Grant Duration
09/22 – 12/25

  1. Characterization of MMex38 Mice and 4-PBA Dosing Paradigms

The project began by establishing the MMex38 murine model (harboring the DYSFL1360P mutation that is synonymous with the human DYSF L1341P pathogenic mutation) as a faithful representation of human dysferlinopathy and to identify histological and physiological endpoints that could be used in downstream experiments. Longitudinal aging studies (3–24 months) identified significant psoas, gluteus, and quadriceps muscle loss beginning at 12 months, which correlated with increased central nuclei, fat infiltration, fibrosis, and functional deficits on the Inclined Balance Beam (IBB) assay.

Therapeutic evaluation of 4-PBA was assessed by several dosing paradigms:

  • Long-Term Longitudinal Study (2.0 mg/mL): One year of treatment (starting at 2 months of age) significantly improved dystrophic phenotypes, reduced fat infiltration in the quadriceps and gluteus, increased psoas weight by 39%, and improved performance on the IBB assay. However, these physical and performance gains were observed only in female mice. This gender disparity may be caused by slower metabolism of 4-PBA in females, leading to greater persistence of the drug and its metabolite, phenyl acetate (PA), in the blood. While IHC confirmed sarcolemmal restoration of dysferlin protein in some of the muscles assessed, levels did not reach WT equivalence. Despite these minimal protein findings, functional membrane repair in EDL muscles was restored to wild-type levels in females, while males showed only partial restoration. Since 10% of wild-type expression has been shown to be sufficient to prevent the dystrophic phenotype in hypomorph models, the minimal dysferlin protein levels observed may have been enough to produce the functional rescue observed.
  • Dose-Protein Relationship and Mechanism: To better determine if therapeutic protein expression could be achieved in humans using approved 4-PBA doses and determine the relationship between dose and about of dysferlin protein produced, a range of concentrations was explored. Single-dose oral gavage was tested at 250, 500, and 1000 mg/kg; a 2000 mg/kg dose was found to be toxic due to sodium overload. IHC detected sarcolemmal dysferlin protein only at the 1000 mg/kg dose in the tibialis anterior at 24 hours, but expression remained undetectable by Western blot in all muscle groups.  These findings indicate that molecular restoration of dysferlin protein expression and localization is not readily detectable following a single dose of 4-PBA in MMex38 mice. Factors such as drug stability, protein turnover, and the rate of protein accumulation may limit detection under these conditions.
  • Short-Term Continuous Dosing (16 mg/mL): In a two-week study, continuous administration of 16 mg/mL 4-PBA achieved consistent sarcolemmal localization across all muscles and restored membrane repair kinetics to wild-type levels. However, dysferlin protein levels remained below the detection threshold for Western blot analysis, emphasizing that functional and IHC assessments maybe be more sensitive indicators of drug efficacy than total protein quantification.
  1. In Vitro Evaluation of 26 Pathogenic DYSF Missense Mutations

The project also assessed the functional rescue of 4-PBA across 26 pathogenic DYSF missense mutations (DYSFPMMs) using immortalized dysferlin-deficient GREG myoblasts. Stable expression was driven via a doxycycline-regulated Tet-on/TRE system, and differentiated myotubes were treated with 1 mM 4-PBA. Using laser-induced damage assays, mutations were stratified into three functional categories:

  • Category I (15 mutations): Variants exhibiting partial baseline repair that 4-PBA significantly enhanced to WT or near-WT levels.
  • Category II (3 mutations): Variants failing to support repair at baseline but achieving full restoration of repair kinetics following treatment.
  • Category III (8 mutations): Variants that displayed improved repair upon overexpression but showed minimal additional benefit from 4-PBA.

In total, 4-PBA enhanced functional repair in 18 of 26 DYSF missense mutations. These results confirm that 4-PBA acts as a broadly applicable chaperone that enhances the functional pool of dysferlin protein.

  1. Investigation of Monocytes and Macrophages as Biomarkers

This aim investigated whether circulating monocytes/macrophages could serve as minimally invasive, quantitative biomarkers for dysferlin protein restoration to support longitudinal monitoring in both mice and humans.

  • Murine Findings: Dysferlin protein expression was not detectable in mouse PBMCs, Ly6C+ monocytes, or the J774A.1 mouse macrophage line using Western blot or flow cytometry. Consequently, murine efficacy assessments of 4PBA still necessitate postmortem muscle tissue analysis.
  • Human Studies: Conversely, human THP-1 cells demonstrated robust dysferlin expression specifically following differentiation into macrophages via PMA stimulation. This finding indicates that human macrophages definitely show dysferlin protein expression, but that the cell state is a critical determinant of expression.

The success of the differentiated human macrophage platform provides a viable proof-of-concept for a minimally invasive biomarker strategy in humans, potentially allowing for longitudinal monitoring of drug response in dysferlinopathy clinical trials of 4-PBA on individuals with 4PBA responsive DYSF missense mutations.