FKRP and Alpha Dystroglycan Related Dystrophy (αDG-RD) Research
FKRP is one of the currently 10 known genes (FKRP, fukutin, LARGE, POMT1, POMT2, POMGnT1, DPM1, DPM2, DPM3, αDG) that can lead to an alpha dystroglycan related dystrophy (αDG-RD, dystroglycanopathy). Research in this field focuses on both FKRP specifically and on the group of disorders subsumed under αDG-RD. Here are currently funded research projects that may provide future insight into both FKRP and αDG-RD, what causes the muscle weakness and potential therapies.
Gene therapy studies in mouse models of LGMD2I
Dr. Qi Lu, at McColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, has published two papers showing pre-clinical evidence that gene replacement therapy for FKRP-related muscular dystrophies could have therapeutic potential. Using a mouse model of LGMD2I that he created in his laboratory, his team first demonstrated, in a paper published last October, that it is possible to improve muscle function after introducing a normal copy of FKRP through the use of a benign viral vector (Adeno-Associated Virus or AAV). Then, more recently, using this same mouse model, they compared the effects of the introduction of FKRP or LARGE gene. They showed that LARGE overexpression, like FKRP, can also correct the glycosylation defect in the FKRP mouse model. Long-term studies are now needed in order to assess the efficacy of LARGE up-regulation in improving muscle function and to determine whether there are any deleterious long term effects of LARGE gene therapy. These studies offer very encouraging animal proof of concept that gene therapy approaches could one day be used to treat FKRP-related muscular dystrophies, such as LGMD2I. Several laboratories are currently pursuing these approaches with the goal of bringing them to clinical trials within the next few years.
Grant to study mutant forms of FKRP
The LGMD2I Research Fund, the Samantha Brazzo Foundation and Cure CMD have jointly funded a research project to support drug screening on mutant forms of FKRP, in the search for compounds that restore normal protein function. It is still not well understood what the function of FKRP is as there have been very few studies that have focused on this protein. This project will try to better understand the role of the FKRP protein and try to find drugs that restore its normal function. The project will be led by Dr. Sebahattin Cirak at the Children’s National Medical Center and will use a novel approach for the analysis of mutated forms of FKRP and will allow for the identification of highly selective drug candidates that could be further tested in patients’ cells and in mice models of FKRP deficiency. This project will help better understand the function of FKRP and could potentially lead to the discovery of therapeutic drugs against LGMD2I.
Developing New Antibodies for the Dystroglycanopathies
Antibodies are used by the body’s immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign object and allows the two structures to bind together with precision. The project will focus on developing and characterizing antibodies that recognize normal and disease forms of alpha-dystroglycan. Existing antibodies can be challenging to use and give inconsistent results. This makes staining of the muscle for both diagnostic and research purposes difficult. This project is being carried out at two centers in the United Kingdom with Dr. Sue Brown in London, and Dr Glenn Morris in Oswestry.
Funding partners: LGMD2I Fund, Stevenson Family Fund at Cure CMD.
Developing AAV-miniagrin for testing in the FKRP mouse model
Mini-agrin is an engineered protein that helps muscle cells attach to their environment. In LGMD2I, as in some other muscular dystrophies, the attachment to the cell surroundings (called matrix) is flawed because of a defect in a protein called alpha-dystroglycan, causing the muscle to be more prone to injury. Data obtained in LGMD2I patient cells shows that mini-agrin restores normal function of these cells. The FKRP AAV-miniagrin project will test whether mini-agrin provides a benefit to the FKRP mouse model and should thus be further tested as a potential treatment for LGMD2I.
Funding partners: LGMD2I Fund, Samantha J Brazzo Foundation, Stevenson Family Fund at Cure CMD.
Development of two FKRP induced pluripotent stem cell lines (iPS)
iPS lines take blood cells and revert them back to an early undifferentiated cell type. This process is somewhat like time travel back in time for an individual cell, and creates a special cell (iPS) that can be directed to become brain cells (neurons, astrocytes), heart cells (cardiomyocytes) and skeletal muscle cells. This allows researchers to study these types of cells from people with a dystroglycanopathy to better understand how these cell types are impacted by the absence of sugars on dystroglycan, and to perform drug screening. This also opens up the door to genetically correct these cells, inject them into mice that have a dystroglycanopathy, and determine if the genetically corrected cells can “treat the disease” in the mouse.
This will be the first time that a stem cell therapy is tested in a mouse model of LGMD2I. If encouraging results are obtained, further testing will be done in order to bring this potential therapeutic approach to the clinic.
Funding partners: Cellular Dynamics, Inc, LGMD2I Fund, Stevenson Family Fund at Cure CMD.
Zebrafish, A Different Kind of Disease Model
Dr. Jim Dowling, MD, PhD, from the University of Michigan has recently been funded to generate an FKRP zebrafish model and compare this model to two different existing αDG-RD zebrafish models (POMT2, POMGnT1). This work builds upon an established program to characterise congenital muscle disease zebrafish models and allows for a comparative analysis of both zebrafish pathology and response to a medium scale drug screen across multiple neuromuscular conditions.
LARGE is the name of one of the αDG-RD genes. Early work from Dr. Kevin Campbell's lab, University of Iowa, has shown that upregulation of LARGE (increasing the expression of LARGE) in patient derived skin cells can restore critical binding of alpha dystroglycan (αDG) to laminin 211, a function that is partially lost in people with an αDG-RD, including FKRP (Barresi et al, Nature Medicine (2004) 10: 696-703). Further work by Dr. Kanagawa, showed that injecting a viral vector containing LARGE into the Fukutin mouse model also improved glycosylation measured by an antibody called 2H6, which correlates with binding of αDG to laminin 211. (Kanagawa et al, Hum. Mol. Genetic. (2009)18(4): 621-631). Several laboratories are currently working on drug screens to identify potential compounds that might upregulate LARGE.
Development of Mouse Models of FKRP
Dr. Sue Brown at the Royal Veterinary College, UK, developed 2 models of FKRP, one with embryonic brain involvement and the other with rescue of the brain phenotype, leading to a mouse model with predominantly skeletal muscle involvement. This mouse model, known as the FKRPMD, is currently being further characterised.
Furthermore, scientists at the McColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, North Carolina, USA, have created several mouse models of muscular dystrophy with mutations in the fukutin-related protein (FKRP). Mutations of the FKRP gene causes diseases ranging from mild to late onset limb-girdle muscular dystrophy type 2I (LGMD2I) to earlier onset, more severe congenital muscular dystrophy (CMD) including Walker-Warburg syndrome and muscle-eye-brain disease with brain and eye defects. McColl-Lockwood Laboratory has generated several mouse models with different mutations in the FKRP gene, leading to mice with different degrees of brain, skeletal muscle and heart involvement. Two of the mouse models, one due to a P448L mutation and one modeling the common L276I mutation, may provide a valuable resource to ongoing understanding of the FKRP disorder. Ongoing efforts to characterize these animal models will define their value in preclinical drug testing.
Magnetic Resonance Imaging: an objective tool to measure disease progression in LGMD2I
A study has been carried out using a technique known as Magnetic Resonance Imaging or MRI, to determine what changes occur in the muscles of patients with LGMD2I, and to also to determine whether MRI can be useful to monitor disease progression compared to standard physical assessment techniques. The study was carried out at four European referral centres: Newcastle, UK; London, UK; Copenhagen, Denmark; Paris, France.
The first part of the study compared two MRI techniques to see if one is better at characterising the muscles of LGMD2I patients and possibly explaining any gender differences. Thirty eight patients (19 male: 19 female) who were all still walking were included in the study, all with the homozygous mutation (c.826C>A) in the FKRP gene. The thigh and lower leg muscles of all patients were assessed using MRI.
This study confirmed that the muscles at the back of the thigh (hamstring muscles) are most affected, which has been shown in previous studies, but also highlighted that there are differences between men and women, in not only in the specific muscles that are affected, but also to what extent. This means that female patients could present and progress differently to males.
The second part of the study was looking to find out if a specific type of MRI could be used to determine the progression of disease compared to conventional assessments, such as muscle strength measurements, the 6 minute walk distance (6MWD), and lung function tests (Forced Vital Capacity – FVC). An MRI was carried out 12 months after the initial MRI, as described above. The change in the ratio of fat to muscle in the legs over the 12 months was determined in all patients. This was then compared with the changes observed using conventional assessments.
Over the 12 month period, a significant increase in the amount of fat, in 9 out of 14 of the leg muscles analysed, was shown using MRI. Such clear changes over the same time period were not seen in any of the conventional assessments.
This study has demonstrated that MRI is more sensitive in determining changes in the course of the disease, otherwise not shown with the standard assessment methods currently used, over a short time period. MRI is non-invasive, objective and does not rely on patient effort compared to the clinical and physical assessments that are currently used. Muscle MRI could be used to assess which muscles are affected and to what extent, and to monitor how effective new therapeutics are in patients with LGMD2I.
Characterisation of FKRPMD mouse model
Characterization of the FKRPMD mouse model and testing of LARGE Over-expression (PI: Dr. Sue Brown). This 2 year project completed the characterization of this important dystroglycanopathy mouse model. Surprising results of LARGE over-expression, meaning increasing LARGE production early in development, will provide new insights and questions that need to be answered prior to pursuing this as a treatment target in the dystroglycanopathies. We look forward to Dr. Brown’s publication of her work.