Research

English

አማርኛ العربية 简体中文 繁體中文 Dansk Nederlands English Français Deutsch עִבְרִית हिन्दी Italiano 日本語 ភាសាខ្មែរ 한국어 كوردی‎ मराठी Norsk bokmål Polski Português ਪੰਜਾਬੀ Русский Español Svenska தமிழ் తెలుగు Türkçe

C3 IS ACCEPTING LETTERS OF INTENT FOR OUR CURRENT REQUEST FOR APPLICATIONS – CLICK HERE FOR INSTRUCTIONS

ACTIVE RESEARCH PROJECTS

• Dr. Elisabeth Barton & Dr. Lan Wei-LaPierre (University of Florida): “Strategies to improve calcium handling in LGMD2A/R1”

Researchers do not fully understand the role of calpain 3 in keeping muscle healthy. Experiments in animal models show one of the consequences of loss of calpain 3 function is a failure of muscles to adapt to increased exercise. This study aims to clarify how calcium signaling, which is a key regulator of exercise response, is affected when calpain 3 is absent. Further, the investigators will use a mouse model of LGMD2A/R1 to test therapeutic strategies that normalize calcium handling in muscle.

• Dr. Pia Elustondo (AGADA Biosciences): “Drug screening in CAPN3b-deficient zebrafish”

Preclinical drug screening is one of the critical initial steps to develop new treatments for rare diseases such as LGMD2A/R1. Several mouse models have been developed to-date. However, the mouse models do not show muscle symptoms until they are older, their symptoms are very mild, and they are slow to reproduce. This makes it difficult for researchers to use mice for drug testing. This research project will use zebrafish as an animal model to test and find new candidate drugs that will potentially improve LGMD2A/R1 disease symptoms. The investigators have generated three unique zebrafish lines that have disrupted calpain 3. They plan to characterize these lines to determine if they have a muscle phenotype, and to use them to screen drugs that have the potential to treat this disease.

• Dr. Svetlana Gorokhova (Aix Marseille University): “A diagnostic functional test to rule out dominant forms of calpainopathy”

Since calpainopathy can be inherited in both recessive and dominant ways, it can be challenging to diagnose an individual with limb-girdle weakness who has only one CAPN3 variant identified: should one continue searching for a second variant or conclude that this patient has the dominant form of the disease? Dr. Gorokhova and her colleagues aim to develop a diagnostic functional assay that could answer if a single variant can cause the dominant form of calpainopathy.

• Dr. Meredith James & Dr. Jordi Díaz-Manera (John Walter Muscular Dystrophy Research Centre at Newcastle University): “Improving clinical trial readiness by investigating the suitability of clinical outcome assessments and documenting the natural progression of LGMD2A/R1 – a retrospective analysis”

This project, a collaboration between the John Walter Muscular Dystrophy Research Centre at Newcastle University and Dr. Linda Lowes and Dr. Lindsay Alfano of the Center for Gene Therapy at Nationwide Children’s Hospital, brings together world leaders in the field of clinical outcome assessment development from two clinics that have cared for many individuals living with LGMD2A/R1. The investigators will examine the clinical records of approximately 100 patients who have already been evaluated at these centers. Analysis of these records will allow the investigators to identify outcome measures that will be suitable for use in clinical trials, as well as to document the rate of disease progression.

• Dr. Nicholas Johnson (Virginia Commonwealth University): “Defining clinical endpoints in LGMD”

This observational study will follow LGMD2A/R1 patients over a period of 12 months to establish clinical outcomes assessments that are sensitive to normal disease progression. These assessments may be used in future clinical trials as tools to determine if investigational drugs are effective.

• Dr. Melissa Spencer (University of California Los Angeles) & Dr. Jeffrey Chamberlain (University of Washington): “Optimization and validation of gene therapy vectors to treat limb-girdle muscular dystrophy”

Previous work has shown that the gene for calpain 3 can be expressed at high levels in skeletal muscle without toxicity, a finding which makes LGMD2A/R1 gene therapy a realistic goal. However, there are unique considerations related to specific features of LGMD2A/R1 that warrant careful development and assessment of gene therapy vectors for this disease. Of particular concern, expression of the gene for calpain 3 in the heart is toxic. Experiments in this proposal will optimize new vectors that are highly selective for all types of skeletal muscle. The new vectors will then be tested in an LGMD2A/R1 mouse model and assessed for toxicity and therapeutic efficacy.

COMPLETED RESEARCH PROJECTS

• Dr. Siri Bolding (Casimir): “A Concept Elicitation/ Feasibility Study for the Limb Girdle Video Assessment”

This concept elicitation and feasibility study proposes to develop a sensitive and reliable outcome measure tool that is specific to LGMD. The aim is to develop an outcome measure tool sensitive enough to detect nuanced, yet meaningful, shifts in functional ability for upcoming LGMD interventional and exploratory clinical trials. As a sensitive, specific, and reliable outcome measure tool to gauge change in the functional ability of clinical participants over time, the Limb-Girdle Video Assessment seeks to contribute to clinical endpoints that incorporate the degree of phenomenological impact of potential interventions, adding the focus of patient-centered outcomes. This project is co-funded by Coalition to Cure Calpain 3 and the Jain Foundation.

• Dr. Michele Calos (Stanford University): “Calpainopathy: DNA-Mediated Gene Therapy”

Dr. Calos proposes to introduce a functional copy of the calpain 3 gene into a calpainopathy mouse model by employing plasmid DNA, or “naked” DNA. This differs from adeno-associated virus (AAV)-mediated delivery in that it does not require viruses to transport genes to muscle cells. Plasmid DNA containing the non-mutated gene will be injected into the bloodstream, where it will then flow out of the microvessels where it will be taken up by muscle fibers. Once inside the muscle cells, DNA will enter the nuclei and express the calpain 3 protein. After various time periods, Dr. Calos’ team will analyze levels of the calpain 3 protein and monitor for potential beneficial effects on muscle structure and function.

• Dr. Antoine Dufour (University of Calgary): “Unbiased systems-wide investigation of calpain 3 in patients with LGMD2A/R1 using mass spectrometry”

Calpain 3, the protein that is missing or defective in LGMD2A/R1, belongs to a class of proteins called proteases. Proteases have the ability to cleave other proteins. Dr. Dufour’s project aims to identify the proteins cleaved by calpain 3. To do this, his team will use a technique called mass spectrometry to screen all of the proteins present in muscle cells to identify which are cut by calpain 3. Muscle samples from LGMD2A/R1 mouse models and patients will be analyzed. Screens will also be performed to identify changes in protein expression and the role of calpain 3 in signaling to other proteins. This project has the potential to help the scientific community more fully understand the biological function of calpain 3, and how its dysfunction leads to LGMD2A/R1. 

• Dr. Louis Kunkel (Harvard University): “Using Transcription Activator-like Nucleases (TALENs) to Create a Calpain-3 Null Zebrafish for Subsequent Characterization and Screening for Small Molecule Corrective Agents”

Dr. Kunkel’s lab specializes in designing assays that facilitate the screening of pharmacotherapeutics to identify existing drugs with the potential to correct muscular diseases. C3 funded Dr. Kunkel to develop a zebrafish that is deficient for calpain 3. This calpainopathy fish model could potentially be used to (1) learn more about why defects in calpain 3 lead to muscular dystrophy, and (2) screen a library of drugs that have already been approved for use in humans, to see if they reverse disease pathology in the fish. If such a screen provides a hit, then it would justify the investigation of its benefit in calpainopathy patients. Dr. Kunkel followed his proposed experimental plan. However, the plan has not yet produced a validated calpainopathy zebrafish model. C3 is currently investigating other approaches to drug screening, such as cell-based assays.

• Dr. Cathleen Lutz (The Jackson Laboratories): “Using CRISPR/Cas9 to create and characterize knock out and null alleles on multiple genetic backgrounds”

This project used innovative gene editing technology to develop new mouse models that are lacking the gene for calpain 3. Dr. Lutz used this approach on 4 different genetic backgrounds. The resulting models were comprehensively characterized to determine if they display the typical features of LGMD2A/R1 seen in human patients, including muscle weakness and histological features consistent with muscular dystrophy. The most relevant lines are available via The Jackson Laboratory’s Mouse Repository, facilitating efficient distribution of specific-pathogen-free live mice to the scientific community where they can be used for drug discovery and therapeutic development. A Study Summary Report detailing the generation and characterization of the lines can be accessed here.

• Dr. Rita Perlingeiro (University of Minnesota): “Gene editing of Calpain 3 in LGMD2A/R1 iPS cells”

Dr. Perlingeiro specializes in the use of gene editing to correct induced pluripotent stem (iPS) cells from muscle diseases. These cells can then be transplanted into the muscles of models of muscular dystrophy. The goal of the current project is to establish methods to utilize the gene editing technology CRISPR-CAS9 to genetically correct mutations in the Calpain 3 gene (CAPN3) in LGMD2A/R1 iPS cells. Gene correction will then be validated in vitro. These studies will determine the feasibility of a gene edited iPS-cell based stem cell therapy. This project is funded in part by a generous grant from Beyond Labels & Limitations.

• Dr. Isabelle Richard (Genethon): “AAV-mediated Transfer of Calpain 3”

Dr. Richard is a leader in the field of gene therapy for muscular dystrophies. She has been the first to utilize gene therapy to deliver the calpain 3 gene into a mouse model for calpainopathy. She was able to achieve efficient and stable expression of calpain 3 in the muscles of these mice, and found that this expression increased muscle size and force. Importantly, Dr. Richard also discovered that systemic delivery of the calpain 3 gene was associated with cardiac toxicity, and identified a strategy using microRNAs to circumvent this adverse effect. These preclinical studies promote gene therapy as a promising approach for calpainopathy, and identifies the importance of restricting gene delivery to skeletal muscles to avoid cardiac toxicity. Dr. Richard is currently testing the safety of calpain 3 gene delivery in primates.

• Dr. Zarife Sahenk (Nationwide Children’s Hospital): “Pre-clinical studies for gene therapy in LGMD2A/R1”

Dr. Sahenk recently demonstrated that LGMD2A/R1 is associated with impaired muscle regeneration after injury, and that this defect can be corrected by injection of the muscle with AAV attached to the calpain 3 gene. In the current project, “Pre-clinical studies for gene therapy in LGMD2A/R1,” Dr. Sahenk will deliver the calpain 3 gene throughout the body of a mouse model for LGMD2A/R1 to test the safety and efficacy of this method, and to provide proof of principal data for a potential calpain 3 gene therapy for LGMD2A/R1 patients.

• Dr. Jaakko Sarparanta (Folkhälsan Research Center, University of Helsinki): “CAPN3-mediated proteolytic processing of C-terminal titin and turnover of titin fragments”

Titin is a large protein that functions as a molecular spring in muscle cells. It has recently been shown that calpain 3, the protein that is associated with LGMD2A/R1, can cut titin, a protein that is associated with several muscle diseases including LGMD2J (a form of titinopathy). However, the physiological importance of this cleavage activity is unknown. Further, the fate of the resulting titin fragments is unclear. Dr. Sarparanta aims to understand the physiological role of calpain 3 in cutting titin and how dysfunction of this activity may lead to muscle disease.

• Dr. Melissa Spencer (University of California Los Angeles): “Exploring the Role of Blunted Signaling Pathways in the Pathogenesis of Calpainopathy”

The basic biological regulation and processes of calpain 3 are not fully understood. Further, the scientific community lacks a complete understanding of the process by which dysfunction of calpain 3 leads to muscular dystrophy. Dr. Spencer is interested in identifying the roles calpain 3 plays in normal muscle maintenance, and how these roles are disrupted in calpainopathy. In normal muscle, genes are turned on and off in response to exercise. Dr. Spencer recently showed that, for certain genes, this response is reduced or absent in muscle deficient for calpain 3. This work provides an important clue to help researchers understand why mutations in the gene for calpain 3 cause calpainopathy.

• Dr. Melissa Spencer (University of California Los Angeles): “Identification of calpain 3 substrates through use of 2D DIGE and mass spectrometry, and testing muscle-building compounds in an LGMD2A/R1 model”

Identification of calpain 3 substrates through use of 2D DIGE and mass spectrometry: Calpain 3 is a type of protein called a protease, which means that it can cleave other proteins. Its role in muscle health has not been fully established. The first goal of Dr. Spencer’s project is to identify the targets of calpain 3’s cleavage activity using a technique called 2D-DIGE to measure and compare relative amounts of proteins in normal muscle and muscle lacking calpain 3. The identification of targets will contribute to our knowledge of calpain 3’s normal role in muscle health and will provide insight into why loss of calpain 3 causes muscular dystrophy.

Testing muscle-building compounds in an LGMD2A/R1 model: The second goal of Dr. Spencer’s project is to test several compounds that are already in the FDA pipeline to determine if they will also be effective in LGMD2A/R1. These compounds promote muscle growth and are currently in development for other forms of muscular dystrophy. If Dr. Spencer’s research results show that these compounds result in improved muscle size, improved function, and lack of toxicity in LGMD2A/R1 models, then this will suggest that these compounds may be beneficial for LGMD2A/R1 patients.

• Dr. Volker Straub (Newcastle University): “Application of next-generation sequencing technologies to a large cohort of patients affected by unexplained limb-girdle weakness: the MYO-SEQ project”

Coalition to Cure Calpain 3 is partnering with several patient organizations and biopharmaceutical companies to support this program, which collaborates with the Broad Institute of Harvard and MIT to apply whole exome sequencing to patients with unexplained limb-girdle weakness. The initial phase of this project found that the gene for calpain 3 was the most likely to contain a disease-causing variant in this group. This project is important because it will increase the number of genetically confirmed limb-girdle muscular dystrophy type 2A/R1 (LGMD2A/R1) patients, and will facilitate better estimates of the number of people living with LGMD2A/R1. It is crucial that we know the prevalence of LGMD2A/R1 so that we can attract the attention of researchers and drug developers. Patients with limb girdle weakness and/or elevated serum creatine kinase levels who do not have genetic confirmation of their disease subtype should have their clinician contact the MYO-SEQ team at myo-seq@newcastle.ac.uk.

• Dr. Kathryn Wagner (The Johns Hopkins School of Medicine): “Targeting Mss51 as a therapeutic option for calpainopathy”

Mitochondria are the part of the cell that break down sugars and fats and turn them into energy that can be used for a variety of cellular processes such as muscle cell contraction. Dysfunction of mitochondria has been suspected to play a role in many kinds of diseases including several types of muscular dystrophy. Abnormal mitochondria have been observed in muscle from patients with calpainopathy and may contribute to disease pathogenesis. Dr. Wagner has recently described a muscle-specific gene, Mss51. When genetically deleted in mice, Mss51 leads to improved mitochondrial activity with increased energy production. Dr. Wagner hypothesizes that reduction in Mss51 will improve the muscle function of a mouse model of calpainopathy. The experiments in this project will determine if Mss51 is a viable target for the treatment of calpainopathy.

PUBLISHED RESEARCH ANNOUNCEMENT

The group used AAV-mediated gene therapy to deliver the human CAPN3 gene to a mouse model of LGMD2A/R1. Mice at 2- and 5-months of age were treated with low and high doses of the gene therapy. The group found that mice treated with either dose and at either age were able to run further on a treadmill than mice that were not treated with gene therapy. Microscopic evaluation of the muscles showed that treatments improved the structure of the muscles. Importantly, safety studies showed no evidence of toxicity in any organs. 

C3 Scientific Director Dr. Jennifer Levy states, “I am encouraged by these promising results. Individuals with LGMD2A/R1 are an underserved population with limited treatment options. This study suggests that gene therapy may be a safe approach to improve muscle function in these patients.”

The paper is available here.