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ACTIVE RESEARCH PROJECTS

  • 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. The DNA will be injected into the bloodstream DNA containing the non-mutated gene, 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. Cathleen Lutz (The Jackson Laboratories): “Using CRISPR/Cas9 to create and characterize knock out and null alleles on multiple genetic backgrounds”

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

  • Dr. Rita Perlingeiro (University of Minnesota): “Gene editing of Calpain 3 in LGMD2A 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 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. 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 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 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. 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 models, then this will suggest that these compounds may be beneficial for LGMD2A 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 (LGMD2A) patients, and will facilitate better estimates of the number of people living with LGMD2A. It is crucial that we know the prevalence of LGMD2A 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.

COMPLETED RESEARCH PROJECTS

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. 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.

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.

PUBLISHED RESEARCH ANNOUNCEMENT

Coalition to Cure Calpain 3 (C3) is excited to announce the publication of important research undertaken by Dr. Melissa Spencer, Dr. Irina Kramverova and the Spencer lab: “Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy).” In normal muscle, genes are turned on or off in response to exercise. This research shows that, for certain genes, this response is reduced or absent in muscle deficient in calpain 3. This work provides an important clue to help researchers understand why mutations in the gene for calpain 3 cause LGMD2A. This work was funded, in part, by a research grant from C3.

An abstract of the paper is available at: http://www.ncbi.nlm.nih.gov/pubmed/27005420