Clinical and translational studies in in-vitro and an in-vivo mouse model of a unique HSPB8 associated vacuolar myopathy

Topic:

Pre-Clinical Research

Poster Number: T405

Author(s):

Lan Weiss, PhD, UCI, Alyaa Shmara, MBCHB, UCI, Pallabi Pal, PhD, University of California Irvine, Barbara Tedesco, PhD, University of Milan, Italy, Hai Zhang, PhD, University of California, Irvine, Philip Farahat, BS, University of California, Irvine, Victoria Boock, University of California, Irvine, Armando Villalta, PhD, University of California, Irvine, Xiangmin Xu, PhD, University of California, Irvine, Angelo Poletti, PhD, University of Milan, Italy, Virginia Kimonis, UC Irvine

Background
HSPB8 associated autosomal dominant rimmed vacuolar myopathy is caused mainly by frameshift (fs) mutations. Patients develop distal myopathy in their thirties, with progressive generalized weakness. Muscle biopsies show fatty replacement, fibrosis, rimmed vacuoles, and aggregates. We have shown that the mutant HSPB8 protein causes increased aggregation and secondary loss of activity of the normal HSPB8.
Objectives
We propose that microRNAs against the toxic HSPB8 mutation in addition to replacement of total endogenous HSPB8 is a logical therapeutic approach to ameliorate muscle weakness and pathology.
Methods
We have generated stem cells from patient skin fibroblasts and transformed them into myoblasts to study the pathogenesis of the disease, and for translational studies. We found reduced HSPB8 protein, increased TDP-43, increased autophagy, and aggregations in patient fibroblasts. Using CRISPR technology, a knock-in Hspb8 mouse model was made of the c.515dupC fs variant. The mice showed muscle weakness starting at 6 months of age. Muscle pathology revealed central nuclei, muscle degeneration, fatty replacement, increased TDP-43, autophagy pathology, and amyloid fibril aggregates recapitulating the clinical phenotype.
Results
For our proof of principle gene therapy delivery studies, we have (1) transfected patient iPSC-derived myoblasts with wild-type HSPB8 and found the increase in HSPB8 protein as well as improved TDP-43 pathology; (2) dosed Hspb8 c515/+ mice with myotropic AAVMYO-CAG-EGFP by systemic delivery to check muscle tropism. Results showed strong expression of the AAVmyo at 1E+12vg dose in muscle vs. AAV9-CAG-EGFP control. Currently, the Hspb8 c515/+ mice are being treated with the AAVMYO-CBH-hHSPB8 construct that incorporates the normal Hspb8. Muscle specific promoters (CK8 and MHCK7) are also tested to select the most potent promoter.
Conclusion
We have demonstrated that our preclinical models recapitulate pathology of patients and gene therapy show good muscle penetration with AAVMYO. These studies pave the way for treatment of the animals with the best combination of microRNA and/or normal Hspb8 constructs using AAVMYO.