The National Institute of Neurological Disorders and Stroke, the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the Office of Rare Diseases sponsored a workshop on the Therapeutic Approaches for Duchenne Muscular Dystrophy (DMD), May 15-16, 2000 in Bethesda, MD. Despite advances in knowledge to date on DMD, the life expectancy and quality of life for a child with DMD has not had substantial improvement since the gene was discovered. The Parent Project for Duchenne Muscular Dystrophy Research requested the help of NIH to promote research relevant to potential therapies, to encourage new scientists to the field of muscle biology research and to research on DMD, and to encourage NIH awareness and promote the importance of muscle research. In examining these issues, the goals of this workshop were to address key questions in improving treatments for DMD and identify areas of needed scientific knowledge, impediments, and critical next steps to promote effective therapy.
An international group of multidisciplinary scientists participated in the May 15-16th workshop. Representatives of the French (AFCM) and US (MDA) muscular dystrophy associations were present, as well as parent representatives from the US Parent Project for Duchenne Muscular Dystrophy Research and a European (German) foundation. The scientific organizers who aided in the development of the agenda were Dr. Louis Kunkel (Harvard), Dr. Lee Sweeney (U. Pennsylvania), Dr. Stanley Froehner (U. North Carolina), and Dr. Thomas Rando (Stanford). The workshop consisted of a round table format composed of 7 sessions, designed to encourage open discussion and develop recommendations to promote future research directions in DMD and muscle biology. (See attached Meeting Agenda and Participant List). A scientist chaired the discussion for each session's topics (topic leader for each session). After the scientific sessions were completed, participants were invited to remain to discuss issues of scientific review with representatives of the Center for Scientific Review (CSR), NIH. Summaries of the May 15-16th workshop presentations and CSR meeting are attached.
On May 17th, following the 2-day workshop of the 15-16th, the scientific organizers, topic leaders, and NIH program directors met to summarize the discussion from each session and formulate the future research priorities. Below is the summary of workshop discussions and identified future research priorities.
A. Expand Molecular Characterization of the Duchenne Muscular Dystrophy (DMD) Population.
Many of the therapeutic approaches presented at this meeting, such as those using chimeraplasts (chimeric RNA/DNA oligonucleotides), antisense oligonucleotides, or amino glycoside antibiotics (see workshop summary) depend on precise knowledge of the dystrophin gene mutation in each patient with DMD. Thus, many of these therapeutic approaches will remain mutation-specific and therefore patient-specific. Current testing by most DMD diagnostic laboratories consists of deletion analysis, which has only about 65% sensitivity. Therefore, molecular characterization of the DMD population should be expanded. Duplication testing by Southern blot analysis or quantitative PCR (polymerase chain reaction) should be offered to all DMD patients. Low-cost, high-throughput DNA-based sequencing should be available to all DMD patients whose dystrophin genes do not have deletions or duplications. The resulting sequence data will not only be absolutely essential for determining specific therapy, but will also improve diagnostic sensitivity.
B. Consider Creatinine Kinase (CK) Screening of Newborns.
Physicians routinely screen at birth for certain metabolic disorders, such as the enzyme deficiency phenylketonuria, because early treatment is essential. Early detection of DMD might enable earlier intervention before the onset of irreversible muscle changes. This suggests the possibility of screening newborns for elevated CK activity, a characteristic of DMD. However, an objection to CK screening of newborns is that specific treatment does not exist for DMD. Furthermore, knowing that a newborn has screened positive might impose a psychological burden that makes it difficult for others in the family to bond with the newborn. Other concerns in newborn CK screening are the sensitivity and specificity of CK testing of samples taken 24 hours after birth, the number and quality of the laboratories doing the testing, and the cost of the test. Given these objections and concerns, it follows that the issue of newborn CK screening should be further considered, such as during a workshop on newborn screening or through a pilot research study.
A. Animal Models and Central Repositories.
Additional studies on DMD pathogenesis are required to develop new areas of therapy that relate to the dystrophin-glycoprotein complex. Animal models might be useful for identifying novel and known genes that affect the phenotype of the muscular dystrophies, specifically DMD. The mouse and other species might be useful. Specifically, model genetic systems might also be helpful to determine the basic biology of the dystrophic process. Animal models or novel strains generated as part of these studies should be made available to all investigators to further DMD therapeutic studies. The role of a central repository for animal models, specifically for new and existing mouse models, should be considered.
B. Further Studies to Elucidate Both the Structural and the Signaling Role of Dystrophin-Glycoprotein Complex.
Studies suggest that the dystrophin-glycoprotein complex has both mechanical and signaling roles. Further studies to uncover downstream mediators associated with the dystrophic process are needed. These mediators might include known or novel signaling pathways. A number of new genes have been discovered that cause different genetic forms of muscular dystrophy. Overlapping disease mechanisms might lead to cell death in many of these similar disorders. Additionally, studies on novel alterations of the cytoskeleton, a critical internal structure of cells, in DMD and new methods to study novel alterations should be encouraged.
C. Role of the Immune System in DMD.
Further studies are needed on the specific immune response and on the nature of the inflammatory changes that accompany degeneration in DMD. Immunomodulatory effects of corticosteroids should be studied, given the effectiveness of these drugs in treating DMD. Additionally, specific studies should be considered on the role of corticosteroids in inflammation, and on the direct effect of corticosteroids on muscle stability and function and on stem cells, immature cells that retain the capacity to multiply and specialize to form new cells.
D. Cardiomyopathy, Vasculopathy, and Other Tissue Involvement.
Studies on the cardiomyopathy that accompanies DMD are needed because cardiac dysfunction significantly contributes to morbidity and mortality in DMD. The pathogenesis of cardiomyopathy--specifically, the role of vascular alterations--requires further study. Along these lines, examination is needed of the role of dystrophin-glycoprotein disruption in tissues other than striated muscle. These studies might include, but should not be limited to retina, brain, smooth muscle and vascular tissues. Additionally, the phenotype in DMD is variable. Identification of modifiers of phenotypic variability might lead to novel approaches to therapy.
A. Delivery (viral mediated; naked DNA)
Results showed that DMD can be partly reversed by delivery of viral vectors into older mice. Presumably, more concentrated or efficient viral vectors could therefore achieve a complete reversal. The extent of reversal that can be achieved in late-stage patients remains unclear. Given the potential for reversal and the clear ability to prevent DMD, gene therapy techniques need to be studied further. However, technological advances to treat this disease need considerable refinement.
Four major areas need to be covered: (1) optimizing expression cassettes; (2) improving vector design; (3) managing immunologic consequences; and (4) optimizing delivery.
1. Optimizing Expression Cassettes
Expression cassettes that are introduced to cells for gene transfer therapies must include not only the gene to be transferred, the transgene, but also DNA elements that control the activity of the gene. A key issue in the organization of expression cassettes is in regulating the transgene, that is, the extent to which the transgene is expressed to produce mRNA which in turn is translated to protein. Regulating the transgene for maximal expression requires understanding which enhancers, promoters, introns, and polyadenylation signals affect the transgene's expression and should be included in the cassette. Tissue-specific gene expression also needs to be understood in designing a cassette.
Another key issue is to decide on which gene or DNA is to be delivered. This is of particular concern in working with large genes such as utrophin and dystrophin where a shorter version of the gene may be more efficient for therapy. The mRNA (messenger RNA) that cells copy from the DNA code includes regions that are cut out before the RNA is translated to make protein. Research is needed on the relationships between structure and function of untranslated regions in dystrophin and utrophin mRNAs and between structure and function in dystrophin and utrophin proteins. The use of the best start and stop codons (the gene code signals to begin and terminate protein translation) and the introduction of heterologous untranslated regions also need to be considered.
2. Improving Vector Design
Vector design clearly needs improvement. The ability of a vector to target or deliver to muscle needs to be considered. This includes whether the vector is or is not infectious, replicating, and integrating. The cloning capacity of vectors needs to be considered, e.g., if vectors can transport small or large genes, or multiple genes. The effect of the persistence of a vector needs to be considered, to include comparing episomal vectors with integrated vectors, measuring stabilization of vector retention, and determining replication competence. Finally, the safety of vectors needs to be determined.
3. Managing Immunologic Considerations
Immunologic considerations include comparisons between normal and dystrophic muscle with regard to the type, presence, and recruitment of immune cells. Immunologic considerations also include characterizing, modulating, and avoiding the response against vectors and against the transgenes themselves. For example, one question is to compare the immunogenicity of transgenes containing the dystrophin gene with the immunogenicity of transgenes containing the utrophin gene.
4. Optimizing Delivery
Delivery is ultimately "the rate-limiting step" in determining the success of gene therapy. It might be difficult to target an expression cassette to all muscles of the body. Route of administration, such as local or systemic, needs to be considered. Targeting vectors to muscle while avoiding other cells might need to be accomplished. Dosing regimens need to be worked out.
B. Alternative (Non-viral) Genetic Therapies
1. Summary of Non-Viral Molecular Mechanisms.
The unifying theme of the session on alternative (non-viral) mediated therapies was that each therapeutic approach uses different molecular mechanisms to target the endogenous dystrophin gene. These approaches include chimeraplasts, antisense nucleotides, and aminoglycoside antibiotics. Chimeraplasts are specifically designed synthetic hybrids of DNA and RNA that can induce the cell's DNA repair machinery to make corrections in a particular sequence of a cell's DNA. Antisense nucleotides, also designed with a sequence that complements a specific region of DNA, can affect which parts of the messenger RNA are skipped and which copied to make protein. Aminoglycoside antibiotics can suppress stop codons introduced by mutations that improperly terminate code for the dystrophin protein. A discussion of peptide translocation domains was presented in the context of delivery mechanisms for oligonucleotides and therapeutic peptides.
The most notable conclusion was that novel therapies might emerge unexpectedly from research that is not directed at developing therapies. Each of the discussed therapies arose from research that had nothing to do with muscular dystrophy or with therapeutics in general. Thus, the emergence of these therapies provides rationale for not focusing too much effort or too many resources solely on research directed at developing therapies. Therapy for DMD in the future is likely to encompass both a combination of approaches currently being studied with approaches not yet even conceived.
2. Research in Delivery and Targeting in Oligonucleotide-Based Therapy.
One specific recommendation is to consider oligonucleotides (used here to include chimeraplasts, antisense oligonucleotides, and plasmid DNA) as drugs and to support research in the emerging area of oligonucleotide-based therapy. As with advances in viral-mediated therapies, advances in non-viral-mediated therapies need to come in two areas. The first is in methods of systemic delivery, including packaging the oligonucleotides for stability, cellular uptake, and bioavailability. The second is in methods of targeting oligonucleotides specifically to muscle, primarily for the purpose of efficiency. Research in systemic delivery and in targeting gene therapy vectors should include studies on oligonucleotide-based therapy.
3. Role of Immune System.
As with viral-mediated therapies, non-viral mediated therapies might enable the production of "new" dystrophin in a boy with DMD, with the ensuing possibility that his immune system will respond to it. The importance of studying the role of immune responses to dystrophin is the same whether or not the therapy is viral-mediated. This further emphasizes the need to include the immunology of DMD within the scope of research efforts.
4. Oligonucleotide-Based Therapy Requires Molecular Genetic Information for Each Individual with DMD.
Finally, each therapeutic modality discussed (chimeraplasts, antisense oligonucleotides, aminoglycoside antibiotics) is applicable to DMD arising only from certain kinds of dystrophin mutations. This emphasizes the need to have accurate genetic data for each patient.
Research needed on stem cell therapy for muscular dystrophy can be summarized in the questions formatted as follows:
What is the nature of the cells? What is their origin and developmental state?
How can the cells be purified? What markers can be used for purification? Is Hoescht's staining a sufficient marker?
What surface markers are the cells expressing? Is there a lineage relationship that needs to be worked out?
How can cells be cultured, their developmental potential expanded and maintained?
Can cells be delivered from circulation? Can cells be recruited in sufficient quantities from the circulation to be efficacious? (Currently, only about 10% can be recruited, which is insufficient.)
What is the developmental potential of cells? Is it necessary to use the earliest stage cells, or will later stage cells suffice? Which is preferable, autologous or heterologous engraftment?
What must be done to make cells therapeutically useful?
A. High-Throughput Screening.
High-throughput screening is an approach to drug development that employs robotics to rapidly screen thousands of chemical compounds in miniaturized assays (tests) to find leads for further testing. High throughput technology is used extensively in industry but less so in academia. A consortium of academic and industrial groups would probably better accomplish development of high-throughput methods for DMD.
B. Assess Steroid Trials and Design Definitive Trials.
Results from smaller trials of prednisone have not been conclusive. Therefore, one strong recommendation is to convene a 1-day workshop to evaluate steroids and design definitive trials. For example, larger, definitive trials with better controls, such as a trial that compares prednisone-dosing regimens, are needed to evaluate the use of prednisone. Trials of deflazacort should also be considered.
C. Understanding the Immunology of DMD.
Research in various basic subjects is needed. Given that prednisone is an immunosuppressive drug, the immunology of DMD needs to be better understood. Further understanding of how prednisone works could lead to the introduction of better drugs. Animal models could be used for studies of various DMD drugs.
A. Non-invasive Muscle Imaging Techniques.
Imaging techniques should be improved so that they better diagnose DMD and monitor disease course during treatment, thus rendering repeated muscle biopsies unnecessary.
B. Development of Efficient Means to Test Potential Therapies in Clinical Trials.
The escalating costs of gene therapy trials might force academic centers to discontinue doing them and result in fewer trials being done. With regards to new therapeutic compounds, an additional concern is that, upon learning about a trial of a test compound, families/patients may forego participating and instead simply try to obtain the compound. Therefore, centers need to cooperate to move trials more quickly.
C. Developing a Centralized Web Base for Accessing Information.
A centralized Web site providing a database should be made available to facilitate knowledge of what clinical information, tissue, reagents, and resources are available for DMD research.
D. General Issues.
Research on muscle biology should be encouraged. Also, much could probably be learned by studying dystrophin and utrophin in tissues other than muscle, such as dystrophin complexes in brain and utrophin in epithelial cells. Vascular abnormalities in the retina should be studied.