Current Grant
09/24 – 12/25

We have established that the clinically approved drug sodium 4-phenylbutyric acid (4-PBA), used for the treatment of patients with Urea Cycle Disorders, is efficacious in restoring a number of patient missense mutations, including hDYSF^L1341P, to the plasma membrane of HEK cells and GREG mouse myoblast and myofibers. Moreover, 4-PBA treatment of GREG myofibers expressing hDYSF^L1341P restores membrane repair capacity of otherwise repair deficient cells. Based on these results we hypothesize that 4-PBA likely acts as a chemical chaperone, one of its known activities, stabilizing misfolded mutant dysferlin proteins preventing their destruction and allowing them safe passage within the endomembrane system allowing for membrane localization and functional restoration of membrane repair.  Please see the 2023-2024 grant summary below for information on the results of preclinical longitudinal study of 4-PBA in dysferlin deficient mice

In the 2024-2025 grant period we are turning our experimental focus towards accruing preclinical data in support of an application for clinical trials to treat dysferlinopathy patient carrying pathogenic missense mutations in dysferlin. To these ends we aim to optimize 4-PBA dose response in MMex38 mice, identify murine and human blood-based biomarkers responsive to 4-PBA treatment, and to identify 4-PBA responsive pathogenic patient missense mutations that support membrane repair.

Given the gender-based differences in 4-PBA efficacy in MMex38 mice, we plan to determine the efficacy of 4-PBA to restore DYSF^L1360P expression in various hind limb muscles (Quad, Glut, Psoas, TA, EDL (used for membrane repair), Gastroc) in MMex38 mice that are delivered 4-PBA by single oral gavage of various doses of 4-PBA. At fixed intervals following various treatment doses blood and muscle will be isolated and utilized to A) determine DYSF localization by immunohistochemical localization of histological sections. B) determine DYSF expression levels by Western blot of various muscles C) determine DYSF expression in peripheral blood mononuclear cells (PBMCs, containing monocytes), and D) determine functional restoration of membrane repair. In addition, we plan to develop a LCMS based protocol to determine levels of 4-PBA and phenylacetate in the blood stream as a function of time post oral gavage treatment to examine drug breakdown and its relationship to DYSF expression in muscle and PBMCs. We expect the results of each iteration of experiments to guide changes in dosage and length of time before analysis of muscle and blood samples.

Human blood monocytes are currently being used as a diagnostic tool in patients, as DYSF is expressed in human blood monocytes, and can be easily isolated from patients in order to determine if monocytes have reduced or in most cases of dysferlinopathy lack DYSF expression, aiding in dysferlinopathy diagnosis.  We plan to explore three different methods using mouse monocytes in order to determine if similar assays can be developed in order to assess effectiveness of 4-PBA treatment without the need to sacrifice animals. To these ends, we will determine DYSF expression levels in blood taken from wild type mice by Western analysis. In order to accurately quantitate DYSF expression and reduce the amount of blood required for detection, we aim to develop a flow cytometry-based assay to quantify DYSF expression in Ly6C positive monocytes from both wild type and MMex38 animals either untreated or treated with 4-PBA. Alternatively, we plan to try and develop a blood smear-based immunocytochemistry assay in order to visually determine DYSF expression levels from small (ca 50-100 mL of blood).

We previously published the results of our 2A-assay system that can quantitively determine the amount of hDYSF found at the membrane surface of HEK cells and found that 4-PBA treatment increases expression and plasma membrane localization of normal human DYSF, by as much as 20%. Based on this finding, we plan to directly assess and compare the amount of hDYSF expression in normal human monocytes that are either untreated or treated with 4-PBA in vitro in order to determine if human monocytes are responsive to 4-PBA treatment. If so, simple blood draws from patients taking 4-PBA may be sufficient to be able to track efficacy of drug treatment during clinical trials.

Project Results

Previous Grant Period
09/23 – 08/24

Sodium 4-phenyl butyrate (4-PBA) is a clinically approved drug for the treatment of Urea Cycle Disorders (UCD) caused by genetic mutations in a number of genes involved in detoxification of ammonia from the blood stream. 4-PBA is also known to act as a histone deacetylase inhibitor, ER-stress reducer, and a chemical chaperone capable of restoring localization and function of proteins containing missense mutations. When consumed orally, 4-PBA is rapidly metabolized by liver and the kidney cells forming phenyl acetate (PA), which is then complexed with ammonia, in the form of glutamine, to form an excretable molecule phenylacetyl glutamine (PAGN), allowing for ammonia detoxification in UCD patients.

During our 2023-2024 funding period we performed a preclinical longitudinal 4-PBA treatment study examining the efficacy of 4-PBA to restore expression, localization, and function to DYSF^L1360P expressed in MMex38 mice, a murine model of dysferlinopathy that mimics a pathogenic patient missense mutation DYSF^L1341P. MMex38 mice were provided 4-PBA (2.0 mg/mL) in drinking water ad libitum for 1-year. We found 4-PBA to be efficacious in improving dystrophic phenotypes in female MMex38 animals, but not male MMex38 animals. 4-PBA treatment increased the hind limb muscle (Quad, Glut, Psoas) weights in female MMex38 mice by as much as 39%; importantly, these muscle groups also have the largest reduction in muscle weights in MMex38 when compared to wild type animals of the same age longitudinally. In addition, at this dosage, female but not male MMex38 animals showed statistically significant improved performance on inclined balance beam assays at 13 and 14 months of age relative to untreated MMex38 mice.

Initial immunohistochemical (IHC) staining data examining DYSF^L1360P expression and localization in muscle explants from 14-month-old (12-months on 4-PBA) study mice revealed that 4-PBA treatment restored substantial sarcolemma localization of DYSF^L1360P in TA muscle in both genders compared to untreated control 4-PBA treated MMex38 mice; however, 4-PBA treated MMex38 animals do not have equivalent DYSF^L1360P sarcolemma localization when compared to similar IHC of TA muscle sections from WT mice, suggesting that 4-PBA does not fully restore sarcolemma expression at the dosage tested. Nevertheless, despite the fact that 4-PBA treatment only partially restores DYSF^L1360P localization, EDL muscles taken from 4-PBA treated mice were capable of restoring membrane repair kinetics to near WT levels following laser-induced damage. Interestingly, EDL muscle from female mice were fully repair proficient similar to EDL muscle form WT mice, while EDL muscle from male mice displayed partial membrane repair kinetics. While DYSF IHC from other muscle tissues from treated and untreated MMex38 animals are ongoing, we have sufficient data indicating that MMEx38 animals on treatment see benefit of DYSF restoration on a molecular level. We surmise that there is a possibility for further improvement of DYSF^L1360P sarcolemma restoration by altering drug dosing. Clinical and preclinical (mice and rats) data examining pharmacokinetics of 4-PBA indicates there are gender differences between male and females related to the breakdown of 4-PBA into PA, and its conversion to PAGN. Both in human clinical and preclinical animal (rat and mice) studies females show greater persistence of 4-PBA and PA in blood samples following 4-PBA treatment compared to similarly treated males. The gender-based pharmacokinetic differences in 4-PBA processing may explain gender-based efficacy differences related to increased muscle weight and improved performance of female animals as compared to males in MMex38 mice.

In summary, we have determined that long-term in vivo 4-PBA treatment improves DYSF^L1360P expression and myofiber sarcolemma localization, as well as, muscle mass of the most affected muscle groups, and physical performance of MMex38 mice harboring a pathogenic dysferlinopathy missense mutation. Going forward, we hope to optimize 4-PBA treatment to maximize its efficacy in improving both molecular and physical dystrophic phenotypes in both male and female MMex38 animals, and to identify potential blood-based biomarkers to aid in determining efficacy of 4-PBA in future clinical trials with dysferlinopathy patients.

Previous Grant Period
09/22 – 08/23

MMex38 mice harbor the murine dysferlin mutation DYSF^L1360P synonymous to the dysferlinopathy patient missense mutation DYSF^L1341P. We previously found that 4-PBA could increase plasma membrane localization and restore membrane repair function to of DYSF^L1341P in vitro and could also do the same after a 2-day treatment of MMex38 mice in vivo (Tominaga et. al. 2021, iScience). During this grant period we began a study to determine if the clinically approved drug, sodium phenylbutyrate (4-PBA), is efficacious in preventing or reducing the severity of dystrophic phenotypes in the MMex38 mouse model of dysferlinopathy.

We first undertook an aging study of MMex38 mice in order to establish a baseline for histological and physiological changes that occur over time in this model in order to be able to establish endpoints for a longitudinal study of 4-PBA. To these ends, we established an aging colony of MMex38 (MUT) and wildtype (WT) control animals ranging from 3 months of age to up to 2 years of age, and isolated hind limb muscles from at least three male mice from each age group; including Quadriceps (Q), Gluteus (GL), Gastrocnemius (GC), Tibialis Anterior (TA), Psoas (P), and Extensor Digitorum Longus (EDL) muscles. We found statistically significant reductions in the weights of P, GL, and Q muscles from MUT animals at or over 12 months, and in GC muscles in MUT mice at or over 16 months. TA muscle show weight reduction at or over 20 months of age, while no differences were noted in EDL. We prepared fixed and fresh frozen tissue samples from all muscle groups for histological analysis, including H&E staining, Mason’s Trichrome staining, Congo Red staining, and Dysferlin immunohistochemistry in order to quantify dystrophic histological markers common to dysferlinopathy such as reduced myofiber size, increased numbers of central nuclei caused by regenerating myofibers, increased fat and collagen content/distribution indicative of fat infiltration and fiber replacement, and fibrosis. Most of these samples have been processed and analysis underway to determine differences between the various WT and MUT muscle groups.

To access the physiological effects on hind limb muscles, we developed an in-house Inclined Balance Beam (IBB) assay that scores the amount of time it takes a mouse to traverse a specified distance on inclined beams of various widths (24mm, 12mm, 10mm, 8mm). We found statistically significant differences in cross times between WT and MUT animals at 13 and 14 months of age on 12mm, 10mm, and 8mm width beams. We therefore plan to use the balance beam assay as a measure of muscle function in our longitudinal 4-PBA drug study.

During this granting period we established a longitudinal 4-PBA drug treatment trial. Thirty-two 2-month-old individually caged MMEx38 animals (16 male and female), were placed on a drug treatment protocol where half receive water containing 4-PBA (2mg/mL) and the remaining are given untreated water ad libitum, with water changed weekly. Animals were placed on protocol as they aged in; full enrollment occurred over three months. All animals were found to consume equivalent amounts of water, and showed equivalent increases in body weight over time; no adverse health events were found. Animals have been/are trained for IBB at 10 months of age, and assayed on the IBB each month thereafter. During our next grant period, we plan to end the longitudinal 4-PBA drug study after IBB assay are performed at 14 months of age, whereupon the various muscle groups listed above will be isolated, weighed, and processed for muscle histology.