Nanomedicine-enhanced delivery of CRISPR-Cas13 for RNA editing in C9orf72-associated ALS

To the Editor,

Amyotrophic lateral sclerosis (ALS) is a fatal neurological degenerative disorder characterized by progressive loss of motor neurons. ALS results in loss of voluntary movement and respiratory failure. The most common inherited mutation, which causes familial ALS and frontotemporal dementia (FTD), is a GGGGCC (GC) hexanucleotide repeat expansion in the first intron of C9orf72^[1]. This expansion triggers pathological pathways, including the development of RNA foci, synthesis of toxic dipeptide repeat proteins (DPRs) via repeat-associated non-ATG translation, sequestration of RNA-binding proteins, impaired nuclear cytoplasmic transport, and loss of function of wild-type C9orf72 protein. C9orf72 expansions account for up to 40% of inherited ALS in European cohorts. Currently, no such therapies that can alter the disease course are available. Rather, management is symptomatic. DNA analysis and clinical examination are the predominant diagnostic approaches^[2].

Latest therapeutic developments concentrate on strategies targeting the RNA using CRISPR-Cas13 variants. Cas13 family nucleases target pathological RNA transcripts instead of editing genomic DNA. This approach reduces the risk of permanent unintended genetic modification. An essential tool is Cas13d (also known as CasRx); it is programmed with guide RNAs, which are engineered to target sense and antisense G₄C₂ repeat transcripts. In animal models, these systems have been administered through adeno-associated viral vectors (AAV). This approach yielded decreased repeat RNA, RNA foci, and DPRs, and it also improved neuronal gene expression patterns. Transport across the blood–brain barrier (BBB) remains challenging. Engineered viral vectors and nanocarriers such as lipid nanoparticles, exosomes, and peptide-functional polymeric nanoparticles are the common approaches that are being employed^[3].

Research reports that in neuronal cultures generated from C9orf72 patients and in iPSCs, CasRx is capable of decreasing both sense and antisense repeat-containing RNAs by roughly 30–70%. This figure varies based on guide design and cell type. Simultaneously, CasRx decreases DRPs by 50–70%, which in turn lowers the risk of excitotoxic injury. Precision Cas13 systems were introduced into the brain through AAV in genetically modified mouse and rat models. These systems markedly suppressed repeat RNAs and DPRs, reduced RNA foci, and reversed some altered gene expression profiles. This was done while preserving physiological C9orf72 transcript levels. To date, safety assessments in preclinical models have demonstrated limited nonspecific transcriptome disturbances and no acute neuronal loss^[4].

However, several challenges restrict clinical application. First, achieving effective and safe delivery of Cas13 across the BBB remains difficult, as the viral vectors may have immunogenicity, have restricted packaging size, and present long-term expression concerns. Second, off-target collateral RNA cleavage is also a significant risk. Even the high-fidelity variants may cause accidental damage to essential RNA transcripts. Third, large-scale and cost-effective production of nanocarriers or viral vector systems that meet clinical-grade quality is also a major challenge. Another difficulty is ensuring reproducibility across manufacturing batches while maintaining safety and stability. Moreover, regulatory hurdles, ethical concerns, long-term risks, and patient safety remain unresolved^[5].

In conclusion, administration of CRISPR-Cas13 therapeutics for C9orf72-linked ALS exhibits considerable potential in suppressing toxic RNAs and DPRs. Future studies should focus on the development of next-generation peptide- or lipid-functionalized nanoparticle platforms that can effectively cross the BBB. More in vivo trials in large animal models are also required. Optimization of guide RNA designs will be crucial to restrict nonspecific activity. Cost reduction strategies for large-scale production are necessary. More clinical awareness and timely genetic diagnosis will play a key role. Robust bioethical oversight will be essential for clinical implementation.

This letter to the editor adheres to the Transparency in the Reporting of Artificial Intelligence in Research (TITAN) guideline^[6]. During the preparation of this manuscript, generative AI tools (DeepSeek, DeepSeek Inc., June 2025 version; temperature parameter = 0.7) were used only as auxiliary aids to assist in formatting standardization, organization of background information, and language consistency checks.

All AI-assisted text was thoroughly reviewed, edited, and refined by the authors. The conceptualization, literature interpretation, synthesis of ideas, and manuscript writing were performed entirely by the authors, ensuring the academic rigor, accuracy, and originality of the review.

Ethical approval

Not applicable – this article does not involve original research on human or animal subjects.

Consent

Not applicable – no patient identifiable data are included.

Sources of funding

The authors received no financial support for the research, authorship, or publication of this article.

Author contributions

E.I. conceptualized the letter, reviewed the literature, and drafted the initial manuscript. R.K.M. contributed to critical revisions, data interpretation, and final editing. S.S. assisted in literature review and manuscript refinement. M.J. contributed to intellectual content and formatting. S.I. reviewed the final version and approved the manuscript for submission. All authors have reviewed and approved the final version of the manuscript.

Conflicts of interest disclosure

None declared.

Guarantor

Raghabendra Kumar Mahato.

Research registration unique identifying number (UIN)

This is new and not registered anywhere.

Provenance and peer review

Not commissioned, externally peer reviewed.

Data availability statement

No datasets were produced or examined for this article; therefore, data sharing is not relevant.

Acknowledgements

Not applicable.