What are the preclinical assets being developed for DMD exon 53?

Overview of Duchenne Muscular Dystrophy (DMD)

Pathophysiology and Genetic Basis
Duchenne muscular dystrophy (DMD) is a severe, progressive X‐linked neuromuscular disorder characterized by the absence of functional dystrophin—a large protein that plays a critical role in maintaining the integrity and stability of muscle cell membranes. Mutations in the DMD gene, which often result in out‐of‐frame deletions, lead to premature truncation of the protein; consequently, muscle fibers become highly susceptible to contraction-induced damage, inflammation, and fibrosis. This results in progressive muscle wasting, loss of ambulation, and ultimately, cardiac and respiratory failure. Because dystrophin is essential for linking the cytoskeleton to the extracellular matrix through the dystrophin–glycoprotein complex, mutations cause instability and an inability to repair damage over time. The genetic basis of the disease is highly heterogeneous, with more than 7,000 different mutations reported. However, the majority of the mutations are deletions that disrupt the reading frame, leading to a complete absence of dystrophin.

Importance of Exon 53 in DMD
Exon 53 represents a critical target in the therapeutic management of DMD for a subset of patients whose specific mutations are amenable to exon skipping. By targeting exon 53, therapies aim to restore the reading frame of the mRNA transcript so that a shorter, yet partially functional, version of dystrophin is produced—a phenotype that resembles the milder Becker muscular dystrophy (BMD). This approach is significant because even modest levels of dystrophin restoration are associated with improved muscle function and delayed disease progression. The rationale behind targeting exon 53 specifically is that skipping this exon can potentially address mutations in approximately 8% of the DMD patient population, offering hope for patients who have mutations that render them ineligible for exon‐51 targeting therapies. The critical importance of exon 53 lies not only in its ability to restore an open reading frame but also in generating dystrophin variants that may better stabilize muscle cell membranes, reduce degeneration, and ultimately improve patient quality of life.

Current Preclinical Assets for Exon 53

Types of Therapeutic Approaches
The preclinical assets being developed for DMD exon 53 primarily revolve around oligonucleotide-based therapeutics with innovative coupling technologies to enhance tissue uptake and activity:

• PepGen Inc. has developed PGN-EDO53, a product candidate that employs their proprietary Enhanced Delivery Oligonucleotide (EDO) technology. While PGN-EDO51, targeting exon 51, has entered clinical trials, their pipeline includes PGN-EDO53 for exon 53 skipping. Preclinical studies performed in non-human primates (NHPs) have demonstrated robust exon 53 skipping following both single and multiple dosing regimens. The EDO technology enhances the cellular uptake and biodistribution of the therapeutic antisense oligonucleotides, hoping to achieve higher exon skipping levels compared with conventional naked oligonucleotides.

• Dyne Therapeutics has been advancing their FORCE™ platform—a modular approach based on a delivery system that couples a phosphorodiamidate morpholino oligomer (PMO) to an antibody fragment targeting the transferrin receptor on muscle cells. Their preclinical data include the in vitro demonstration of exon 53 skipping using a FORCE conjugate. In particular, preclinical results showed dose-responsive exon 53 skipping and restoration of dystrophin expression. This approach not only improves the delivery of exon-skipping molecules to muscle tissues but also opens the door to a broader DMD franchise by rapidly identifying candidates for other exons such as 45 and 44.

• Several patents have also disclosed methods involving small molecules that can enhance exon skipping. These small molecules, when co-administered with antisense oligonucleotides or used on their own, aim to modulate splicing machinery and facilitate exon exclusion. Although these approaches are generally not exclusive to exon 53, they offer alternative strategies and potential combination therapies that could improve the overall efficacy of exon skipping in DMD patients, including those with exon 53 amenable mutations.

Mechanisms of Action
The various therapeutic modalities under development for exon 53 share a common goal: to modulate RNA splicing in order to exclude exon 53 from the mature mRNA transcript. This allows muscle cells to produce a truncated but functional dystrophin protein. The mechanism of action can be described from several perspectives:

• Antisense Oligonucleotides (AONs):
In the majority of these preclinical assets, AONs are chemically modified nucleotide sequences designed to bind to specific splicing regions on the pre-mRNA. For exon 53, they typically hybridize to sequences within exon 53 or at the flanking intronic regions to mask splicing signals. This masking forces the splicing machinery to skip the exon, restoring the open reading frame. The chemical modifications—such as those used in PMOs—enhance stability and bioavailability while minimizing off-target effects.

• Enhanced Delivery Technologies:
The innovation in many of these preclinical assets is not solely in the design of the AON itself but also in its delivery. For instance, the EDO technology employed by PepGen couples the oligonucleotide with a peptide that enhances cellular uptake and facilitates efficient delivery to muscle tissue. Similarly, Dyne Therapeutics’ FORCE platform attaches the oligonucleotide to an antibody fragment that binds the transferrin receptor 1, highly expressed on muscle cells. This improves the pharmacokinetic and pharmacodynamic profile of the therapeutic by increasing muscle tissue concentrations and ensuring that the drug reaches both skeletal and potentially cardiac muscles.

• Small Molecule Adjuvants:
A third mechanism, identified in the patents, involves the use of small molecules that modulate splicing factors or enhance the activity of oligonucleotides. These molecules may act by altering the conformation of the pre-mRNA or by interacting with regulatory proteins in the spliceosome. Such co-therapies could potentiate exon skipping efficacy, thereby increasing the amount of dystrophin protein produced.

Developmental Status and Challenges

Current Progress in Preclinical Studies
Recent preclinical studies for the exon 53 assets have shown promising efficacy results in both in vitro and in vivo models. Specifically:

• PepGen’s PGN-EDO53 has demonstrated robust exon 53 skipping in non-human primate models. Preclinical studies used both single dose administrations and repeated dosing protocols, showing a dose-dependent increase in exon skipping levels. In multiple dose experiments, there was a notable accumulation of exon-skipped transcripts in muscle tissue, which significantly surpassed levels observed with chemically similar naked oligonucleotides administered in a comparable fashion. These data provide a strong rationale for advancing PGN-EDO53 to IND-enabling preclinical studies in the near future.

• Dyne Therapeutics has presented foundational in vitro data for its FORCE™ platform conjugate targeting exon 53 at major scientific congresses such as the World Muscle Society meeting. The in vitro assays demonstrated that the FORCE conjugate can induce exon 53 skipping in muscle cell cultures, with a dose-responsive effect. The data also suggested that the mechanism is robust enough to lead to the expression of a truncated dystrophin protein, a finding critical to meeting the clinical thresholds for efficacy. Although these preclinical data are still in early stages compared to clinical study candidates like DYNE-251, the modularity of the FORCE platform allows for rapid expansion into other exons, which includes exon 53, thereby broadening the potential impact of the technology.

• Preclinical development is also ongoing in the domain of small molecule enhancers as described in several patents. While details regarding such compounds specific for exon 53 skipping are less abundant compared to oligonucleotide conjugates from PepGen and Dyne, the existence of reliable preclinical models has enabled researchers to evaluate these compounds in vitro. The quantitative assessments involve evaluating exon skipping percentages using PCR-based methods and densitometric analyses after dosing muscle cells with candidate molecules. These studies indicate that with appropriate chemical modification, small molecules can serve as valuable adjuvants to enhance the efficiency of antisense therapeutics for exon 53 skipping.

Challenges in Development
Despite the promising preclinical data, several challenges remain in advancing exon 53 assets from bench to bedside:

• Delivery to Target Tissues: One of the key challenges is the efficient delivery of these oligonucleotide therapies to all muscle types affected by DMD—including skeletal, respiratory, and cardiac muscles. While enhanced delivery technologies such as EDO and FORCE are promising, consistent and uniform biodistribution remains difficult to achieve. For instance, lower uptake in cardiac muscle compared with skeletal muscles is a recurrent issue, and strategies to overcome this are continuously being optimized.

• Dose Optimization and Toxicity: Achieving a balance between therapeutic efficacy and toxicity is critical. Preclinical studies in NHP models have highlighted the importance of dosing regimens that yield sufficient levels of exon skipping without inducing off-target effects or immune responses. In addition, kidney toxicity is a concern for several antisense oligonucleotides, as seen with related products like golodirsen, though these toxicities were not observed in all studies. Monitoring kidney function and adjusting dosing protocols accordingly is an essential component of preclinical safety evaluations.

• Translational Consistency: There exists a known challenge in translating the results obtained from preclinical models (mice and non-human primates) to human patients. Variability in splicing machinery and differences in muscle physiology can contribute to inconsistent efficacy. In some cases, the magnitude of exon skipping observed in animal models does not fully predict the level of dystrophin restoration in human trials, raising concerns over the predictive accuracy of current in vitro and in vivo models.

• Manufacturing and Scale-up Challenges: As with any advanced therapeutic modality, scaling up production of these modified oligonucleotides (especially those coupled with peptide or antibody components) presents significant manufacturing challenges. This includes ensuring chemical consistency, purity, and stability of the final product, all of which must meet stringent regulatory standards before moving into clinical trials.

• Regulatory Considerations: Given that exon skipping is a personalized medicine approach, regulatory pathways must accommodate a multiplicity of condition-specific therapies. Each exon-specific asset—such as those targeting exon 53—must demonstrate not only safety and efficacy in a defined patient subgroup but also a meaningful clinical benefit over existing standards of care. This creates an additional layer of complexity in clinical trial design, endpoint selection, and ultimately, market approval.

Future Directions and Potential Impact

Prospective Clinical Applications
If preclinical assets for exon 53 continue to demonstrate their efficacy and safety, the prospects for future clinical applications are highly encouraging:

• For patients with DMD mutations amenable to exon 53 skipping, therapies such as PGN-EDO53 and the FORCE™ conjugate have the potential to restore dystrophin expression to therapeutically meaningful levels. The ultimate goal is not complete normalization of dystrophin levels, but rather enough production to stabilize the muscle membrane, attenuate disease progression, and enhance overall quality of life. Given that even small improvements in dystrophin production have correlated with clinical benefit, these assets could open new avenues for treatment for approximately 8% of DMD patients.

• Exon 53 skipping therapies could be used as stand-alone treatments for DMD or in combination with other therapies (such as corticosteroids or gene therapies) to produce synergistic benefits. The modularity of both the EDO and FORCE platforms suggests that, in the future, multi-exon skipping strategies may be developed to provide broader coverage for various mutations, further personalizing the treatment approach for DMD patients.

• The development of small molecule enhancers, as an adjunct to antisense oligonucleotides, holds promise not only as a potentiator of exon skipping but also as a possible means to reduce dosing frequency and improve overall patient compliance. Such combination regimens could represent a new standard of care in DMD management if they prove effective in enhancing dystrophin production in clinical settings.

Innovations and Research Directions
Ongoing research in the field of DMD exon skipping is rapidly evolving, with several exciting innovations on the horizon:

• Optimization of Oligonucleotide Chemistry: Researchers continue to explore new chemical modifications that could further improve the stability, efficacy, and tissue penetration of antisense oligonucleotides. These include next-generation PMOs and alternative backbone chemistries that could lower the risk of toxicity and facilitate more uniform distribution to cardiac and respiratory muscles.

• Advanced Delivery Systems: Innovations in vector design, such as the incorporation of tissue-specific ligands or receptor-targeting moieties (as exemplified by the FORCE platform’s targeting of transferrin receptor 1), are being refined. Future research will focus on engineering conjugates that optimally target difficult-to-reach tissues, ensuring that both skeletal and cardiac muscles receive adequate therapeutic concentrations.

• Combination Therapies: An emerging area of research is the evaluation of combination treatment strategies. This may involve the concomitant use of exon skipping agents with small molecule enhancers, other gene therapies, or even CRISPR-based editing tools. Combination regimens are being investigated in preclinical studies to determine if synergistic effects can overcome the limitations of single-agent therapies and produce more robust dystrophin expression.

• Preclinical Model Improvement: To better predict clinical efficacy, efforts are underway to refine animal models of DMD, including advancing hiPSC-derived muscle cells and humanized mouse models. These models are expected to provide more accurate assessments of pharmacokinetics, toxicity, and overall therapeutic benefit, thereby streamlining the transition from preclinical studies to clinical trials.

• Biomarker Development: A critical aspect of future research involves the identification of reliable biomarkers that can be used to measure therapeutic efficacy. This includes not only quantifying dystrophin protein levels but also evaluating functional outcomes such as muscle strength and contractility. Improved biomarkers will aid in the early prediction of clinical benefit and facilitate regulatory approval processes.

• Regulatory Strategy Evolution: As the field of precision genetic medicine grows, regulatory authorities are adapting their guidelines to account for exon-skipping therapies. Future research directions include the development of standardized protocols for evaluating these therapies, as well as the design of clinical trials that incorporate natural history data and surrogate endpoints. This will be crucial in bringing effective exon 53 therapies to market in a timely manner.

In summary, the preclinical assets for DMD exon 53 being developed today represent a convergence of advanced oligonucleotide chemistry, innovative delivery technologies, and novel co-therapeutic strategies. The efforts by companies such as PepGen and Dyne Therapeutics highlight the promise of their respective platforms—EDO for targeted enhanced delivery and FORCE for receptor-mediated uptake—in achieving robust exon skipping and dystrophin restoration. These technological innovations are underpinned by rigorous preclinical studies in both in vitro systems and non-human primate models, which have demonstrated significant levels of exon 53 skipping and the accumulation of exon-skipped transcripts in muscle tissues.

However, challenges remain. Delivering consistent therapeutic concentrations to all affected muscle types, particularly the heart, constitutes a major hurdle. Moreover, optimizing dosing regimens to maximize efficacy while minimizing toxicity, as well as scaling up production under stringent regulatory standards, are issues that continue to demand innovative solutions. Despite these challenges, the ongoing progress in preclinical development and the emergence of combination strategies using small molecule enhancers underscore the potential impact of these therapies on the DMD patient community.

Looking ahead, as additional preclinical studies refine these approaches and address the current obstacles, there is a strong potential for the translation of these assets into clinical trials. The eventual goal is to offer a life-changing therapy to DMD patients whose mutations are amenable to exon 53 skipping, thereby complementing existing therapies for other exons and broadening the scope of personalized treatment options available. Innovations in biomarker development, refined preclinical models, and evolving regulatory frameworks will collectively drive this transition, ultimately bringing exon 53-targeted therapies into clinical practice and improving outcomes for a significant subset of DMD patients.

In conclusion, the preclinical assets for DMD exon 53 are diverse and innovative. They range from peptide-conjugated antisense oligonucleotides utilizing enhanced delivery technologies to modular antibody fragment platforms and the exploration of small molecule adjuvants. These multifaceted approaches attempt to overcome the inherent challenges of delivering therapeutic agents systemically, achieving robust exon skipping, and ensuring that the resultant dystrophin expression is sufficient to produce clinical benefit. Continued preclinical research is essential to optimize these platforms and address the remaining challenges related to dosing, delivery, and long-term safety. If these assets progress successfully through the preclinical phase, they hold the promise of significantly altering the treatment landscape for DMD patients, especially those with mutations amenable to exon 53 skipping, paving the way for improved patient outcomes and a better quality of life.