Date of Graduation

5-2024

Document Type

Thesis

Degree Name

Bachelor of Science in Biomedical Engineering

Degree Level

Undergraduate

Department

Biomedical Engineering

Advisor/Mentor

Nelson, Christopher

Abstract

Gene therapies are emerging as powerful tools for treating genetic diseases and cancers, offering the potential for a permanent cure. Hemophilia B, affecting approximately 6,000 men in the U.S., results from a deficiency in coagulation factor IX protein (FIX), which is crucial for blood clotting. A substantial portion of patients, over 40%, suffer from severe hemophilia B, experiencing spontaneous, prolonged bleeding. Current prophylactic treatments involve frequent coagulation factor infusions, yet a curative approach would alleviate this burden. Gene editing technologies like CRISPR-Cas systems show promise for correcting genetic mutations, but the diverse nature of factor IX gene (F9) mutations poses challenges for a universal therapy. An alternative approach involves delivering a complete F9, eliminating the need for personalized treatments. However, existing therapies like Hemgenix, which delivers a modified F9 gene to the liver, face limitations such as immune reactions, liver health restrictions, and the need for high intravenous doses. This study investigates skeletal muscle (SM) as a promising site for gene therapy delivery, given its regenerative nature and ability to express proteins essential for FIX processing. Three promoters—cytomegalovirus (CMV), creatine kinase (CK8), and a synthetic muscle-specific promoter (SPc5-12)—were examined for their efficacy in driving F9 expression in SM. Furthermore, two F9 variants, Padua (R338L) and K5A, which increase bioactivity and enhance exportation respectively, were studied. We transfected plasmids containing these promoters and mutations into mouse myoblast cells (C2C12) for analysis. Results from RNA analysis revealed significantly higher F9 gene expression under the CK8 promoter compared to CMV and SPc5-12 promoters. F9 variants did not show significant differences in expression. The study also compared the expression of all plasmid constructs, showing a trend of higher expression with CK8 plasmids. This research provides valuable insights into optimizing non-endogenous protein production in SM, laying a foundation for future gene therapies utilizing SM as a protein factory.

Keywords

Gene therapy; genetic promoters; hemophilia B; factor IX; protein replacement therapy

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Available for download on Saturday, April 25, 2026

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