Public Abstract:
Facioscapulohumeral muscular dystrophy (FSHD) is the third most common of the major muscular dystrophies affecting 1 in 20,000 individuals. In most cases, FSHD is associated with a reduction in the number of repeat sequences on human chromosome (hchr) 4q. Loss of DNA organization in the region is thought to affect expression of nearby genes causing FSHD. The gene encoding the transcription factor REX-1 is located in this region. The role of REX-1 in the etiology of FSHD or skeletal muscle development has not been fully explored. The hypothesis to be tested is that over-expression of REX-1 affects muscle development and/or regeneration, and contributes to the pathology observed in FSHD. To determine whether REX-1 could be a key regulator of muscle development it will be necessary to assay REX-1 expression through all stages of muscle development. But many stages of human muscle development that occur during embryonic and fetal development are inaccessible. Therefore, to examine embryonic and fetal stages of muscle development we will use a novel approach taking advantage of newly developed methods for modeling muscle development by isolating muscle precursors from human embryonic stem cells (ESCs). The results of the studies proposed here will delineate the role of REX-1 in muscle development and its role in regulating a signaling pathway required in muscle development. To test the novel hypothesis that inappropriate expression of REX-1 during development of skeletal muscle contributes to the pathology observed in FSHD, we will also make use of induced pluripotent stem (iPSCs) made from FSHD and control fibroblasts and myoblasts. These cells will then be induced to differentiate to compare muscle development and gene expression in FSHD to that of normal controls. To further test whether REX-1 over-expression contributes to FSHD pathology, we will reduce REX-1 expression in FSHD iPS cells to attempt to normalize FSHD pathology and increase REX-1 expression in control iPS cells to test the idea that REX-1 contributes to FSHD pathology. Determination that REX-1 regulates muscle development and altered expression of REX-1 causes muscle pathology would be a major step forward in our understanding of a molecular mechanism underlying cellular differentiation and human disease. FSHD iPSCs will also provide an important reagent to fill vital gaps in our knowledge of the molecular mechanism of the disease as well as provide a novel reagent to identify drugs to treat FSHD.
Statement of Benefit to California:
Facioscapulohumeral muscular dystrophy (FSHD) is third most common type of muscular dystrophy affecting about 1 in 20,000 individuals. The age of onset is about age 20. The disease begins with weakness and wasting of muscles around the eyes, mouth, shoulder blades and upper arms and progresses relatively slowly spreading to the legs and abdominal region. In most cases, affected individuals have a normal life span. The impact of this disease on affected individuals and their families is significant and long-lived, both financially and emotionally. The cost to the state of California directly is equally long-lived. At the present time, an effective treatment does not exist, in part, because even after many years of investigation, the disease mechanism is still unknown. This lack of understanding of the disease mechanism has made development of effective treatments difficult. The studies in this proposal will fill vital gaps in our knowledge of the molecular mechanisms that control muscle development and how these mechanisms could go awry in neuromuscular diseases such as FSHD. We will use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to dissect the molecular pathways that regulate muscle development. These pluripotent cells differentiate into all cells in the body providing a unique tool to identify, isolate and characterize specific muscle precursor populations in human development. The results of our studies could constitute a major step forward in our understanding of a molecular mechanism underlying cellular differentiation and human disease. In addition, the FSHD iPSCs generated in this study will provide an important reagent to develop drugs to treat FSHD.
Review Summary:
EXECUTIVE SUMMARY
This proposal aims to test two hypotheses: (i) that the transcription factor Rex-1, plays a role in skeletal muscle development, and (ii) that altered Rex-1 expression in muscle precursors contributes to the pathology of one of the muscular dystrophies, facioscapulohumeral dystrophy (FSHD). These hypotheses will be tested in human cells by studying skeletal muscle differentiation from human embryonic stem cells (hESC) as well as human adult primary myoblasts and FSHD patient-derived induced pluripotent stem cells (iPSC). In Aim 1, the applicant will determine if Rex-1 is regulated during skeletal muscle differentiation by monitoring changes in Rex-1 expression at various time points during the differentiation process; in Aim 2, the effects of Rex-1 over-expression and knock down on a signaling pathway known to play a role in muscle differentiation will be investigated; and in Aim 3, in order to determine whether Rex-1 expression contributes to FSHD muscle pathology, muscle differentiation from normal-iPSC versus FSHD-iPSC will be compared to identify potential abnormalities in in vitro muscle development. Rex-1 expression will then be manipulated to investigate the role of Rex-1 in contributing to such abnormalities.
Reviewers agreed that the hypothesis that Rex-1 plays a role in skeletal muscle development and in FSHD etiology is novel. There is no existing cure for FSHD and little information about disease instigators. If the proposed studies indeed implicate Rex-1 as a major player, then this information could have an impact on the future course of FSHD research. Furthermore, the generation of FSHD-iPSC will provide a novel and important resource for investigations into the pathology and therapeutic possibilities for this rare disease. However, although some aspects of the proposal are in scope with this RFA as they focus on molecular mechanisms that control differentiation of hESC to skeletal muscle, reviewers pointed out that the FSHD-iPSC aspect of the proposed research utilizes stem cells as a tool in muscular dystrophy research rather than to study basic stem cell biology.
Despite their appreciation for the potential impact of the proposed studies, reviewers had major reservations about their feasibility due to several weaknesses in the rationale and study plan. A concern was voiced about the logic for the proposed Rex-1 knock down in adult myoblasts when those cells do not appear to express that gene. Some reviewers described the study plan as logical but not well developed, commenting that it is short on specifics such as markers to be used to identify specific stages of differentiation. Although the preliminary studies provided evidence that the required technical expertise exists in the laboratory, the data lacked maturity, and did not provide sufficient support for the main hypotheses pursued in this application. For example, reviewers noted that a key finding cited by the applicant to support the rationale for Aim 2 was misrepresented. With respect to Aim 3, some questioned whether FSHD-iPSC are likely to display a differentiation phenotype in the dish, which would indicate a global deficiency in skeletal myogenesis, when the disease is characterized by a highly specific and limited pattern of muscle involvement. In other words, since the FSHD mutation appears to affect only a subset of muscles, it is not clear that this would be revealed in an in vitro hiPSC differentiation assay. This potential shortcoming is not addressed in the proposal. Likewise, the applicant does not provide a compelling argument that FSHD pathology is attributable to a muscle cell-autonomous defect, which is a necessary premise of the proposed experiments. Alternative plans are discussed to some degree.
The principal investigator (PI) has established expertise in hESC/hiPSC biology, participates in a multi-PI collaborative group aimed at the study of FSHD and other muscular diseases, and has a reasonable publication record as an independent researcher, with many collaborative studies but few senior authorships. The PI will dedicate 30% effort to this project and has recruited a number of appropriate expert collaborators on stem cell biology and FSHD to assist with these studies. Overall, some reviewers felt the track record of the assembled team indicates suitable experience exists, whereas others expressed some doubts. The available research facilities are appropriate for the proposed project.
In summary, reviewers appreciated the potential impact of the proposed studies on the field of human skeletal muscle development in general, and on a rare genetic disorder, FSHD, specifically, but thought that the proposal fell short in experimental support for the rationale and the quality of the research plan.
