong to critical biological pathways of ACT. Further, these molecular mechanisms also provide a unique opportunity to develop safe and effective interventions to mitigate ACT.

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Chronic primary systemic vasculitis (PSV) describes a diverse group of debilitating and potentially life-threatening diseases, characterized by inflammation of blood vessels within various organs such as the kidneys, lungs, brain, eyes, and skin. Subtypes of the small- to medium-sized vessel vasculitides are particularly challenging to classify due to many overlapping clinical symptoms. Their differentiation is important, however, as there is evidence that different subtypes benefit from different treatment approaches. The rarity of vasculitis in children has limited pediatric-specific PSV studies and the clinical approach to pediatric PSV is adapted primarily from adult studies. Not surprisingly, adult-derived classification criteria are imperfect for children and consequently, fail to classify up to two thirds of children with small- to medium-vessel PSV. The objective of this dissertation is to identify genetic markers and select, circulating biomarkers to better our understanding and clinical approach to managing the disease and to improve outcomes of children with chronic PSV. Using biological samples and clinical metadata from a worldwide cohort of children with chronic PSV, this dissertation reports (1) genetic associations specific to pediatric autoimmune vasculitis through employing a genome-wide association study; (2) a high prevalence of autoantibodies to lysosome associated membrane protein-2 in pediatric PSV that correlate to vasculitis-associated kidney dysfunction; and (3) the identification of nine patients, originally diagnosed with chronic PSV, harbouring novel and/or rare variants in adenosine deaminase 2 (ADA2) – these genetic data alongside having abrogated ADA2 enzyme activity have led to their reclassification as having a new monogenic form of vasculitis, deficiency of adenosine deaminase 2. This dissertation reports the first large-scale genotype and biomarker study of primary vasculitis focusing solely on pediatric cases. Improved classification is critical for timely therapeutic intervention, for identification of appropriately classified children for clinical trials, and for research, all of which will improve our understanding of the disease and the quality of life of children suffering from chronic PSV.

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Genetic diseases are a leading cause of death and disability worldwide with immense economic and societal burdens. However, currently fewer than 5% of genetic diseases have approved treatments, often with limited therapeutic benefit to patients. Genome editing provides a new therapeutic strategy to treat genetic diseases. In the last few years, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has emerged as a promising gene editing approach due to its ease in design and flexibility to target different genetic diseases. One factor limiting its broader application is that it will introduce double-stranded deoxyribonucleic acid (DNA) breaks (DSBs). New advances in the CRISPR/Cas9 system are “base” and “prime” editing, which do not generate DSBs. Base editing has shown great potential to precisely repair the majority of pathogenic point mutations in the patient’s DNA. Prime editing, while currently less efficient, can introduce a broader range of possible edits. However, there remains a major challenge: delivery of genome editors to the affected tissues.To begin to address this challenge, two reporter gene mouse models were developed. Both models introduce a point mutation to knockout the reporter gene activity. Upon gene editing, the mutation is corrected back to the wildtype sequence, thereby restoring the reporter gene activity. The efficiency of gene editing can be visualized and measured with an in vivo whole animal imaging system, which permits repeated and long-term assessments of gene editing. To validate both mouse models, base editor mRNA and a mutation-specific single guide RNA (sgRNA) were encapsulated with lipid nanoparticles (LNPs) and intravenously administered into the reporter mice. This resulted in 83.5% restoration of wildtype reporter gene activity in the liver. One major application of these mouse models will be to evaluate and optimize gene editor delivery systems targeting various tissues. As an example, intramuscular administration of LNPs encapsulated with base editing components led to significant genome editing in the injected muscle, which could be readily visualized in the live imager. These novel reporter gene mouse models will provide critical tools for the in vivo evaluation and optimization of genome editor delivery and improved genome editor formulations.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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There are currently no effective treatments for 95% of the over 7,000 known genetic diseases [1, 2]. The use of CRISPR/Cas9 has recently emerged as a powerful approach to directly repair disease-causing mutations [3-8]. The goal of this project was to optimize a novel CRISPR/Cas9 ‘base editing’ approach to treat genetic diseases by specifically repairing the disease-causing mutation in situ in the patients’ DNA. This technology uses a fusion of a partially deactivated Cas9 (nCas9) protein coupled with a nucleotide modifying enzyme to achieve 15-30% editing, and in some cases up to 75% editing in vitro [9-13]. Two-thirds of all genetic diseases are caused by point mutations [1] and base editors have now been developed that can target more than 68% of all disease-causing point mutations [8]. Despite the growing potential of base editing, limitations in the ability to quantify gene editing quickly and precisely have resulted in a lack of robust high throughput studies to compare and optimize base editors. We hypothesized that developing base editor reporter model systems would enable us to efficiently test and optimize the safety and effectiveness of base editing. To this end, we successfully developed both in vitro and in vivo reporter models and utilized these models to test the safety, delivery, and effectiveness of base editing. This series of novel model systems address a critical gap in the field of CRISPR/Cas9 gene editing. Using these, we were able to significantly increase the efficiency of base editing in vitro. We were also able to compare the safety of different base editors using a new application of my in vitro reporter model. We also demonstrated the therapeutic feasibility of our base editing approach in vitro to repair three prevalent mutations that cause a debilitating genetic disease: Lipoprotein Lipase Deficiency. Lastly, using the in vivo reporter model, we were able to demonstrate sustained correction and efficient delivery of base editors using lipid nanoparticles. Overall, these experiments have demonstrated critical steps towards the implementation of new treatment options for patients with a wide variety of genetic diseases.

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