By Alex Neupauer, Genetics and Genomics, ’23
Author’s Note: As a Genetics and Genomics major and a person with Duchenne muscular dystrophy (DMD), I was compelled to write a review on how to alleviate the suffering imposed by this devastating genetic disease. I consulted various scholarly articles and interviewed five experts on DMD. Approaches to improve patient outcomes will involve a mixture of researching cures and improving the care of patients’ symptoms. While we may be far away from truly curing DMD, we are not currently powerless against it. With this article, I hope to promote awareness of DMD among both scientists and the public, as the DMD community relies on outside support to improve patient outcomes.
Duchenne muscular dystrophy (DMD) is a devastating disease resulting in muscle degradation. DMD results from mutations to the dystrophin gene that impair the function of the dystrophin protein, an important structural component of muscle tissues. Individuals with the disease suffer from the impacts of weakened muscles on skeletal, circulatory, and pulmonary systems, often shortening their lifespan. The main solution consists of restoring dystrophin expression in cells. CRISPR/cas9 and gene replacement therapy can in theory restore dystrophin expression, while other methods can restore some of its function. To implement CRISPR or gene therapy as cures, researchers must figure out how to deliver them safely and effectively to cells. They must also improve and expand treatments already on the market or on trial to target a wider variety of DMD patients. Exon-skipping therapies only exist for skipping the exons most often implicated in DMD. And nonsense mutation readthrough appears to work better in theory than practice. Implementing and improving methods of dystrophin restoration will also rely on better modeling of DMD and more basic research in molecular and cellular biology. Improved care for the symptoms of DMD is also vital while researchers find and develop cures. To better care for patients’ DMD-related complications, we must create better standards of comprehensive care and facilitate their access to it. Finally, education about DMD is key. Public education will lead to increased funding, allowing for more necessary research. Education will also promote parents’ awareness of screening for DMD and clarify complex care considerations for patients and their families, making quality care easier to secure.
DMD results from the lack of a functional gene encoding dystrophin. Muscle tissues must constantly bear the force of contraction and need proteins like dystrophin to reinforce their strength. Dystrophin ensures muscle fibers and their membranes are not damaged during contraction . Specifically, it links actin filaments inside muscle cells to membrane proteins, which in turn link the membrane to the extracellular matrix . Without dystrophin, actin filaments lack a stable connection to the extracellular matrix, resulting in muscle cell damage.
Dystrophin attaches f-actin to membrane-bound proteins on the muscle cell membrane (sarcolemma). These membrane proteins attach to the extracellular matrix. Without dystrophin, this membrane-bound complex is abolished and f-actin is not anchored to the extracellular matrix. Image from Mbakam et al .
The cell damage induces cellular stress, inflammation, and eventual cell death , and improper muscle function ensues. As a consequence, individuals with this disease typically become wheelchair-bound in their mid-teens. The lessened use of skeletal muscles results in increased bone fragility and the development of contractures  – permanent stiffening of the joints. People with DMD also develop complications in cardiac and respiratory muscles, the most serious complications as a result of the disease . As such, DMD has a negative impact on lifespan and quality of life. And there is no present cure for the disease.
In addition to the lack of a cure, patients often suffer from inadequate care of their symptoms. As the disease impacts patients beginning from a young age, critical care decisions are those of their parents. Despite their best intentions, patients’ families sometimes make choices that negatively impact their outcomes. For example, families refuse steroid treatments, which do not cure DMD but significantly improve patient outcomes. Jessica A. Guzman, an RN for the pediatric neuromuscular clinic at Lucile Packard Children’s Hospital, laments that parents may hold off on steroids out of fear of relatively minor side effects such as shortened stature or in anticipation of a ‘miracle drug’ . Parents may also fail to work through the labyrinth of caring for the health of their child, as the disease impacts many body systems, requiring the attention of many specialist doctors. Scheduling appointments with cardiologists, pulmonologists, orthopedic doctors, and more can be overwhelming. Parents’ uninformed decisions and difficulty navigating the needs of DMD demonstrate the need for increased education on DMD and more support for patients from the medical community.
Stopping the suffering inflicted by DMD has medical, scientific, and social barriers. And it leaves behind it a path of destruction that humanity should not accept. The best approach to end the devastation of DMD consists of researching and implementing methods to restore dystrophin expression to all muscle tissues while improving the care of patients’ symptoms and overall health, employing public education, better models, and basic research to overcome implementation challenges.
Restoring Dystrophin Expression
One of the best solutions is to tackle the problem at the root: the lack of dystrophin protein. There are several approaches to restore the expression of this essential protein: CRISPR/cas9, gene replacement therapy, exon-skipping, and post-transcriptional methods.
Gene Replacement Therapy
One of the most noteworthy challenges in treating or curing DMD relates to the fact that many types of mutations can lead to dysfunction of the dystrophin gene. Patients can have deleted or duplicated exons or point mutations of a single nucleotide . And there is much variability with respect to which exons are affected or which nucleotides are mutated. Gene replacement therapy sidesteps the need for mutation-specific cures by providing all cells with a brand-new copy of the dystrophin gene. Adeno-associated viruses (AAVs) can deliver the replacement dystrophin gene. However, the dystrophin gene is over 2 million bases while the capacity of AAVs is only around 4,700 bases . Micro-dystrophin overcomes this capacity issue, encoding a 1,001 amino acid long protein compared to the 3,684 amino acid long wild-type protein . Carly Siskind, MS, CGC from Stanford Health Care, believes the approach could be helpful, so long as all necessary parts of the protein are encoded by the miniature gene . However, micro-dystrophin does not replace the original protein, which is much longer. In other words, micro-dystrophin appears to solve the problem as a mediocre substitute, which poses the question of whether this mechanism can wholly cure DMD or not.
Furthermore, Claudia Senesac, PT, PhD, PCS from the University of Florida, laments that gene replacement therapy does not appear to last forever, requiring eventual redosing . Unless gene therapy targets satellite cells, the therapy can be lost as muscle cells are replaced. Muscle tissues naturally have turnover rates, which are elevated in the context of gene therapy, as it does not tend to remedy 100 percent of all muscle cells . Satellite cells divide to replace damaged muscle cells, so they must also contain functional dystrophin genes to ensure replaced muscle cells also have a functional copy of the gene. Unfortunately, AAVs do not target satellite cells very effectively . Gene replacement therapy avoids having to consider a patient’s particular mutation, and thus sounds like an excellent solution. However, gene replacement therapy in its current form does not constitute a cure. To make gene therapy a cure, we must develop delivery systems of higher capacity that target a wide array of muscle cell types and create longer-lasting therapies.
The mechanism of CRISPR/cas9 originates as a bacterial defense against viruses. The CRISPR array locus contains a catalog of sequence fragments from previously infecting viruses. These fragments are known as spacer sequences, which alternate with invariable repeats: spacer, repeat, spacer, repeat, etc. When a novel virus infects bacteria, they sequester fragments of viral DNA and insert them into their CRISPR array locus. When the virus infects again, bacteria transcribe their entire CRISPR array. The mRNA of each spacer sequence is cleaved from the array and loaded onto a cas9 protein. The cas9-RNA complexes containing RNA complementary to segments of the invading viral DNA bind to the viral DNA. Then cas9 cuts at the binding site. Thus, the CRISPR system has high specificity with regard to where it cuts. In the lab, researchers can create their own short RNA sequences called small guide RNA (sgRNA) to guide cas9 to specific sites in DNA.
Mechanism of CRISPR in a bacterium. Viral DNA sequences are saved in the CRISPR array locus and transcribed during future infection to target cas9 to cut viral DNA. Image from “A Tool for Genome Editing: CRISPR-Cas9,” BioRender in collaboration with the Doudna Lab at UC Berkeley.
Researchers have adapted the system to accomplish a variety of functions that may be applicable to curing/treating DMD. Three useful applications are exon knock-in, base editing, and prime editing. Exon knock-in consists of CRISPR cutting the DNA between exons and inserting the missing exon, both restoring lost information and the reading frame . Base editing uses enzymes that chemically convert one base into another. These enzymes are attached to a CRISPR system to guide the modifications to specific nucleotides . Prime editing uses reverse transcriptase, guided by CRISPR, to replace a short segment of DNA with new DNA synthesized from an RNA template . As these templates can contain different, extra, or missing nucleotides relative to the original DNA sequence, prime editing can change, insert, or delete particular nucleotides at particular sites. Taken together, prime and base editing can resolve point mutations. Thus, the diversity of CRISPR editing methods fits well with the diversity of mutations causing DMD. One big issue remains: targeting every single muscle cell with this treatment.
AAVs can deliver the materials necessary for the cell to complete CRISPR editing . However, this delivery mechanism would succumb to the same issues of targeting satellite cells. Lipid nanoparticles may be another possibility for delivery. Together, cas9 (cationic) and sgRNA (anionic) form an anionic ribonucleoprotein (RNP) complex, which is placed inside a cationic lipid nanoparticle . The RNP enters the cell via endocytosis , the fusion of the lipid nanoparticle envelope with the phospholipid membrane. It could be a promising avenue given the success of COVID-19 vaccines designed with lipid nanoparticles. Unfortunately, CRISPR also must overcome the challenge of off-target cuts. Permanent changes to DNA can be dangerous if done incorrectly, as the cell’s genome is forever changed. Jacinda Sampson, MD, PhD, adds that CRISPR can now be modified to target mRNA instead . Thus, permanently altering the genome is no longer a concern. Collectively, all experts interviewed express hope of CRISPR as a future treatment option but recognize the aforementioned challenges in achieving that goal [4, 5, 6, 7, 8].
CRISPR gene editing is an exciting possibility for a future cure. To use CRISPR as a cure, we must overcome its delivery challenges and investigate its safety in humans. Additionally, researchers should further investigate RNA CRISPR and lipid nanoparticle delivery as potential cures.
Small Molecules and Exon Skipping
There are other possible methods to restore dystrophin expression that do not involve changing the gene or delivering new copies. Rather, they work with the cell to use the mutated code in a way that allows for the production of dystrophin. One such approach uses small molecules to induce ribosomes to read through a nonsense mutation in dystrophin mRNA. Govardhanagiri et al define small molecules as molecules less than 900 Da, including drugs and biological molecules not including proteins, nucleic acids, or polysaccharides . The fact that cells selectively degrade transcripts with premature stops suggests that premature and normal translation termination may have different mechanisms. Aminoglycosides have been able to promote nonsense mutation readthrough but are toxic after continued use . Pharmaceutical company PTC Therapeutics used high throughput screens to identify nontoxic compounds that promote readthrough of exclusively premature stops in mammalian cells and found ataluren as their top candidate .
Chemical structure of ataluren.
While ataluren restored some fully functional dystrophin expression in mouse models, only a small proportion of patients in their study showed increases in dystrophin expression . In theory, the right small molecule drug can allow ribosomes to skip over premature stops to make full length, fully functional dystrophin, halting disease progression. It seems that in practice, this reality does not yet exist. Dr. Sampson hopes that small molecule treatments will improve in the future . However, point mutations like premature stops only represent a fraction of all patients .
Another method uses antisense oligonucleotides (AONs) to skip over problematic exons . During splicing, exons must contain an exonic splicing enhancer (ESE) to be retained in the mRNA transcript. ESEs recruit splicing factors that ensure exon inclusion. AONs complementary to an ESE bind to the ESE, abolishing the binding of splicing factors and causing the exon to be spliced out . Exon-skipping may partially restore dystrophin expression in a variety of contexts. Exons could have problematic point mutations that need to be skipped over. Or, an exon deletion could disrupt the reading frame, such that skipping neighboring exons would restore the reading frame. However, exon skipping produces shortened dystrophin, which significantly improves but cannot eliminate the disease phenotype entirely. Furthermore, because each exon is unique, a different treatment must be developed for each specific exon to be skipped. Some already exist on the market, but only for the most commonly problematic exons.
However, AON exon-skipping and nonsense mutation readthrough have already emerged in clinical trials or on the market, making them a more present solution than gene therapy or CRISPR. Nonsense mutation readthrough must improve and exon-skipping treatments must come to include patients with a larger variety of mutations. Exon-skipping remains a good way to mitigate the effects of DMD while more effective treatments are developed. Additionally, if nonsense mutation readthrough therapies become effective, they will be complete cures in their own right.
CRISPR/cas9, gene replacement therapy, and nonsense mutation readthrough can restore fully functional dystrophin, but require much development before patients can use them. Exon-skipping therapies are more readily available but do not wholly cure DMD. Restoring dystrophin attacks the problem at the source, making it an integral part of the fight against DMD.
Even without a present cure, we still have power against the disease. Patients can fight back with quality health care targeting the damage inflicted by DMD. Seeing DMD patients daily as a nurse, Guzman stresses the importance of comprehensive health care:
While research is crucial, we must not forget that while studies are ongoing, [patients] will get weaker. We need to make sure that the patient is well cared for in [their] cardiac function […] There is quite a difference between our patients that have parents championing them with good care and follow up, vs. patients that are not being compliant with care recommendations .
Guzman urges that while a cure remains on the minds of most patients and their families, they must not forget to care for the symptoms of DMD in the present. Furthermore, the fact that patients with good care fare much better than those without it demonstrates the importance of quality care. Similarly, Dr. Sampson notes the importance of monitoring patients’ bone and pulmonary health and preventing contractures . Unfortunately, scheduling all these appointments with various specialists is difficult and overwhelming for patients and their families. Additionally, many DMD clinics do not have all specialists present at once . DMD patients need more standardized care programs, which integrate multiple specialists into a single check-up. Patients would benefit from a healthcare liaison who helps them comply with care standards and schedule appointments, especially if clinics cannot schedule all specialists into one appointment.
But care encompasses much more than tending to physical health. If our culture becomes more supportive of folks with disabilities, the outcome for DMD patients will improve. While this article focuses on DMD, we must remember that social justice for folks with disabilities at large remains an important goal to improve the lives of countless people, not just those with DMD. The motivation for cures of serious diseases must originate from a desire to reduce suffering, rather than a desire to remedy resulting inabilities out of the belief that they reduce the value of the people who have them. Neither should we accept the personal ‘shortcomings’ of folks with disabilities as the source of their barriers; social attitudes and infrastructure are the real shortcomings we must resolve. Siskind argues that we “need to have a better society for people who are differently-abled,” filled with improved infrastructure, more considerations, and better mindsets for those using wheelchairs . Alleviating the challenges of DMD in the present includes lessening the burden of wheelchair use. If we ease the daily lives of those with disabilities and provide infrastructure that expands their freedom, their quality of life will improve. Even if supporting the disability-related needs of DMD patients does not directly benefit their physical health, it improves their emotional state, which in turn improves physical well-being. If we look after the whole person – mind, body, and spirit – we will reduce much suffering from DMD right now.
Addressing Scientific Gaps in Knowledge
Improved modeling of the disease will help tackle the challenges of implementing new biological discoveries as treatments and better care standards. Human induced pluripotent stem cells (iPSCs) provide a more accurate cellular model in which to study DMD than mouse models. iPSCs are formed by extracting a patient’s cells and converting them to stem cells . From there, they could be turned into muscle cells to study DMD. Vera et al describe one study, which found an herbal compound that reduced oxidative stress in DMD heart cells derived from iPSCs . Thus, iPSCs are already clarifying disease processes and elucidating treatments to alleviate the damage caused by DMD. Artificial intelligence can improve conceptual models of DMD. In particular, natural language processing (NLP), can comb through existing literature to find possible treatment combinations or characterize processes of DMD that humans cannot . Thus, AI could find new information about DMD within existing studies and published work. Even modeling at the stage of clinical trials is important. According to a doctorate of neurology from Stanford University who prefers to remain unnamed, “improved genotype:phenotype information before and after various treatments will provide real world data that will clarify the disease process, response to treatment and cause of unmet needs .” More modeling of the disease will reveal more about the processes of and players implicated in DMD. Thus we will be able to better predict what we need to remedy and how diseased muscle cells will respond to a given treatment. When improved models fill gaps in knowledge, solutions to the implementation of treatments become more clear. In the case of iPSCs, patients can even have personalized models, allowing them to receive more targeted treatments. Furthermore, these increases in knowledge will inspire brand new treatment approaches in addition to perfecting current ones for clinical implementation.
Basic research improves the general understanding of biology, eventually filling gaps in knowledge necessary to implement CRISPR, gene replacement therapy, and nonsense mutation read-through as cures. The anonymous researcher expressed that studying molecular and cellular biology, particularly in diseased cells, shows promise in the fight to cure DMD . For example, improved knowledge of cell biology can better illuminate how cells import materials from their surroundings and respond to exogenous DNA. Such knowledge could lead to more effective deliveries to muscle cells and reveal why replacement genes in gene therapy lose effectiveness over time. We must also increase our knowledge specifically on the cell types that will be the target of cures. Even basic research in seemingly distant fields could bring forth unforeseen possibilities. For example, CRISPR/cas9 would not exist without having studied bacterial immunity. And computer science led to AI and NLP, which may one day lead the fight against DMD. Basic research provides the raw materials from which new cures are built.
Public Education and Outreach
Finally, public education will be necessary to both secure proper funding for research and promote screening to limit the disease’s toll. Dr. Senesac calls for public education to increase awareness of the disease . Although the disease is devastating, the public will stand still until they become aware of its existence. As a small community, the DMD community can only make progress with the support of the public. Without knowledge, the public cannot provide support for movements, especially those securing more funding from corporations and governments. Three of five expert respondents directly stated that more money was needed for research [5, 7, 8]. Given that these respondents have direct ties to research, their personal opinion that more funding is needed speaks volumes. A better-educated public also results in parents who are more aware of screening options. Nurse Guzman explains that early screening can “not only identify the child that is affected, and [allow families to] start making interventions early, but can… have an impact on family planning .” She recalls that some families discovered their status as carriers too late, having multiple children with DMD. Had they known of their first kid’s diagnosis earlier, they would have not had more kids or screened embryos . Educating parents or prospective parents on screening expands the ways they can look out for the health of their kids. An old adage suggests knowledge is power. In the case of DMD, education is an integral weapon in the fight.
Duchenne muscular dystrophy is responsible for immense suffering, but we have tools with which to fight the disease: research to restore dystrophin expression, improved care, improved modeling, and public education. There are various promising approaches to treatments requiring varying amounts of development to become a reality. For example, patients are already using exon-skipping therapies, while CRISPR requires much more research to be safely applied to humans. In addition to attacking the root cause of DMD, caring for the whole person results in better health and improved quality of life in the face of devastation. Basic research and modeling will close the necessary gaps in biology to develop improved treatments and care regimens. Finally, public education brings awareness to the issue to instill action against DMD, potentially overcoming the issue of funding. We have many tools to fight DMD, and winning the battle requires the use of all of them. Likewise, genetic counselor Siskind remarks that “we are a long way from truly curing DMD, and people with DMD need resources from many different avenues .”
In reality, the problem is not DMD alone. The approaches outlined here could apply to other monogenic diseases. But the issue of disease in general extends far beyond science. In addition to being a biological issue, disease is a social, economic, and political issue. We cannot act on any disease without the support of entire societies worldwide. Fighting disease requires many people working together to not only find a cure but to care for those ailing from them in the present. Science is a necessary component of fighting disease, but it is not sufficient. When we fight disease, we fight with our heads and our hearts.
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- Sampson, Jacinda, MD, PhD. Interview conducted by Alex Neupauer. May 13, 2022.
- Guzman, Jessica A., RN. Interview conducted by Alex Neupauer. May 13, 2022.
- Siskind, Carly, MS, CGC. Interview conducted by Alex Neupauer. May 13, 2022.
- Senesac, Claudia, PT, PhD, PCS. Interview conducted by Alex Neupauer. May 13, 2022.
- Anonymous. Interview conducted by Alex Neupauer. May 13, 2022.
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