| Description |
HSPB5 (aka αB-crystallin or CRYAB) is a small molecular weight heat shock protein that functions as a key chaperone in striated muscle. Mutations in HSPB5 are linked with human disease including cardiomyopathy, skeletal myopathy, and cataracts. Abnormal accumulation or protein aggregation including the mutant form of HSPB5 in muscle is a hallmark of the majority of known disease-associated HSPB5 variants, though it is yet unclear mechanistically how this mutant chaperone contributes to myopathy (reviewed in Chapter 1). This dissertation focuses on molecular studies of one such mutation, 343delT, which is associated with severe early-onset skeletal myopathy requiring ventilation to sustain life. Induced pluripotent stem cells (iPSCs) derived from the patient harboring the 343delT mutation along with genome edited isogenic control cell lines are utilized in this work as a mechanism to study the endogenous form of the protein in cell types of interest (i.e., skeletal and cardiac muscle). Molecular studies of 343delT HSPB5 demonstrate extreme insolubility of the mutant protein and suggest a loss of function mechanism for disease, though gain-of-toxic function cannot be excluded (Chapter 3). Chapter 2 is included as an addendum to the Introduction on HSPB5 (Chapter 1) to provide relevant background information on iPSCs and genome editing. The fast-paced genome editing field is currently hampered by inefficient means of isolating cells containing modifications of interest. Previously published approaches employed in Chapter 3 were highly efficient, though required a two-step editing process and were not readily scalable. Chapter 4 presents a strategy for genome editing, termed cotargeting with selection (CTS) that involves simultaneous targeting of two loci, where selection for incorporation of a selection cassette into a safe-harbor locus enriches many fold for a separate modification at a gene of interest. CTS streamlines the genome editing process compared with previous techniques. Chapter 5 of this dissertation includes a combined discussion and Chapter 6 presents future experiments planned for the study of 343delT HSPB5. Altogether, this dissertation affords molecular insights into myopathy causing 343delT HSPB5 using cutting edge technology of iPSCs and genome editing, as well as providing technical advancement to the field of genome editing. |
