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FSH Society Research Fellows and Grantees
For a list of current FSH Society Research Fellows and Grantees, granted in the August 2011 and February 2011 grant cycles and a description of the research projects, please click here for PDF or MS-Word For a list of current FSH Society Research Fellows and Grantees, granted in the previous three grant cycles (August 2009, February 2010, August 2010) and a description of the research projects, please click here for PDF or MS-Word For a list of past FSH Society Research Fellows and Grantees, and a description of the research projects, please click here for PDF or MS-Word The FSH Society Scientific Advisory Board (SAB) met in October 2011 to review grant applications received for the August 2011 round of FSH Society grants funding. Below is a list of the funded projects, including project descriptions as submitted by grant applicant. 1. Identification of the epigenetic mechanisms that regulate DUX4 activity in skeletal muscle Summary (Provided by Applicant): D4Z4 repeat array chromatin relaxation and transcriptional de-repression of the non-polyadenylated double homeobox 4 (DUX4) gene unifies D4Z4 contraction-dependent FSHD1 and contraction-independent FSHD2. Only from FSHD-permissive genetic backgrounds the DUX4 transcript originating from the most telomeric unit of the array can be stabilized by a polyadenylation (polyA) signal outside the array. Non-permissive chromosomes fail to stabilize DUX4 in the absence of this polyA signal. Somatic DUX4 derepression in FSHD1 and FSHD2 leads to bursts of DUX4 protein in sporadic nuclei of cultured FSHD myotubes. 2. Resonance Imaging and Spectroscopy Biomarkers in FSHD Summary (Provided by Applicant): Facioscapulohumeral muscular dystrophy (FSHD) is the most prevalent hereditary progressive muscle disorder in humans. It is an autosomal dominant disease that causes wasting and weakness in multiple muscle groups (face, shoulders, and upper arms initially, and legs later in the disease) as well as significant disability in affected individuals. Recent advances in understanding the pathophysiology of FSHD have led to the identification of therapeutic targets. However, we lack appropriate biomarkers that reflect the degree of muscle degeneration and regeneration in these patients. Such biomarkers will be necessary for the successful completion of clinical trials. The proposed study will test the hypothesis that proton magnetic resonance spectroscopy (MRS) and quantitative magnetic resonance imaging (MRI) can be used to define unique metabolic profiles in the skeletal muscle of patients with FSHD and controls with normal muscles. These profiles can then be used as biomarkers of disease severity and surrogate outcome measures in therapeutic clinical trials in FSHD. Summary (Provided by Applicant): This $12,500 per year grant is supplementary support linked to an NIH R21 1R21NS076671-01 (2-year) application directed towards identifying chemical inhibitors of DUX4. We have previously screened 200,000 compounds and identified approximately 600 inhibitors of cell death induced by DUX4, and current work is directed towards identifying the most promising leads within this set of 600. The NIH application was recently selected for funding and the grant was initiated. This support from the FSH Society will be used to supplement the NIH project, principally by providing additional medicinal chemistry support (allowing us to increase the number of compounds that we follow-up by purchase/synthesis. These compounds help us to understand pathways that can indirectly inhibit toxicity associated with DUX4. See 1R21NS076671-01 [ http://projectreporter.nih.gov/project_info_description.cfm?aid=8225734&icde=10980193 ] for more information on the R21. Awards for February 2011 Cycle The FSH Society Scientific Advisory Board (SAB) met in June 2011 to review grant applications received for the February 2011 round of FSH Society grants funding. Below is a list of the funded projects, including project descriptions as submitted by grant applicants. 1. Antisense strategies against DUX4 as therapeutic approaches for FSHD Summary (Provided by Applicant): FSHD is a muscle degeneration disease genetically linked to contractions of the D4Z4 repeat array on the 4q35 subtelomeric region. Our group has identified the double homeobox 4 (DUX4) gene within each unit of the D4Z4 repeat array and shown that the encoded protein was expressed in primary myoblasts and biopsies of patients with FSHD but not in non-affected individuals. We found that the only stable DUX4 messenger RNAs derive from the last unit and extend to the flanking pLAM sequence that provides a polyA addition signal. This signal is required to develop FSHD as independently confirmed by an eight-laboratory consortium which studied genetic polymorphisms in hundreds of patients and thousands of healthy individuals. In aggregate our collaborative studies with four different groups have shown that the DUX4 protein is a transcription factor that targets a large set of genes, some of which encode other transcription factors that in turn target additional genes. Globally, DUX4 activation at the FSHD locus initiates a transcription cascade leading to muscle atrophy, inflammation, decreased differentiation potential and oxidative stress, the key features of the disease. By differential protein, RNA and gene studies we keep identifying additional FSHD biomarkers and define whether they are direct or indirect DUX4 targets. 2. Humanized mouse model for the study of Facioscapulohumeral Dystrophy FSHD is genetically caused by the contraction of D4Z4 DNA repeats located on chromosome 4 in 4q35. Although the genetic defect was identified 20 years ago, the exact molecular mechanism causing the disease is unknown, and there is currently no mouse disease model. To provide such a valuable tool, we will develop a humanized mouse model for FSHD, obtained by the engraftment of FSHD patient-derived myoblasts into mouse muscle. Engrafted human cells are able to form muscle fibers in the host mouse muscle, thus allowing pioneering studies in an in vivo context. Because of the dominant nature of FSHD, we hypothesize that the engrafted fibers will display a disease phenotype and recapitulate pathological molecular mechanisms associated with FSHD that will allow us to study the development of the disease. Our preliminary studies have already established the feasibility of this project. Through the cell repository of the Boston Biomedical Research Institute (BBRI) Wellstone Center, we have the unique opportunity to access early passage myoblast cells from cohorts of FSHD probands and their appropriate controls, i.e., a first degree relative. We will graft these standardized cultured cells into mouse muscle to obtain the FSHD humanized mouse model, thereby generating a well-controlled in vivo model for the study of FSHD. The very pressing issue in the field today is the verification of the current DUX4 model. The humanized mice produced will be used to investigate the hypothesis that DUX4 gene expression is a major cause of FSHD pathogenesis. In the obtained model, DUX4 expression will be evaluated during in vivo regeneration, and the consequence of its expression on fiber turnover and satellite cell renewal will be assessed. This work will contribute to the understanding of the role of DUX4 in vivo, thus providing a better understanding of FSHD pathogenesis. 3. Testing a therapeutic approach for FSHD: evaluation of the efficacy of AOs blocking DUX4 in a mouse model of isolated myofibres FSHD is considered the most frequent hereditary muscle disorder in adults, affecting one individual in 20,000. It is associated with contractions of the D4Z4 repeat array in the 4q35 subtelomeric region. In non-affected individuals, this array comprises 11-100 tandem copies of the 3.3-kb D4Z4 element while in patients, only 1-10 D4Z4 copies are left (Wijmenga et al., 1992). Our group has identified the double homeobox 4 (DUX4) gene within each unit of the D4Z4 repeat array (Gabriels et al., 1999) and several studies have now demonstrated the causative role of DUX4 in FSHD. We have demonstrate that the stable full-length DUX4 messenger RNA (mRNA) is produced from the last D4Z4 unit in FSHD, using a polyadenylation signal in the flanking pLAM region, located telomeric to the distal repeat (Dixit et al., 2007) as recently confirmed by a study of genetic polymorphisms in hundreds of patients and thousands of non-affected individuals (Lemmers et al., 2010). This polyadenylation site is necessary to develop FSHD on a contracted allele therefore called “permissive chromosome” (Lemmers et al., 2010). The mRNA from this distal D4Z4 unit contain the entire DUX4 open reading frame (ORF) and 1 or 2 alternatively spliced introns in the 3’UTR (DUX4-fl). In addition, a short DUX4 mRNA terminates at the previously described polyadenylation site in the pLAM region but uses a cryptic splice donor site within the DUX4 ORF (DUX4-s). DUX4-fl was only detected in FSHD muscle cells and biopsies, whereas DUX4-s is detected both in control and some FSHD samples (Snider et al., 2010). A long DUX4 mRNA was detected in induced pluripotent stem cells (iPS cells) and human testis where the gene contains 4 additional exons and a more distal polyadenylation signal. Expression of this DUX4 mRNA was suppressed during differentiation of control iPS cells to embryoid bodies whereas expression of full length DUX4 mRNA persisted in differentiated FSHD iPS cells (Snider et al., 2010). These data, together with the conservation of the DUX4 ORF through evolution (Clapp et al., 2007) suggests a possible role of DUX4 in human development. 4. Investigating mouse models of FSHD There is a great need for a valid mouse model for FSHD. Such an animal model would provide a valuable tool for exploring the effects of newly cloned genes and novel proteins on the pathophysiology of this disease. It would also greatly facilitate research towards the development and testing of new therapeutic approaches to FSHD. We propose to examine two possible mouse models of FSHD, the FRG1 over-expressor, from Drs. Davide Gabellini and Rossella Tupler, and mu-crystallin over-expressor, developed by Drs. Patrick Reed and Robert Bloch. I will breed these mice and test them for their physiological and morphological characteristics, and their susceptibility to injury and ability to recover from injury. I will also initiate xenografting studies to create mice with humanized normal and FSHD ankle dorsiflexor muscles, combining methods that are routine in the Bloch laboratory with unique reagents provided by collaborators in the Wellstone Muscular Dystrophy Cooperative Research Center (MDCRC), “Biomarkers for Therapy of FSHD.” These experiments should reveal the usefulness of available transgenic models for the study of FSHD, and promote the development of humanized mouse muscles for the study of the pathophysiology of FSHD in situ. 5. Epigenetic abnormality in FSHD Our preliminary findings indicate that D4Z4 repeat regions indeed interact with other genome regions, and that these interactions are indeed disrupted in FSHD. With a three-month extension of my fellowship, I plan to perform a high-throughput identification of potential target genes that interact with D4Z4 using the recently developed “Chromatin Interaction Analysis using a Paired-End Tag” (ChIA-PET) technique. This strategy enables the genome-wide detection of chromatin interactions mediated by specific factors that are normally assembled at D4Z4. Identification of additional FSHD pathogenic genes other than FRG1 and DUX4 is important to explore future therapeutic targets to improve or prevent the clinical 6. Analysis of DUX4-fl expression We request support from the FSH Society for our pilot project investigating DUX4 expression in unaffected and FSHD subjects. The DUX4-fl expression model for FSHD has not been independently validated, likely due to the lack of quality clinical resources in the field. At this point in FSHD research, validating and expanding upon the DUX4-cytotxicity model for pathogenesis is vital to the entire field and we are best positioned to do the necessary experiments with the unique set of highly controlled reagents being generated by the NIH Wellstone Muscular Dystrophy CRC for FSHD at BBRI. Each Wellstone cohort consists of an FSHD affected subject and an unaffected first-degree relative. Each subject donated two biopsies, one from the biceps and one from the deltoid. A portion of each biopsy was used to derive myogenic cell cultures. Quite surprisingly, in our initial preliminary results using 4 cohorts we found some inconsistencies with the published DUX4 expression results that have warranted further investigation. Therefore we have begun a much larger effort to analyze DUX4-fl mRNA and protein expression in a larger set of Wellstone cohorts using RT-PCR and immunostaining (ICC). However, this project is not funded at all in my lab or in the original Wellstone budget and my lab receives no financial support from the Wellstone Center. The Wellstone has supported us by providing us with cells, which we culture, and RNA which the Louis Kunkel lab purified from biopsies (we do not actually work with the biopsies) and we have been fortunate to receive these Wellstone samples. At this point, to ensure that our results are statistically meaningful, we need to analyze many more cells and biopsy RNAs and it has become cost prohibitive. Therefore I am requesting financial support for consumables and services (DNA sequencing) to conduct these experiments.
Awards for August 2010 Cycle The Scientific Advisory Board (SAB) met on November 22, 2010, to review grant applications received for the August 2010 round of FSH Society grants funding. Below are: 1.) a listing of the funded projects by grant applicants along with project descriptions as submitted by grant applicants. 1. “Small Molecule Screen to Identify Inhibitors of DUX4-mediated Toxicity, Therapeutic Approach for FSHD" PROJECT SUMMARY We and others have shown that DUX4 is toxic to different cell types, and induces FSHD-associated morphological and transcriptional changes in vitro.As a first step towards developing a targeted therapy for FSHD, we have taken advantage of conditional toxicity of DUX4-inducible myoblasts and we developed a small molecule screening platform for identifying inhibitors of DUX4. In our iC2C12-DUX4 inducible myoblasts, we incorporated full length of the last D4Z4 repeat, so prior its induction, we can not exclude that besides DUX4, some other products are not expressed (RNA, spliced proteins). Assay based on rapid cell death within 24 hours induced by high levels of DUX4 was used for high throughput screen of 200,000 chemicals, part of UT Southwestern HTS compound library. We identified more then 586 compounds with significant rescue ability (60 to >100% cell survival). To identify direct inhibitors, we have conducted serial follow up assays, including secondary screens to eliminate compounds which interfere with the rtTA/TRE inducible gene expression system, to distinguish anti oxidants, to confirm reversion of toxicity in other DUX4-expressing cell types. Several classes of compounds reverted toxicity indirectly, including antioxidants. After these secondary screens, we have narrowed down the list to 82 potentially direct DUX4 inhibitors. The goal of this proposal is to discover a chemical compound/s which efficiently inactivates the DUX4 protein and build on that discovery to develop a drug for a therapeutic approach to FSHD. To achieve this we will have to filter our current list (82 compounds) with additional secondary screens. Among them will be an analysis of MyoD expression and stability as well as cellular localization of the DUX4 protein (Aim 1). We reported that DUX4 is a potent inhibitor of MyoD expression. Therefore, a compound that will rescue MyoD expression in DUX4 induced cells is likely to be a therapeutically effective DUX4 inactivator. We assume that compounds which will be able to inactivate DUX4 in our iC2C12-DUX4 system most likely will be able to rescue FSHD myoblast phenotype. FSHD myoblasts were reported to have impaired differentiation, missregulation of myogenic transcription factors and increased susceptibility to oxidative stress. For that reason, as a functional in vitro study, we will test selected compounds for reversion of FSHD myoblast phenotype (Aim 2). Furthermore, we will test whether selected compounds exhibit their effect on inactivation of DUX4 protein or inhibition of DUX4 transcription or translation (Aim 2). At the end the most potent compound /s will be test for pharmacokinetic and pharmacodynamic properties (Aim 3). The aims of our proposed study target the most crucial topic and urgent needs of FSHD patients: specific and direct pharmacological therapy. Aim 1. To narrow our focus to the most promising direct DUX4 inhibitors. + 2. “Defining the Tissue and Cell Specificity of the Human DUX4 promoter in Mice” PROJECT SUMMARY FSHD was formally classified in 1954, and the primary genetic defect, D4Z4 contraction, was identified in 1992, but the pathogenic mechanisms underlying the disease have only recently started to come into focus. One reason for the difficulties in understanding FSHD biology is the lack of a relevant animal model expressing FSHD-permissive D4Z4 arrays. Since animal models, particularly mice, are crucial tools for studying disease pathogenesis and developing potential therapeutics, the absence of an FSHD mouse model is a fundamental problem in the FSHD field. A major goal of the Harper lab is to generate an FSHD mouse model expressing a single FSHD-permissive human D4Z4 repeat, and to use this model to understand the role of the D4Z4-resident gene, DUX4, in FSHD pathogenesis, and develop RNAi therapeutics targeting DUX4. In preliminary data, supported by previous FSH Society Fellowships to the Harper Lab, we delivered DUX4 to mouse muscle using adenoassociated viral vectors (AAV). DUX4 over-expression in muscle caused myopathy, but DUX4 is generally toxic to many non-muscle cells as well. Thus, we hypothesized that if DUX4 over-expression is an underlying pathogenic event in FSHD, it must be preferentially expressed only in affected muscles. We therefore developed transgenic mice expressing the green fluorescent protein (GFP) gene from the human DUX4 promoter (DUX4p-GFP mice), to determine the tissue and cell specificity of DUX4. In preliminary studies, we observed gross GFP expression in the face, shoulder girdle, and limbs of three independent DUX4p-GFP mouse lines. In this proposal, we will more carefully define the developmental and cellspecific expression patterns of DUX4p-GFP mice, and develop an AAV vector to determine whether a viral-mediated vascular delivery approach can produce the same expression patterns. Ultimately, these studies will be important first steps toward developing an AAV-mediated D4Z4 mouse model. Specific Aim 1: To define the developmental and cell-specific expression patterns of the human DUX4 promoter in mice. Mounting evidence supports the hypothesis that over-expression of the D4Z4-resident DUX4 gene is an underlying pathogenic event in FSHD. DUX4 is generally toxic to many cell types, and since FSHD is characterized by dystrophy of very specific muscle groups, we hypothesized that DUX4 is preferentially expressed only in affected muscles. Our newly generated DUX4p-GFP reporter mice grossly express GFP in areas that are preferentially affected in FSHD. In this Aim, we will perform a detailed characterization of GFP expression in our DUX4p-GFP mice. These results will help define the expected expression patterns of DUX4, and ultimately increase our understanding about the role of DUX4 FSHD pathogenesis. Specific Aim 2: To develop an AAV vector-mediated DUX4p-GFP mouse model. Previous endeavors to generate D4Z4 or DUX4 FSHD mouse models using traditional transgenic approaches have been unsuccessful. Although the previous attempts are not published in peer-reviewed literature, the difficulties encountered in generating these models were discussed in abstracts and talks at various scientific meetings over the last several years, including at the FSH Society’s 2008 International Research Consortium and Research Planning Meeting held in Philadelphia, Pennsylvania (http://www.fshsociety.org/assets/pdf/FSHD_ASHG_IRC2008_Philadelphia_11Nov_ProgramAndAbstract s_proof.pdf). Vascular delivery of AAV vectors carrying FSHD-permissive D4Z4 repeats to adult mice may circumvent the early embryonic death or developmental defects arising from germline transmission of D4Z4 repeats using traditional methods. In this Aim, we will test the feasibility of using AAV vectors to drive D4Z4-specific expression patterns in mouse muscle using an AAV.DUX4p-GFP reporter vector. + 3. “Identification of a Novel FSHD Biomarker [an unknown 50 kDa polypeptide highly expressed in FSHD samples]" PROJECT SUMMARY Screening FSHD patient-derivedmyoblasts, control myoblast, and muscle samples for expression changes at the proteomic level produced an unknown 50 kDa polypeptide highly expressed in FSHD samples compared to controls. Interestingly, this polypeptide is equally expressed in both normal and FSHD-patient derived myoblasts and early myotubes, however, unlike in control cells where its expression decreases, this unknown polypeptide remains highly expressed in differentiated muscle suggesting it is developmentally regulated and this regulation is disrupted in FSHD. This proposal will utilize standard biochemical techniques including column chromatography and mass spectrometry to purify and identify this 50 kDa putative FSHD biomarker. Subsequently, specific antibodies will be gerneated and characterized for further use to screen FSHD-derived cells to establish the universality of this biomarker. In addition, regardless of what its eventually identification turns out to be, identifying this protein will provide insight into FSHD pathophysiology, will be a useful FSHD biomarker, and may be one of the first proteins consistently and specifically upregulated in viable FSHD muscle. Therefore, generating specific and standardized antibodies to this protein will provide a useful resource for clinicians and basic FSHD researchers. + 4. “Toward Therapeutics for FSHD: Understanding mRNA Processing" PROJECT SUMMARY Recently, the group of van der Maarel reported in the journal Science their findings of the high These findings suggest a direct line to a novel therapeutic approach. The toxicity leading to + 5. “A multicenter collaborative study on the clinical features, expression profiling, and quality of life of pediatric facioscapulohumeral muscular dystrophy” PROJECT SUMMARY Fascioscapulohumeral dystrophy (FSHD) is the third most common type of muscular dystrophy, with an estimated prevalence of 1 in 15,000 to 20,000 (Kissel, 1999) (Flanigan et al., 2001). It is an autosomal dominant disorder due to a deletion within the D4Z4 repeat region located on the subtelomeric region of chromosome 4q35. FSHD causes progressive atrophy and frequently asymmetrical weakness involving the face, shoulder girdle, upper arm, abdominal, and lower limb muscles. Most affected individuals develop symptoms during their second or third decade, with 20% eventually become wheelchair dependent (Padberg, Lunt, Koch & Fardeau, 1991) (Zatz et al., 1998). Early childhood onset of FSHD may be associated with more severe weakness as well as extra-neuromuscular manifestations such as mental retardation, retinal vasculopathy, and sensorineural hearing loss (Jardine et al., 1994) (Klinge et al., 2006). Although the majority of cases of FSHD are inherited, about 20 - 30% of sporadic cases may occur as a result of spontaneous mutation or mosaicism (van der Maarel & Frants, 2005). Despite recent advances in the understanding of the molecular genetics of FSHD, the exact mechanism responsible for the disease remains unknown, and presently there is no cure (Tawil & Van Der Maarel, 2006) (van der Maarel, Frants & Padberg, 2007). As well, the prevalence, clinical variability, cross cultural presentation, and the psychosocial impact of FSHD on affected individuals constitute a significant public health concern. Emerging therapeutic trials will benefit from the availability of natural history data and reliable outcome measures (Rose & Tawil, 2004) (Tawil, 2008) for both children and adults with FSHD. Purpose of Study The main objectives of this study are: 1) to establish a standardized muscle testing protocol for use in children and youth with FSHD; 2) to describe the clinical phenotypes of pediatric onset FSHD; 3) to evaluate the impact of FSHD on health-related quality of life and disability across different age groups; and 4) to explore potential genetic modifiers of clinical phenotypes and disease progression in FSHD. TEXT SIZE
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