A Translational Roadmap for Neurological Nonsense Mutation Disorders

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. You wrote: Readthrough efficiency is profoundly influenced by the nucleotide sequence surrounding the PTC (the "codon context"), the identity of the stop codon (UGA, UAG, UAA),... write the percentage of the stop codons.
2. You wrote: Next-generation mass spectrometry now allows for the direct detection of low-level, full-length protein in patient cerebrospinal fluid or via extracellular vesicle. Can you explain a little bit about Extracellular Vesicle Applications and Clinical relevance
3. You wrote: Next-generation capsid engineering, using directed evolution in primate models, is yielding novel AAV variants with enhanced CNS tropism and reduced seroprevalence. can you explain more? For example: bypassing blood-brain barrier (BBB) limitations.
4. Which type of the AAV are used?
5. You wrote: Pharmacological agents that induce translational readthrough represent a promising, therapeutic approach for diseases caused by nonsense mutations. These compounds promote the insertion of near-cognate tRNAs at PTCs, thereby allowing ribosomes to bypass the stop signal and produce full-length, potentially functional proteins. Can you list here in text some of the small molecules? I know you have written below, but I mean listing here will be better.

Author Response

Comments 1: You wrote: Readthrough efficiency is profoundly influenced by the nucleotide sequence surrounding the PTC (the "codon context"), the identity of the stop codon (UGA, UAG, UAA),... write the percentage of the stop codons.

Response 1: We thank the reviewer for this helpful suggestion. We have added the relative frequencies of nonsense mutations arising from each stop codon to provide greater context to the reader. Revised Text: “Among disease-causing nonsense mutations, the relative frequencies are approximately 38.5% for UGA, 40.4% for UAG, and 21.1% for UAA [16]. Generally, UGA is the 'leakiest' and most susceptible to pharmacological readthrough, followed by UAG and UAA [17–20].” References Added: We have ensured these statistics are supported by the following citations, which have been included in the reference list:

Mort, M., et al. (2008). A meta-analysis of nonsense mutations causing human genetic disease. Human Mutation, 29(8), 1037–1047.

Loughran, G., et al. (2014). Evidence of efficient stop codon readthrough in four mammalian genes. Nucleic Acids Research, 42, 8928-38.

 

Comments 2: You wrote: Next-generation mass spectrometry now allows for the direct detection of low-level, full-length protein in patient cerebrospinal fluid or via extracellular vesicle. Can you explain a little bit about Extracellular Vesicle Applications and Clinical relevance

 Response 2: We appreciate this suggestion. We have expanded the text to clarify that CNS-derived EVs serve as a non-invasive "liquid biopsy" tool, allowing for the monitoring of biomarkers that would otherwise require invasive procedures. Revised text: "Extracellular vesicles (EVs) are lipid-bilayer nanoparticles secreted by all CNS cell types that can traverse the blood-brain barrier (BBB) into the peripheral circulation. These vesicles carry molecular cargo—including proteins, lipids, and RNAs—that mirror the physiological state of their parent cells. In the context of nonsense suppression, mass spectrometry-based detection of full-length protein within CNS-derived EVs in blood or plasma provides a 'liquid biopsy' to monitor therapeutic efficacy and protein restoration without the need for invasive brain biopsies."

 

Comments 3: You wrote: Next-generation capsid engineering, using directed evolution in primate models, is yielding novel AAV variants with enhanced CNS tropism and reduced seroprevalence. can you explain more? For example: bypassing blood-brain barrier (BBB) limitations.

Comments 4: Which type of the AAV are used?

 Response 3&4: We thank the reviewer for requesting these details. We have revised the section to explicitly describe the mechanism of receptor-mediated transcytosis and to specify the AAV serotypes currently under investigation, including primate-tropic variants. Revised text: “To bypass the restrictive BBB, these engineered capsids are designed to utilize receptor-mediated transcytosis, allowing them to cross from the vasculature into the brain parenchyma. While early variants like AAV-PHP.eB showed high CNS tropism in specific mouse strains by targeting the LY6A receptor—a mechanism not conserved in primates—recent efforts have identified primate-tropic variants such as AAV.CAP-B10 that exhibit superior distribution in the non-human primate brain. Furthermore, new capsids are being engineered to target human-specific receptors like Carbonic Anhydrase IV, which is highly expressed in brain endothelial cells, ensuring more reliable clinical translation.”

 

Comments 5: You wrote: Pharmacological agents that induce translational readthrough represent a promising, therapeutic approach for diseases caused by nonsense mutations. These compounds promote the insertion of near-cognate tRNAs at PTCs, thereby allowing ribosomes to bypass the stop signal and produce full-length, potentially functional proteins. Can you list here in text some of the small molecules? I know you have written below, but I mean listing here will be better.

 Response 5: We agree that listing specific examples earlier improves the flow and clarity of the section. We have updated the introductory sentence of Section 4.1 to include these compounds. Revised text: “These compounds, such as aminoglycosides (e.g., Gentamicin, G418), next-generation synthetic aminoglycoside derivatives (e.g., ELX-02), and non-aminoglycoside small molecules like Ataluren (PTC124) promote the insertion of near-cognate tRNAs at PTCs, thereby allowing ribosomes to bypass the stop signal and produce full-length, potentially functional proteins.“

Reviewer 2 Report

Comments and Suggestions for Authors

Comments:

1. The statement that nonsense mutations account for “~11% of inherited genetic disorders” requires a clear citation and clarification of whether this refers to all Mendelian disorders or monogenic diseases only. Please justify the estimate and its scope.
2. Duchenne muscular dystrophy is primarily a neuromuscular disorder, not strictly a CNS disease. Please clarify whether the review focuses on CNS involvement, motor neuron pathology, or neurological manifestations broadly defined.
3. The authors should clarify how the proposed “4 D’s of Readthrough Therapy” differs from or advances beyond existing translational or precision medicine frameworks. Is this model conceptual, operational, or predictive?
4. Consider briefly defining readthrough therapy earlier for readers outside the RNA biology field.
5. What specific biomarkers (molecular, imaging, or transcriptomic) are currently validated for patient stratification in nonsense-mediated disorders of the CNS?
6. Figure quality is bad. Make it clean and clear.
7. English grammar mistake. Please edit by native English speaker

Author Response

Comment 1: The statement that nonsense mutations account for “~11% of inherited genetic disorders” requires a clear citation and clarification of whether this refers to all Mendelian disorders or monogenic diseases only. Please justify the estimate and its scope.

 Response 1: We thank the reviewer for this important suggestion to improve the precision of our manuscript. The estimate that nonsense mutations account for ~11% of disease-causing mutations refers specifically to described gene lesions causing human monogenic (Mendelian) diseases, as cataloged in the Human Gene Mutation Database (HGMD). This figure is supported by the seminal meta-analysis by Mort et al. (2008), which found that nonsense mutations accounted for approximately 11% of all described gene lesions causing human inherited disease. We agree that "inherited genetic disorders" is too broad and could be misinterpreted to include complex polygenic traits. Therefore, we have revised the text to specify "monogenic" disorders. Revised Text (Abstract): "Nonsense mutations, responsible for approximately 11% of gene lesions causing human monogenic diseases, introduce premature termination codons (PTCs) that lead to truncated proteins and nonsense-mediated mRNA decay (NMD)."

 

Comment 2: Duchenne muscular dystrophy is primarily a neuromuscular disorder, not strictly a CNS disease. Please clarify whether the review focuses on CNS involvement, motor neuron pathology, or neurological manifestations broadly defined.

 Response 2: This is a crucial clarification. While DMD is primarily characterized by muscle pathology, we included it in this review because it is a multi-systemic disorder with significant CNS comorbidities, including cognitive deficits, which are often targets for readthrough therapy. We have revised the text to acknowledge the primary pathology of DMD while justifying its inclusion. Revised Text (Section 1): "Nonsense mutations affect a significant proportion of genetic disorders with major neurological or neuromuscular manifestations, including spinal muscular atrophy (SMA), Rett syndrome, Duchenne muscular dystrophy (DMD), and genetic epileptic encephalopathies..."

 

Comment 3: The authors should clarify how the proposed “4 D’s of Readthrough Therapy” differs from or advances beyond existing translational or precision medicine frameworks. Is this model conceptual, operational, or predictive?

 Response 3: We appreciate the request to articulate the framework's novelty. The "4 D’s" framework is designed specifically as an operational model for the unique challenges of nonsense suppression, rather than a general diagnostic model. We have added a clarifying statement to the Introduction. Revised text: “Unlike general precision medicine frameworks that focus on broad diagnostic-therapeutic pairings, the '4 D's' model is primarily an operational and predictive framework specifically designed for the unique molecular hurdles of nonsense suppression. It advances beyond existing translational models by explicitly linking the molecular detection of codon context and NMD efficiency with the selection of a decoding modality and the necessity for brain-penetrant delivery systems.”

 

Comment 4: Consider briefly defining readthrough therapy earlier for readers outside the RNA biology field.

Response 4: We agree and have added a concise definition to the Introduction section to ensure accessibility for a broader audience. Revised Text: "To address this, readthrough therapy has emerged as a promising precision medicine approach. It utilizes molecular agents to 'suppress' the ribosome into bypassing these premature stop signals, allowing the cell to continue translation and produce a full-length, functional protein."

 

Comment 5: What specific biomarkers (molecular, imaging, or transcriptomic) are currently validated for patient stratification in nonsense-mediated disorders of the CNS?

 Response 5: We have added a discussion on current and emerging stratification tools to address this query. Revised Text: "Current patient stratification relies heavily on genomic sequencing to identify the specific PTC and its surrounding 3' context. Emerging biomarkers for clinical trials include the measurement of NMD efficiency via transcriptomic analysis and the quantification of residual protein levels in patient-derived iPSCs or CNS-derived EVs."

 

Comment 6: Figure quality is bad. Make it clean and clear.

Response 6: We apologize for the low resolution of the initial figures. We have regenerated and processed all figures at high resolution (300 DPI) to ensure they are crisp and legible, meeting standard publication quality requirements.

 

Comment 7: English grammar mistake. Please edit by native English speaker

Response 7: The manuscript has undergone a thorough professional edit for grammar, syntax, and flow to ensure it meets the standards for publication.