Date of Graduation
5-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Cell & Molecular Biology (PhD)
Degree Level
Graduate
Department
Cell & Molecular Biology
Advisor/Mentor
Nelson, Christopher E.
Committee Member
Wolchock, Jeffrey C.
Second Committee Member
Murach, Kevin A.
Third Committee Member
Balachandran, Kartik
Keywords
CRISPR; endogenous-promoter; gene therapy; next generation sequencing; skeletal muscle; targeted integration
Abstract
This dissertation presents the development and evaluation of a CRISPR/Cas9-based platform for targeted gene integration as a therapeutic strategy for loss-of-protein diseases. By repurposing highly expressed muscle loci, such as Ckm, this platform demonstrates potential for stable therapeutic RNA expression. In vitro and in vivo studies were conducted to validate its efficiency and assess the safety of the selected integration sites, with particular focus on the effects of double-strand break (DSB)-mediated targeted gene insertion.
The first aim establishes proof-of-concept for the targeted integration of therapeutic genes into high-expression muscle loci using Homology-Independent Targeted Integration (HITI), achieving high RNA expression with minimal adverse effects on the global transcriptome. The second aim examines the transcriptomic consequences of HITI-mediated gene editing, using short-read and long-read sequencing to evaluate integration precision, transcript diversity, RNA modifications, and unintended integration events. The third aim applies similar orthogonal short- and long-read sequencing approaches to investigate Duchenne Muscular Dystrophy (DMD), the most common genetic disorder. In this section, we investigate the genomic and transcriptomic safety of AAV-CRISPR delivery for DMD therapy, identifying complex structural variations and unexpected AAV insertions that might be overlooked by a single sequencing method.
Collectively, these studies demonstrate the potential and challenges in developing targeted integration mediated by CRISPR-based therapeutics and highlight the value of comprehensive sequencing methodologies to advance gene editing safety and reliability.
Citation
Padmaswari, M. (2025). Repurposing High-Expressing Muscle Loci with CRISPR Gene Editing: A Multi-Platform Analysis at Efficiency and Safety. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/5645
