Investigating Modified Acid-α-Glucosidase Histidine 201 Variants for Enhanced Pompe Disease Gene Therapy

Topic:

Pre-Clinical Research

Poster Number: LB462

Author(s):

Prasad Trivedi, MS, University of Florida, Madhurima Saha, PhD, University of Florida, Denise Cloutier, University of Florida, Radhika Bhake, MS, University of Florida, Matthieu Colpaert, PhD, University of Florida, Shandra Trantham, PhD, University of Florida, Ramon Sun, PhD, University of Florida, Matthew Gentry, PhD, University of Florida, Manuela Corti, PhD, University of Florida, Barry Byrne, MD, PhD, University of Florida

Hypothesis: Modifying the acid-α-glucosidase (GAA) gene at histidine 201 (H201) with hydrophobic (isoleucine, leucine) and amphipathic (tyrosine, serine) residues would improve lysosomal processing and increase enzymatic activity, enhancing Pompe disease gene therapy efficacy.
Methods: To test our hypothesis, we generated various GAA variants by substituting the wild-type H201 with specific amino acid residues. Subsequently, we transfected multiple immortalized cell lines (HEK293T, C2C12, Neuro2A, HepG2) with CMV-driven-H201 variant plasmids per 1 x 10^6 cells. After a 48-hour incubation period, we assessed acid-α-glucosidase protein expression and enzymatic activity using anti-GAA western blot assay, glucose content (Glc-Glo) assay, and GAA enzyme activity assay. Results were compared to wild-type H201.
Results: Our findings revealed notable quantitative and enzymatic activity differences between the engineered H201 variants (I/L/Y/S) and the wild type. The H201 variants exhibited higher basal expression levels and comparable enzymatic activity to the wild-type GAA gene. Intriguingly, the amino acid Serine at 201 plays a crucial role in converting from the 76-kDa to the 70-kDa form of GAA. We observed a significant increase in glucose content in cells expressing H201S compared to the wild type. Western blot revealed a distinct 76kDa band for a more efficient GAA enzyme.
Conclusion: Our study shows enhanced enzymatic activity in engineered H201 variants, with H201S being notably potent. These findings support their potential in improving Pompe disease gene therapy through sustained GAA activity. Next steps include testing in patient-derived fibroblasts and packaging optimized variants into AAV vectors for in vivo analysis of long-term GAA activity in key tissues.