RNA Therapeutics Targeting Skeletal Muscle: Emerging Antisense and Gene-Modifying Strategies

Response to Reviewer 1 Comments

1. Summary

We sincerely thank the reviewer for the constructive and insightful comments. We have carefully revised the manuscript accordingly.

Comments 1: The use of "FCMD" to refer to facioscapulohumeral muscular dystrophy is incorrect; the appropriate abbreviation is "FSHD." "FCMD" conventionally refers to Fukuyama congenital muscular dystrophy.

Response 1: Thank you for pointing this out. We fully agree with the reviewer. We have corrected all instances of “FCMD” to “FSHD” throughout the manuscript. (Page 9, Section 3.3, Lines 333)

Comments 2: In discussing the use of ASO, the authors state that they activate "minimal innate immune response" (page 2, section 1.2). However, this may be too broad a statement as the activation of the innate immune response can depend on conjugates, AOS sequence and dose.

Response 2: We appreciate this important clarification. We have revised the sentence to provide a more accurate and nuanced description, acknowledging that innate immune activation depends on chemistry, sequence, dose, and conjugates.

Page 2, Section 1.1, Lines 67–69

“PMOs generally exhibit low innate immune activation and limited nonspecific protein binding; however, this can vary depending on sequence, dose, and conjugated ligands.”

Comments 3: Similarly, the statement that "AAVs are widely used and relatively safe" does not convey current concerns regarding the safety of AAV gene therapies. The use of high-dose systemic administration for neuromuscular diseases has shown number of dose-dependent, immune-mediated, and multi-organ toxicities such as hepatotoxicity, DRG toxicity, and immune responses to the capsid. This section should be revised to provide a more balanced discussion of these safety considerations.

Response 3:

Thank you for your valuable suggestion. We agree with the reviewer and have revised the AAV section to include a balanced discussion of dose-dependent and immune-mediated toxicities with multiple organs.

Page 5, Section 2.3, Lines 177–181

“Although extensively utilized in gene therapy applications, recent clinical experience with AAV vectors—particularly with high‑dose systemic administration—has revealed toxicities including immune responses to the capsid, complement activation, hepatotoxicity, and dorsal root ganglion (DRG) toxicity.”

We have also added multiple references to support this statement.

References

Hawley, Z.C.E.; Pardo, I.D.; Cao, S.; Zavodszky, M.I.; Casey, F.; Ferber, K.; Luo, Y.; Hana, S.; Chen, S.K.; Doherty, J.; et al. Dorsal Root Ganglion Toxicity after AAV Intra-CSF Delivery of a RNAi Expression Construct into Non-Human Primates and Mice. Molecular Therapy 2025, 33, 215–234.

51.        Mingozzi, F.; High, K.A. Immune Responses to AAV Vectors: Overcoming Barriers to Successful Gene Therapy. Blood 2013, 122, 23–36.

52.        Hordeaux, J.; Lamontagne, R.J.; Song, C.; Buchlis, G.; Dyer, C.; Buza, E.L.; Ramezani, A.; Wielechowski, E.; Greig, J.A.; Chichester, J.A.; et al. High-Dose Systemic Adeno-Associated Virus Vector Administration Causes Liver and Sinusoidal Endothelial Cell Injury. Molecular Therapy 2024, 32, 952–968.

Comments 4: The authors inconsistently use RNA-guided gene modification and genome editing technologies. This is especially unclear in sections 1.3 and 3.1. ADAR-mediated RNA editing is grouped together with DNA-editing systems in 1.3. ADAR does not mediate gene modification at the genomic level; rather, it edits RNA transcripts.

Response 4:

We apologize for this ambiguity and thank you for highlighting this point.

We have revised Sections 1.3 and 3.1 to clearly distinguish:

DNA-level genome editing (CRISPR–Cas, base editing, prime editing) vs.

RNA-level editing (ADAR-mediated A-to-I editing)

Page 2, Section 1.3, Lines 83–88

“RNA-guided gene-modifying technologies encompass both DNA-level genome editing and RNA-level transcript editing. DNA-level genome editing approaches include the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR–Cas) systems, which introduce double-strand breaks (DSBs) at target DNA sequences complementary to a guide RNA.”

Page 3, Section 1.3, Lines 96–98

“In contrast, RNA-level editing is mediated by adenosine deaminases acting on RNA (ADAR) are endogenous enzymes that mediate adenosine-to-inosine (A-to-I) RNA editing within double-stranded RNA structures.”

Additional clarification were also added in Section 3.1.

Page 8, Section 3.1, Lines 279–280

“RNA-guided RNA-editing approaches have also been investigated for the treatment of DMD.”

Comments 5: This manuscript heavily cites other reviews and is missing a number of seminal foundational papers. For example, in DMD exon-skipping studies these should include Dunckley et al (1998), Mann et al. (2001), and Aartsma-Rus et al. (2003 and 2004). The authors should reference appropriate primary papers such as first demonstrations and landmark discoveries, throughout this review.

Response 5:

We appreciate this valuable suggestion. We have added the foundational primary papers.

Added references:

DMD:

Dunckley, MG.; Manoharan, M.; Villiet, P.; Eperon, I.C.; Dickson, G. Modification of Splicing in the Dystrophin Gene in Cultured Mdx Muscle Cells by Antisense Oligoribonucleotides. Hum Mol Genet. 1998, 7(7):1083-90.

Wilton, S.D.; Lloyd, F.; Carville, K.; Fletcher, S.; Honeyman, K.; Agrawal, S.; Kole, R. Specific Removal of the Nonsense Mutation from the Mdx Dystrophin MRNA Using Antisense Oligonucleotides. ; Neuromuscul Disord. 1999, 9(5):330-8.
Mann, C.J.; Honeyman, K.; Cheng, A.J.; Ly, T.; Lloyd, F.; Fletcher, S.; Morgan, J.E.; Partridge, T.A.; Wilton, S.D. Antisense-Induced Exon Skipping and Synthesis of Dystrophin in the Mdx Mouse. Proc Natl Acad Sci U S A. 2001, 98(1): 42-7.

Aartsma-Rus, A.; Janson, A.A.M.; Kaman, W.E.; Bremmer-Bout, M.; Van Ommen, G.-J.B.; Den Dunnen, J.T.; Van Deutekom, J.C.T. Antisense-Induced Multiexon Skipping for Duchenne Muscular Dystrophy Makes More Sense. Am J Hum Genet. 2004, 74(1), 83-92.

Aartsma-Rus, A.; Janson, A.A.M.; Kaman, W.E.; Bremmer-Bout, M.; den Dunnen T., J.T.; Baas, F.; van Ommen, G.J.B.; van Deutekom, J.C.T. Therapeutic Antisense-Induced Exon Skipping in Cultured Muscle Cells from Six Different DMD Patients. Hum. Mol. Genet. 2003, 12, 907–914.

Goyenvalle, A.; Vulin, A.; Fougerousse, F.; Leturcq, F.; Kaplan, J.C.; Garcia, L.; Danos, O. Rescue of Dystrophic Muscle through U7 SnRNA-Mediated Exon Skipping. Science. 2004, 306, 1796–1799.

DM1:

Wheeler, T.M.; Leger, A.J.; Pandey, S.K.; Mac Leod, A.R.; Wheeler, T.M.; Cheng, S.H.; Wentworth, B.M.; Bennett, C.F.; Thornton, C.A. Targeting Nuclear RNA for in Vivo Correction of Myotonic Dystrophy. Nature 2012, 488, 111–117.

van Agtmaal, E.L.; André, L.M.; Willemse, M.; Cumming, S.A.; van Kessel, I.D.G.; van den Broek, W.J.A.A.; Gourdon, G.; Furling, D.; Mouly, V.; Monckton, D.G.; et al. CRISPR/Cas9-Induced (CTG⋅CAG)n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing. Molecular Therapy 2017, 25, 24–43.

Piasecka, A.; Szcześniak, M.W.; Sekrecki, M.; Kajdasz, A.; Sznajder, Ł.J.; Baud, A.; Sobczak, K. MBNL Splicing Factors Regulate the Microtranscriptome of Skeletal Muscles. Nucleic Acids Res. 2024, 52, 12055–12073.

FSHD:

Lemmers, R.J.L.F.; Van Der Vliet, P.J.; Klooster, R.; Sacconi, S.; Camaño, P.; Dauwerse, J.G.; Snider, L.; Straasheijm, K.R.; Van Ommen, G.J.; Padberg, G.W.; et al. A Unifying Genetic Model for Facioscapulohumeral Muscular Dystrophy. Science. 2010, 329, 1650–1653.

Comments 6: The reference list requires additional editing. Citation formatting is inconsistent, and several references contain placeholder metadata and/or incomplete bibliographic information.

Response 6:

Thank you for pointing this out. We apologize for the inconsistent reference information. We have thoroughly revised the entire reference list to ensure correct formatting, removal of placeholder metadata, and completion of all bibliographic details. The updated reference list now follows the journal’s required style consistently.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1:    (in red)

5. Additional clarifications

We have removed all corrected articles (Refs 46, 53, 82, 96) as requested by the editor and replaced them with new or existing references.

Ref. 46

(Original)

49.      Birnkrant, D.J.; Bushby, K.; Bann, C.M.; Apkon, S.D.; Blackwell, A.; Brumbaugh, D.; Case, L.E.; Clemens, P.R.; Hadjiyannakis, S.; Pandya, S.; et al. Diagnosis and Management of Duchenne Muscular Dystrophy, Part 1: Diagnosis, and Neuromuscular, Rehabilitation, Endocrine, and Gastrointestinal and Nutritional Management. Lancet Neurol. 2018, 17, 251–267.

(Replaced)

Messina, S.; Vita, G.L. Clinical Management of Duchenne Muscular Dystrophy: The State of the Art. Neurological Sciences 2018, 39, 1837–1845.

Ref. 53

(Original)

Frank, D.E.; Schnell, F.J.; Akana, C.; El-Husayni, S.H.; Desjardins, C.A.; Morgan, J.; Charleston, J.S.; Sardone, V.; Domingos, J.; Dickson, G.; et al. Increased Dystrophin Production with Golodirsen in Patients with Duchenne Muscular Dystrophy. Neurology 2020, 94, E2270–E2282.

(Replaced)

Wagner, K.R.; Kuntz, N.L.; Koenig, E.; East, L.; Upadhyay, S.; Han, B.; Shieh, P.B. Safety, Tolerability, and Pharmacokinetics of Casimersen in Patients with Duchenne Muscular Dystrophy Amenable to Exon 45 Skipping: A Randomized, Double-Blind, Placebo-Controlled, Dose-Titration Trial. Muscle Nerve 2021, 64, 285–292.

Ref. 82

(Original)

49.      Rowel, K.; Lim, Q.; Maruyama, R.; Echigoya, Y.; Nguyen, Q.; Zhang, A.; Khawaja, H.; Chandra, S.; Jones, T.; Jones, P.; et al. Inhibition of DUX4 Expression with Antisense LNA Gapmers as a Therapy for Facioscapulohumeral Muscular Dystrophy. Proc Natl Acad Sci U S A. 2020, 117(28), 16509-16515.

(Replaced)

Zhang, A.; Lim, K.R.Q.; Chen, Z.; Yokota, T.; Chen, Y.-W. DUX4 Reduction and Muscle Function Improvement by Subcutaneous Delivery of Gapmer Antisense Oligonucleotides. Mol. Ther. Nucleic Acids 2026, 37, 102791.

Bouwman, L.F.; den Hamer, B.; van den Heuvel, A.; Franken, M.; Jackson, M.; Dwyer, C.A.; Tapscott, S.J.; Rigo, F.; van der Maarel, S.M.; de Greef, J.C. Systemic Delivery of a DUX4-Targeting Antisense Oligonucleotide to Treat Facioscapulohumeral Muscular Dystrophy. Mol. Ther. Nucleic Acids 2021, 26, 813–827.

Ref. 96

(Original)

Coenen-Stass, A.M.L.; Mcclorey, G.; Manzano, R.; Betts, C.A.; Blain, A.; Saleh, A.F.; Gait, M.J.; Lochmüller, H.; Wood, M.J.A.; Roberts, T.C. Identification of Novel, Therapy-Responsive Protein Biomarkers in a Mouse Model of Duchenne Muscular Dystrophy by Aptamer-Based Serum Proteomics. Sci. Rep. 2015, 5.

(Replaced)

Molinaro, M.; Torrente, Y.; Villa, C.; Farini, A. Advancing Biomarker Discovery and Therapeutic Targets in Duchenne Muscular Dystrophy: A Comprehensive Review. Int. J. Mol. Sci. 2024, 25.

108.      Coenen-Stass, A.M.L.; Sork, H.; Gatto, S.; Godfrey, C.; Bhomra, A.; Krjutškov, K.; Hart, J.R.; Westholm, J.O.; O’Donovan, L.; Roos, A.; et al. Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD. Mol. Ther. Nucleic Acids 2018, 13, 1–15.

Response to Reviewer 2 Comments

1. Summary

We sincerely thank the reviewer for the constructive and insightful comments. We have carefully revised with additional explanation and further elaboration of different therapeutic approaches.

Comments 1: Line 57: The sentence should be corrected, as a word appears to be missing, making it difficult to understand.

Response 1: Thank you for pointing this out. We have corrected the sentence to ensure clarity and to specify the molecular processes affected by PMOs.

Page 2, Section 1.1, Lines 64–65

“PMOs are synthetic oligonucleotides designed to bind specific RNA sequences and sterically block pre‑mRNA splicing or translation.”

Comments 2: ASO chemistries besides PMOs should also be discussed.

Response 2: We agree with the reviewer. We have expanded Section 1.1 to include additional ASO chemistries, including 2′‑O‑methoxyethyl (2′‑MOE), locked nucleic acids (LNA), and phosphorothioate (PS) backbones, along with their pharmacological properties and safety considerations.

Page 2, Section 1.1, Lines 54–63

“Gapmer ASOs consist of a central DNA “gap” flanked by chemically modified nucleotides, typically 2′‑O‑methoxyethyl (2′‑MOE) or locked nucleic acids (LNA), to increase binding affinity to the target RNA [6]. Gapmer ASOs also normally contain phosphorothioate (PS) internucleotide linkages instead of natural phosphodiester bonds to enhance nuclease stability. However, PS linkages can contribute to toxicity through nonspecific protein binding, which remains a concern in clinical applications [8]. Another major class is RNase H–independent splice-switching oligonucleotides (SSOs), which bind to pre-mRNA and modulate splicing. SSOs commonly incorporate 2′‑MOE or other sugar modifications and typically include PS linkages for stability.”

Comments 3: An explanation of the AGO2 enzyme and its mechanism of action should be included.

Response 3: We appreciate this suggestion. We have added a concise explanation of AGO2 as the catalytic component of the RNA-induced silencing complex (RISC), including its endonucleolytic “slicer” activity.

Page 2, Section 1.2, Lines 73–78

“The antisense strand is incorporated into the RNA-induced silencing complex (RISC), which guides the complex to complementary target mRNA. Within RISC, the core catalytic component is Argonaute‑2 (AGO2), an endonuclease that cleaves the target mRNA through its “slicer” activity. This AGO2‑mediated cleavage generates site‑specific mRNA degradation, leading to effective gene silencing through the RNA interference (RNAi) pathway.”

Comments 4: detailed description of LNPs

Response 4: We agree. Section 2.2 has been expanded to include LNP composition (ionizable lipids, helper lipids, cholesterol, PEG-lipids), endosomal escape mechanisms, and recent advances in LNP engineering.

Page 5, Section 2.2, Lines 157–168

“Lipid nanoparticles (LNPs) are nanoscale vesicles composed of four key components: ionizable lipids, helper lipids, cholesterol, and PEG‑lipids, which together enable efficient encapsulation and delivery of nucleic acids. After cellular uptake by endocytosis, protonation of ionizable lipids in the acidic endosome promotes membrane destabilization and release of the RNA cargo into the cytosol.

The clinical utility of LNPs was demonstrated by their use in COVID‑19 mRNA vaccines, and LNPs are now considered one of the most promising platforms for RNA therapeutics. Patisiran, an LNP‑formulated siRNA targeting transthyretin (TTR) mRNA, became the first FDA‑approved RNAi therapeutic, validating systemic LNP delivery in humans. Recent engineering advances, including selective organ targeting (SORT) lipids, have broadened the applicability of LNPs to additional RNA modalities such as antisense oligonucleotides and guide RNAs.”

Comments 5: description of aptamers,

Response 5: We have added a brief explanation of aptamers, their SELEX-based discovery, and their emerging use as targeting ligands for RNA therapeutics.

Page 3, Section 1.4, Lines 103-122

“1.4. Aptamers

Aptamers are short, structured single-stranded RNA or DNA oligonucleotides that bind target molecules with high affinity and specificity, functioning similarly to antibodies. They are typically identified through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process, an iterative in vitro selection method that enriches oligonucleotides capable of binding a desired protein or receptor. Because of their small size, chemical tunability, and low immunogenicity, aptamers are increasingly used as targeting ligands to enhance the cell‑type specificity of RNA therapeutics, including siRNAs, ASOs, and oligonucleotide conjugates. Recent studies have demonstrated aptamer‑mediated delivery to muscle cells, highlighting their potential as modular components in next‑generation RNA delivery platforms for skeletal muscle diseases.”

Page 9, Section 3.2, Lines 327–331

“Aptamer‑based targeting strategies are also being explored to enhance the delivery of RNA therapeutics to skeletal muscle. Muscle‑homing RNA aptamers have been shown to increase the uptake of siRNAs and antisense oligonucleotides in muscle cells, demonstrating their potential as modular ligands for precision delivery in DMD models.”

Comments 6: description of transferrin receptor 1 (TfR1), especially in connection with peptide- and antibody-conjugate approaches

Response 6: We agree. We have added a dedicated explanation of TfR1 biology, receptor-mediated transcytosis, and its relevance to peptide- and antibody–oligonucleotide conjugates (AOCs).

Page 4, Section 2.1, Lines 145–154

“Another important strategy involves targeting transferrin receptor 1 (TfR1), a highly expressed iron‑transport receptor on skeletal and cardiac muscle. TfR1 undergoes receptor‑mediated endocytosis and transcytosis, enabling efficient intracellular trafficking of ligand‑bound cargo. This pathway has been leveraged for peptide‑ and antibody–oligonucleotide conjugates (AOCs), in which TfR1‑binding antibodies or peptides facilitate tissue‑specific delivery of ASOs and siRNAs to muscle. Recent preclinical studies have demonstrated robust uptake and exon‑skipping activity in skeletal muscle using TfR1‑targeted AOCs, highlighting the receptor’s value as a delivery gateway for neuromuscular RNA therapeutics.”

Comments 7: Recent advances in miRNA-based and circRNA therapeutics should also be briefly discussed

Response 7: Thank you for your valuable comment. We have added a new paragraph summarizing miRNA inhibitors (e.g., Miravirsen), miRNA mimics, and emerging circRNA-based therapeutic platforms.

Page 3, Section 1.5, Lines 115–122

“1.5. MicroRNA (miRNA)‑based and circularRNA (circRNA) therapeutics

MicroRNA (miRNA) therapeutics include antisense inhibitors such as LNA‑modified antimiRs (e.g., Miravirsen targeting miR‑122) and miRNA mimics that restore downregulated miRNA activity. These approaches modulate post‑transcriptional gene regulation and are being explored across metabolic, cardiovascular, and neuromuscular diseases. Circular RNAs (circRNAs) have recently emerged as a stable RNA platform capable of long‑lasting protein expression or regulatory functions, and synthetic circRNAs are now being developed as next‑generation RNA therapeutics”

Comments 8: Challenges like immunogenecity, allele specificity and benefits of combinatorial platforms in therapeutic approaches

Response 8:

We agree with your suggestion. Section 4.3 (Safety considerations) has been expanded to address additional challenges, including immunogenicity and the need for allele‑specific discrimination in dominant‑negative or toxic‑gain‑of‑function disorders.

To address the reviewer’s point regarding the benefits of combinatorial platforms, we added a new subsection (Section 2.5, Future delivery approaches using combinatorial platforms) that summarizes emerging strategies such as antibody and peptide conjugates, AAV‑delivered RNAi, and ligand‑decorated nanoparticles, and highlights their advantages over single‑modality delivery systems.

Page 11, Section 4.3, Lines 423–429

“In addition to these platform‑specific toxicities, immune activation remains a major challenge for oligonucleotide therapeutics, particularly for unmethylated motifs or double‑stranded RNA structures that can stimulate innate immune sensors. Allele specificity is another critical safety consideration for dominant‑negative or toxic‑gain‑of‑function disorders, where incomplete discrimination between mutant and wild‑type alleles may lead to unintended loss of essential gene function.”

Page 5, Section 2.5, Lines 194–5

“2.5. Future delivery approaches using combinatorial platforms

Next‑generation delivery strategies aim to overcome the limitations of single‑modality platforms. Antibody‑ and peptide‑based conjugates provide enhanced tissue specificity, while AAV‑delivered RNAi enables long‑lasting gene silencing in post‑mitotic tissues. Cell‑penetrating ligands and engineered receptor‑binding peptides further improve intracellular trafficking and endosomal escape. Combinatorial platforms—such as AOC‑PMO, AOC‑siRNA, and ligand‑decorated LNPs—offer additive benefits by integrating the potency of oligonucleotides with the targeting precision of biologics, representing a promising direction for future RNA therapeutic development.”

Comments 9: Other clinical endpoints such as the Hammersmith Functional Motor Scale and not only the North Star Ambulatory Assessment

Response 9: We appreciate your suggestion. We have added the Hammersmith Functional Motor Scale (HFMS) and clarified its relevance for SMA patients.

Page 10-11, Section 4.2, Lines 407–411

“Functional endpoints such as the 6‑minute walk test, motor function measure (MFM), North Star Ambulatory Assessment (NSAA) for DMD, myotonia/respiratory assessments for DM1, reachable workspace for FSHD, and the Hammersmith Functional Motor Scale (HFMS) for spinal muscular atrophy remain essential for linking molecular effects to clinical benefit.”

Comments 10: Future approaches should also be discussed briefly, including topics such as: * Antibody conjugates * Peptide conjugates * AAV-delivered RNAi * Cell-penetrating ligands

Response 10: We have added a new subsection summarizing these emerging modalities and their translational potential.

Thank you for the suggestion. We have added a new subsection (Section 2.5) that briefly discusses emerging delivery approaches, including antibody conjugates, peptide conjugates, AAV‑delivered RNAi, and next‑generation cell‑penetrating ligands. This section also outlines how these modalities function as combinatorial platforms that expand the translational potential of RNA therapeutics.

Page 5, Section 2.5, Lines 194–5

“2.5. Future delivery approaches using combinatorial platforms

Next‑generation delivery strategies aim to overcome the limitations of single‑modality platforms. Antibody‑ and peptide‑based conjugates provide enhanced tissue specificity, while AAV‑delivered RNAi enables long‑lasting gene silencing in post‑mitotic tissues. Cell‑penetrating ligands and engineered receptor‑binding peptides further improve intracellular trafficking and endosomal escape. Combinatorial platforms—such as AOC‑PMO, AOC‑siRNA, and ligand‑decorated LNPs—offer additive benefits by integrating the potency of oligonucleotides with the targeting precision of biologics, representing a promising direction for future RNA therapeutic development.”

Comments 11: Table 1. may be omitted, as the information is already adequately covered in the text.

Response 11: We appreciate the suggestion. We have removed Table 1 as the relevant information is already included in the text.

Comments 12: Figure 1. upper part: Overview of major RNA-based therapeutic modalities – This figure should be replaced with a more informative and detailed illustration

Response 12: Thank you for your important suggestion. We have replaced Figure 1 with a more detailed schematic illustrating RNA-based therapeutics modalities.

Figure 1. Overview of major RNA‑based therapeutic modalities and delivery strategies. RNA therapeutics act at multiple levels of gene expression, including DNA or RNA‑targeting RNA‑guided genome‑editing systems, pre‑mRNA splice modulation by antisense oligonucleotides (ASOs), post‑transcriptional gene silencing mediated by small interfering RNAs (siRNAs) or microRNAs (miRNAs), and protein‑level modulation through aptamer binding. These modalities can be paired with diverse delivery platforms—including chemical conjugation using peptide or antibody ligands, lipid nanoparticles (LNPs), adeno‑associated virus (AAV) vectors, and U7 small nuclear RNA systems—to enhance cellular uptake, endosomal escape, nuclear access, and overall therapeutic activity. Figures were created with BioRender.com.

Comments 13: Recent reviews on therapeutic approaches for neuromuscular disorders should also be cited (see Beck Siet al, 2024, Byrne BJ et al, 2026, etc.)

Response 13: We appreciate your suggestion. We have added the recommended recent reviews and incorporated them into the Introduction and Discussion sections.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1:    (in red)

5. Additional clarifications

We have removed all corrected articles (Refs 46, 53, 82, 96) as requested by the editor and replaced them with new or existing references.

Ref. 46

(Original)

49.      Birnkrant, D.J.; Bushby, K.; Bann, C.M.; Apkon, S.D.; Blackwell, A.; Brumbaugh, D.; Case, L.E.; Clemens, P.R.; Hadjiyannakis, S.; Pandya, S.; et al. Diagnosis and Management of Duchenne Muscular Dystrophy, Part 1: Diagnosis, and Neuromuscular, Rehabilitation, Endocrine, and Gastrointestinal and Nutritional Management. Lancet Neurol. 2018, 17, 251–267.

(Replaced)

Messina, S.; Vita, G.L. Clinical Management of Duchenne Muscular Dystrophy: The State of the Art. Neurological Sciences 2018, 39, 1837–1845.

Ref. 53

(Original)

Frank, D.E.; Schnell, F.J.; Akana, C.; El-Husayni, S.H.; Desjardins, C.A.; Morgan, J.; Charleston, J.S.; Sardone, V.; Domingos, J.; Dickson, G.; et al. Increased Dystrophin Production with Golodirsen in Patients with Duchenne Muscular Dystrophy. Neurology 2020, 94, E2270–E2282.

(Replaced)

Wagner, K.R.; Kuntz, N.L.; Koenig, E.; East, L.; Upadhyay, S.; Han, B.; Shieh, P.B. Safety, Tolerability, and Pharmacokinetics of Casimersen in Patients with Duchenne Muscular Dystrophy Amenable to Exon 45 Skipping: A Randomized, Double-Blind, Placebo-Controlled, Dose-Titration Trial. Muscle Nerve 2021, 64, 285–292.

Ref. 82

(Original)

49.      Rowel, K.; Lim, Q.; Maruyama, R.; Echigoya, Y.; Nguyen, Q.; Zhang, A.; Khawaja, H.; Chandra, S.; Jones, T.; Jones, P.; et al. Inhibition of DUX4 Expression with Antisense LNA Gapmers as a Therapy for Facioscapulohumeral Muscular Dystrophy. Proc Natl Acad Sci U S A. 2020, 117(28), 16509-16515.

(Replaced)

Zhang, A.; Lim, K.R.Q.; Chen, Z.; Yokota, T.; Chen, Y.-W. DUX4 Reduction and Muscle Function Improvement by Subcutaneous Delivery of Gapmer Antisense Oligonucleotides. Mol. Ther. Nucleic Acids 2026, 37, 102791.

Bouwman, L.F.; den Hamer, B.; van den Heuvel, A.; Franken, M.; Jackson, M.; Dwyer, C.A.; Tapscott, S.J.; Rigo, F.; van der Maarel, S.M.; de Greef, J.C. Systemic Delivery of a DUX4-Targeting Antisense Oligonucleotide to Treat Facioscapulohumeral Muscular Dystrophy. Mol. Ther. Nucleic Acids 2021, 26, 813–827.

Ref. 96

(Original)

Coenen-Stass, A.M.L.; Mcclorey, G.; Manzano, R.; Betts, C.A.; Blain, A.; Saleh, A.F.; Gait, M.J.; Lochmüller, H.; Wood, M.J.A.; Roberts, T.C. Identification of Novel, Therapy-Responsive Protein Biomarkers in a Mouse Model of Duchenne Muscular Dystrophy by Aptamer-Based Serum Proteomics. Sci. Rep. 2015, 5.

(Replaced)

Molinaro, M.; Torrente, Y.; Villa, C.; Farini, A. Advancing Biomarker Discovery and Therapeutic Targets in Duchenne Muscular Dystrophy: A Comprehensive Review. Int. J. Mol. Sci. 2024, 25.

108.          Coenen-Stass, A.M.L.; Sork, H.; Gatto, S.; Godfrey, C.; Bhomra, A.; Krjutškov, K.; Hart, J.R.; Westholm, J.O.; O’Donovan, L.; Roos, A.; et al. Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD. Mol. Ther. Nucleic Acids 2018, 13, 1–15.