Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Lek, Monkol
Abstract
The journey towards finding a cure for rare disease patients is hindered by two significant obstacles: the challenge of obtaining a definitive diagnosis and the difficulty in accessing effective treatment. This dissertation research aims to address both issues in rare muscle diseases. Firstly, a highly adaptable workflow called Saturation Mutagenesis-Reinforced Functional assays (SMuRF) was developed to enhance variant interpretation and aid in clinical decision-making. To validate its utility, SMuRF was applied to a group of rare diseases called dystroglycanopathies, which are caused by mutations in enzymes involved in alpha-dystroglycan glycosylation, including FKRP and LARGE1. Over 99.9% of all possible single nucleotide variants (SNVs) of these two enzymes were evaluated. SMuRF recapitulated and significantly expanded the knowledge gained from clinical reports and population databases, which aided in definitive variant classification. SMuRF also expanded the training datasets of the computational predictors, further improving their capability. Lastly, SMuRF highlighted the critical regions in the enzyme structure which shed light on different disease mechanisms associated with the same enzyme and provided a reference for treatment decisions. Secondly, a gene therapy was customized for a Duchenne Muscular Dystrophy (DMD) patient with dystrophin muscle isoform exon1 deletion, employing a strategy called Substitute Isoform Rescue (SIR). SIR utilized redundancy present in genomes for therapeutic purposes. Specifically, the SIR approach involved up-regulating the cortical isoform of dystrophin, a nearly identical substitute for the muscle isoform, using a CRISPR-activation (CRISPRa) construct. The efficacy of this therapeutic strategy was demonstrated in pre-clinical experiments in human model cell lines, patient-specific cell lines, and a hDMD/mdx2 mouse model. The U.S. Food and Drug Administration (FDA) issued a “Safe to Proceed” letter for the Investigational New Drug (IND) application for AAV9-mediated delivery of the construct, enabling an n-of-1 clinical trial (NCT05514249) at UMass Medical School (UMMS). Tragically, the patient passed away 8 days after the AAV infusion. The family consented for an extensive post-mortem analysis to be performed and shared, the results of which suggested the adverse events were attributed to the patient's advanced disease condition and the reaction to high-dose AAV9, rather than the expression of the CRISPRa construct. The knowledge gained from this post-mortem analysis will help design improved high dose gene therapy trials in older patients and patients with more advanced conditions. Ongoing DMD gene therapy trials led by pharmaceutical companies are not available to every patient because of the strict inclusion/exclusion criteria. Despite the unexpected outcome, our exploration of the non-traditional pathway instilled hope within the patient community, demonstrating the feasibility and accessibility of rapid and precise therapy development. In conclusion, this thesis centers on two pivotal facets aimed at expediting the pursuit of cures for rare muscle diseases: the improvement of diagnostics and the exploration of new pathways for treatment development. This dissertation introduces an innovative workflow for developing simple and cost-effective variant interpretation methods that strive for greater accessibility across laboratories in the field. Additionally, a comprehensive account of an individualized gene therapy development process is presented. These contributions offer insights that may hold value for the entire rare disease community and make a modest addition to the existing body of work in this domain. Meanwhile, the projects outlined in this dissertation also inspire a set of thought-provoking questions that motivate further investigation. Potential avenues for unraveling these questions were also proposed in this thesis, and they await further exploration in future research.
Recommended Citation
Ma, Kaiyue, "Improving Genetic Diagnostics and Developing Gene Therapies in Rare Muscle Diseases" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1205.
https://elischolar.library.yale.edu/gsas_dissertations/1205
