Dean J. Burkin - US grants

embryonic stem cell; biomedical facility; clinical research; genetically modified animals;

integrins; vascular smooth muscle;

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is a severe muscle wasting disease that leads to premature death. Currently, there is no cure or effective treatment for this devastating neuromuscular disease. Expression of the 17 integrin is increased in the muscle of DMD patients and the mdx mouse model. Mice lacking both dystrophin and the 17 integrin exhibit severe muscular dystrophy and die before 4 weeks of age. Recently we demonstrated that enhanced transgenic expression of the 17 integrin in skeletal muscle can increase the viability of dystrophic mice. Together these studies show that the 17 integrin is a major genetic modifier in the muscle of DMD patients and mdx mice and suggest that compounds that up-regulate 17 integrin gene expression may serve as a potential therapy for DMD. In this study we will initiate a small-molecule discovery program to identify compounds that target 17 integrin gene expression. We will use a clonal muscle cell line isolated from transgenic mice to develop a fluorescent assay system for integrin gene expression. This system will allow an assessment of 17 integrin promoter activity in its normal cellular and genomic context. This cell-based system will be tested with compounds known to increase 17 integrin gene expression. The validated cell-based assay will then be applied to an automated high throughput drug screen to identify compounds that increase 17 integrin gene expression in cultured mouse muscle cells. Positive compounds will be tested in cultured murine and human muscle cells to determine drug efficacy, toxicity and ability to translate to human muscle cells. Together these aims will allow us to test the hypothesis that small compounds can increase 17 integrin gene expression in murine and human muscle cells. The identification of small compounds that increase 17 integrin gene expression will form the basis of future studies which will translate our basic biomedical research studies to the development of drugs for future human clinical trails.Project Narrative Duchenne Muscular Dystrophy is a devastating muscle disease that affects nearly 50,000 children in the United States. Increased expression of 17 integrin has been shown to be enormously beneficial to mouse models for this disease. This study aims to identify drugs that increase 17 integrin gene expression which may prove to be of therapeutic value to patients that suffer from DMD.

[unreadable] DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is a devastating lethal muscle wasting disease that affects nearly 50,000 patients in the US alone and for which there is no effective treatment or cure. Both DMD patients and mdx mice (the mouse model for DMD), have dystrophin gene mutations and lack the dystrophin protein. Although damaged muscle is capable of repair, muscle regeneration in DMD patients is unable to keep pace with muscle damage. Satellite cells are a population of adult muscle stem cells that mediate muscle regeneration and therapeutic strategies have been aimed at increasing the capacity of these cells to repair damaged muscle. The 1721 integrin is a major laminin receptor expressed in satellite cells, myoblasts and vascular smooth muscle. Our laboratory has recently shown that loss of the 17 integrin in mdx mice leads to severe muscular dystrophy which is caused in part by a reduced capacity for muscle repair. These data suggest the 1721 integrin is a major genetic modifier in dystrophic muscle. In addition DMD patients and mdx mice exhibit reduced vascular function. Given the close association of muscle microvasculature and satellite cells, reduced vascular function may negatively impact satellite cell activation, survival and muscle regeneration. The 1721 integrin is highly expressed in vascular smooth muscle cells and myoblasts; however it is unknown if enhancing 17 integrin expression in these cells will further prevent muscle disease in mouse models for DMD. This proposal will test the hypothesis that targeting 17 integrin expression in myoblasts or vascular smooth muscle can serve as an effective integrin-based therapy for muscular dystrophy. We will test this hypothesis in three Specific Aims. First, using mice and isolated primary myoblasts that lack the 17 integrin we will determine the role of the 1721 integrin in muscle repair. Second, we will determine if enhancing the expression of 17 integrin in myoblasts can improve myoblast survival, engraftment and repair of damaged and dystrophic muscle. Finally we will determine if enhanced 17 integrin expression in vascular smooth muscle can improve vascular function and reduce muscle pathology in dystrophic mice. Determining if enhanced 17 integrin expression improves vascular function and muscle regeneration may provide new targeted integrin-based approaches in the treatment of Duchenne and other muscular dystrophies. PUBLIC HEALTH RELEVANCE. Duchenne Muscular Dystrophy is a devastating muscle disease that affects nearly 50,000 children in the United States. Increased skeletal muscle expression of 17 integrin has been shown to be enormously beneficial to mouse models for this disease. This study will use transgenic mice to investigate the role of the 1721 integrin in muscle repair and determine if increasing integrin expression in muscle repair cells or vascular smooth muscle may further improve integrin-based therapies for muscular dystrophy. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is a lethal muscle wasting disease for which there is currently no cure or effective treatment. DMD is caused by mutations in the gene encoding dystrophin resulting in an absence of the dystrophin protein, a critical component of the laminin binding dystrophin glycoprotein complex in muscle. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The 1721 integrin is also a major laminin receptor in muscle that is increased in DMD patients and the mdx mouse model. These observations led to the hypothesis that the 1721 integrin is a major modifier of disease progression in DMD. In support of this idea, transgenic over-expression of the 17 integrin in muscle alleviates muscle disease in dystrophic mice, while loss of 17 integrin in dystrophic muscle results in more severe disease. Together these results indicate pharmacological interventions which promote increased 17 integrin expression in muscle may be an effective therapeutic approach for the treatment of DMD. Recently, using a novel muscle cell-based assay and drug discovery technology, we have identified several compounds that increase 17 integrin expression in cultured muscle cells. In this study we will initiate preclinical testing of these compounds in mouse models of DMD. Our studies will determine the optimal dose of these compounds and determine if they increase longevity and prevent muscle disease progression in DMD mouse models. Compounds will also be tested to determine if they prevent the development of dilated cardiomyopathy associated with muscular dystrophy using echocardiography and if they prevent deterioration in muscle function. Together these aims will allow us to test the hypothesis that compounds which increase 17 integrin gene expression can prevent muscle disease progression in mouse models of DMD. The identification of compounds that increase 17 integrin expression and prevent muscle disease in mouse models of DMD will form the basis of future studies that will translate our basic biomedical research into the development of drugs for clinical trials for DMD. PUBLIC HEALTH RELEVANCE: Duchenne Muscular Dystrophy (DMD) is a devastating muscle disease that affects nearly 50,000 children in the United States and European Union. Increased expression of 17 integrin has been shown to be enormously beneficial in DMD mouse models. This study aims to conduct preclinical testing of novel drugs that enhanced 17 integrin expression in muscle to determine if they maybe of therapeutic value to DMD patients.

DESCRIPTION (provided by applicant): This application seeks NIH funding to support a conference titled Congenital Muscular Dystrophy: From Clinical Pathology to Underlying Scientific Mechanisms, Exploring the Role of the Myomatrix which will be held at the University of Nevada School of Medicine from Sunday April 22 to Tuesday April 24, 2012. This conference will bring together basic scientists, clinicians, advocacy groups and pharmaceutical industry representatives with expertise in the matrix and muscular dystrophies in general, those with expertise in forms of CMD more specifically, and those from outside the immediate neuromuscular field but with expertise that is relevant to the CMDs. This is a unique conference dedicated to the intersection of muscle and extracellular matrix both in development and disease states, specifically muscular dystrophy and CMD. The conference will combine two keynote addresses with seven core sessions: matrix in development, matrix and regeneration, matrix and intracellular organelles, matrix biomechanical and physical properties, matrix and signaling receptors, matrix and the neuromuscular junction. The conference will end with a panel discussion to build a myomatrix roadmap, unifying key themes from prior sessions, providing opportunities for productive collaborations and identifying pathways towards translational research for the congenital muscular dystrophies. PUBLIC HEALTH RELEVANCE: This application seeks funding to support a conference titled Congenital Muscular Dystrophy: From Clinical Pathology to Underlying Scientific Mechanisms, Exploring the Role of the Myomatrix. The primary conference goal is to address, evaluate and achieve consensus on contributing mechanisms of the myomatrix that drive key clinical pathology relevant to the quality of life and life span of patients with congenital muscular dystrophies. Conference deliverables include defining new focus areas, bringing together researchers that may otherwise not have a chance to communicate or collaborate and using the congenital muscular dystrophy clinical context to frame research queries into translational targets.

DESCRIPTION (provided by applicant): Merosin Deficient Congenital Muscular Dystrophy type 1A (MDC1A) is a devastating neuromuscular disease caused by mutations in the LAMA2 gene and loss of laminin-?2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance to breathe and have reduced life expectancy. There is currently no effective treatment or cure for MDC1A and all affected children will die from this genetic disease unless treatments are soon discovered. Laminin-?2 is required for the formation of heterotrimeric laminin-211 (?2, ?1, ?1) and laminin-221 (?2, ?1, ?1) which are major constituents of skeletal and cardiac muscle basal lamina. Laminin-111 (?2, ?1, ?1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-?2 deficient muscle but is absent from adult skeletal muscle. We have recently shown that mouse laminin-111 protein can be systemically delivered to the muscle of laminin-?2deficient mice to prevent muscle pathology, maintain muscle strength and dramatically increase life expectancy. These findings demonstrate that laminin- 111 is a highly potent therapeutic in the dyW-/- mouse model of MDC1A. At the time of diagnosis, children with MDC1A have developed significant muscle disease and it is unclear if laminin-111 protein therapy is effective at preventing disease progression after it has already started. This proposal will investigate if laminin-111 protein therapy can prevent disease progression after onset, determine if recombinant human laminin-111 can prevent muscle disease and investigate the mechanism of protection conferred by laminin-111 protein treatment. We will test the hypothesis that laminin-111 protein can functionally substitute for laminin- 211 and serve as an effective therapeutic for MDC1A. We will test this hypothesis in three Specific Aims. First we will determine if laminin-111 prevents further muscle damage after disease onset, preserves muscle function and improves survival in the dyW-/- mouse model of MDC1A. Second we will determine if transgenic expression of human laminin-111, recombinant human laminin-111 or recombinant human laminin-211 improves preclinical outcomes in the dyW-/- mouse model of MDC1A. Finally we will determine scalability of laminin-111 protein therapy and define the pharmacokinetic and pharmacodynamic profiles in mouse and dog models. Results from this study will pave the way towards developing laminin-111 protein therapy as a novel therapeutic for MDC1A.