Recent advances in RNA-targeting therapy for neurological diseases

Advances in sequencing and molecular technology now allow us to understand the genetic underpinnings of complex diseases such as neurological disorders. Genetic variations (or mutations) in the DNA sequence of single genes have been implicated in neurological diseases such as Huntington’s disease and spinal muscular atrophy. As a result, the development of gene therapies for neurological diseases is now a feasible endeavor. Indeed, gene therapy for neurological diseases has recently been invigorated by the market approvals of Zolgensma® (onasemnogene abeparvovec) in 2019 and Upstaza™ (eladocagene exuparvovec) in 2022. These gene therapies deliver a transgene to compensate for an aberrant or missing gene for the therapeutic benefit of neurological diseases and have demonstrated significant clinical potential. However, current gene therapy is limited to loss-of-function genetic diseases, and the delivery approach for large genes has been a challenge.

Recent advances in genetic technologies have fuelled the gene therapy field by overcoming the limitations of conventional gene therapy. In particular, post-transcriptional engineering with RNA-targeting tools has garnered significant attention. Changing the RNA that produces proteins offers even more flexible therapies for various diseases. Recently, RNA-targeting therapies have even become more attractive due to advanced chemical modifications in antisense oligonucleotide (ASO) and small interfering RNA (siRNA) strategies, as well as highly precise RNA editing techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas). Therefore, RNA-targeting therapy has the potential to become a fast-growing avenue of gene therapy that will change the way many diseases are treated and enable personalized medicine.

The current clinical landscape of RNA-targeting therapeutics: To date, RNA-targeting therapies such as ASO, siRNA, and small molecule strategies have been clinically proven for the treatment of neurological diseases. Antisense oligonucleotides (ASOs) are short chemically modified chains of nucleotides that are complementary to a certain region of target mRNA and are capable of altering mRNA expression through a variety of mechanisms (Figure 1A). Six market-approved ASO drugs (nusinersen, casimersen, eteplirsen, golodirsen, viltolarsen and inotersen) have been clinically used for the treatment of spinal muscular atrophy, Duchenne muscular dystrophy, and hereditary transthyretin amyloidosis. Apart from these, Huntington’s disease, being a monogenic disorder, is also an appealing target for central nervous system (CNS) gene therapy. Notably, one completed Phase 3 clinical trial and one ongoing clinical trial (NCT03761849 and NCT03842969, respectively) use ASOs to target HTT for mRNA silencing. SOD1 is also a common candidate in the development of CNS gene therapy for the treatment of amyotrophic lateral sclerosis and ASOs targeting SOD1 for silencing have completed Phase 3 trials (NCT02623699). Notably, a New Drug Application has been recently filed for the drug known as Tofersen with the United States Food and Drug Administration (US FDA) for approval of treating amyotrophic lateral sclerosis. With various applications possible and three clinical trials in progress (Additional Table 1) investigating their use, ASOs emerge as the clear front-runner for RNA-targeting therapy of neurological disease.

Figure 1.

Schematic of the various RNA-targeting therapies.

(A) Short interfering RNAs (siRNA), siRNAs recruit an RNA-induced silencing complex (RISC) for targeted cleavage of mRNA. (B) Antisense oligonucleotides (ASOs), ASOs can cleave mRNA through recruitment of RNase H or modify splicing by blocking splicing factors or inhibit translation through blocking ribosome binding. (C) Adenosine deaminases acting on RNA (ADAR)-mediated base editing, ADAR enzymes can be recruited by modified RNA sequences, such as circular RNAs, to induce adenosine to inosine change at target bases. (D) Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas)13, the CRISPR-Cas13 system can be used for targeted mRNA cleavage, fused with deaminases like ADAR for targeted RNA base editing, translation blocking, or splicing modulation. Created with BioRender.com.

Additional Table 1.

List of ongoing clinical trials for RNA-targeting therapies against neurological diseases

Drug	Company	Indication	Strategy/ Mechanism	NCT number	Status	Study details
Inotersen	Ionis Pharmaceuticals	Hereditary transthyretin-mediated amyloidosis	ASO/ Inhibition of TTR	NCT03702829	Phase II	50 participants will receive weekly subcutaneous injection of Inotersen to study efficacy and safety at 3-, 6-, 12-, 18-and 24-month time periods. No study results posted.
Renadirsen (DS-5141b)	Daiichi Sankyo Co., Ltd	Duchenne muscular dystrophy	ASO/ Skipping of dystrophin exon 45	NCT04433234	Phase II	8 participants will receive weekly subcutaneous injections of Renadirsen to evaluate long-term (< 2 years) safety and efficacy. No study results posted.
RO7234292 (RG6042)	Hoffmann-La Roche	Huntington’s disease	ASO/ Inhibition of HTT	NCT03842969	Phase III	236 participants will receive intrathecal injections of RO7234292 every 8 or 16 weeks up to 6 years to evaluate the long-term safety and tolerability. No study results posted.
Patisiran	Alnylam Pharmaceuticals	Hereditary transthyretin-mediated amyloidosis	siRNA/ Inhibition of TTR	NCT03997383	Phase III	360 participants with hATTR will be administered intravenously with Patisiran to evaluate safety and efficacy up to 12 months. No study results posted.
Vutrisiran	Alnylam Pharmaceuticals	Hereditary transthyretin-mediated amyloidosis	siRNA/ Inhibition of TTR	NCT04153149	Phase III	655 participants with hATTR with cardiomyopathy will receive subcutaneous injections of Vutrisiran once every 3 months for up to 36 months to evaluate safety and efficacy. No study results posted.
Vutrisiran	Alnylam Pharmaceuticals	Hereditary transthyretin-mediated amyloidosis	siRNA/ Inhibition of TTR	NCT03759379	Phase III	164 hATTR amyloidosis participants will receive subcutaneous injections of Vutrisiran once every three months for 18 months, and every six months thereafter to evaluate efficacy and safety. Significantly lower (P < 0.0000001) scores were recorded from the Modified Neurologic Impairment Score +7 assessment when compared with placebo control.
Branaplam	Novartis Pharmaceuticals	Spinal muscular atrophy	Small molecule/ Splice-switching of SMN2	NCT02268552	Phase I/II	40 infants with Type 1 SMA will receive once weekly oral administration of Branaplam up to 13 weeks to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics and efficacy. No study results posted.
Branaplam	Novartis Pharmaceuticals	Huntington's disease	Small molecule/ Inhibition of HTT	NCT05111249	Phase II	75 participants with HD will receive once weekly oral administration of Branaplam for up to 2 years to determine the optimal dose required to lower mutant HTT protein levels in the cerebrospinal fluid. No study results posted.

ASO: Antisense oligonucleotide; hATTR: hereditary transthyretin-mediated amyloidosis; HD: Huntington's disease; HTT: huntingtin; SMA: spinal muscular atrophy; SMN2: survival of motor neuron 2; TTR: transthyretin

siRNAs have also rapidly advanced RNA-targeting therapy. It is highly specific and efficient and interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation (Figure 1B). Three siRNA drugs, namely patisiran, givosiran and vutrisiran, have been approved for the treatment of familial amyloid polyneuropathy, acute hepatic porphyria, and hereditary transthyretin amyloidosis, respectively. In addition, one clinical trial is in progress while another is currently recruiting participants to investigate the siRNA approach for the treatment of hereditary transthyretin amyloidosis and Huntington’s disease, respectively (NCT03759379 and NCT04120493). With vutrisiran gaining market approval in 2022 and Atalanta therapeutics launching to develop novel siRNA therapeutics against neurodegenerative diseases, the clinical applicability and potential of siRNAs against neurological diseases may be expanded in the near future.

Apart from these sequence-based approaches in targeting RNA, the structure-based approach is also being actively pursued in the form of small molecule drugs. A prominent example is that of Evrysdi (risdiplam), an FDA-approved orally administered drug for spinal muscular atrophy. Unlike the ASO and siRNA drugs that target specific sequences in the target gene for silencing or splice modulation, Evrysdi targets specific RNA structures to stabilize the splicing machinery and promote exon inclusion. With most bioactive small molecules able to target stable, functional structures, only RNA structural knowledge is required to determine druggable targets, offering a promising front for the development of small molecule drugs against neurological disease. One small molecule drug, known as branaplam is currently in clinical trials for the treatment of spinal muscular atrophy and Huntington’s disease (NCT02268552 and NCT05111249). However, notably, Novartis has suspended branaplam trials for Huntington’s disease, citing nerve damage in treated participants.

New frontier of RNA-targeting therapeutics: With an increased understanding of neurological disease etiology and the advancement of gene editing techniques, new technologies are being explored to develop highly precise therapeutics.

An emerging avenue for RNA-targeting therapy is the use of adenosine deaminase acting on RNA (ADAR) enzymes for targeted base editing due to the high expression of the catalytically active ADAR1 and ADAR2 in the CNS (Figure 1C). ADAR enzymes recognize dsRNA and catalyze adenosine to inosine modification, allowing the correction of G-to-A mutations. The deaminase domains of ADAR1 and ADAR2 have been delivered with guide RNAs (gRNAs) for targeted base editing. The ADAR recruiting system, known as MCP-ADAR, was able to restore protein expression in a Duchenne muscular dystrophy mouse model by efficiently correcting a pathogenic nonsense mutation (Katrekar et al., 2019). ADAR2 has also been co-expressed with Mecp2-targeting gRNAs in a Rett syndrome mouse model for efficient recovery of Mecp2 protein and prolonged survival (Sinnamon et al., 2022). Alternatively, endogenous ADAR enzymes may also be recruited for RNA-targeted therapy using engineered gRNAs. While this strategy is yet to be studied against neurological disease and in vivo studies are limited, high editing efficiencies have been reported across multiple cell lines, with minimal off-target effects (Qu et al., 2019). Circular RNAs have also proven useful to recruit endogenous ADAR and efficient editing was reported in PCSK9 transcripts. This strategy was subsequently also demonstrated in mouse models of Hurler syndrome (Katrekar et al., 2022).

CRISPR-Cas technology is another attractive and emerging avenue that has seen tremendous leaps over the last decade since its first description. Initially described for genome editing applications, the characterization of novel Cas enzymes and repurposing of the currently popular enzymes is paving the way for novel therapeutics and diagnostics. CRISPR-Cas13 nucleases, in particular, are attractive for their exclusive RNA-targeting capacity, allowing reversible genetic alterations (Figure 1D). Compact Cas13 enzymes, such as Cas13d and Cas13e, are also available for adeno-associated virus delivery. Notably, the hyper-compact Cas13e has been shown to possess high efficiency and specificity (Xu et al., 2021). The current literature boasts several studies that have employed CasRx (also known as RfxCas13d) for disease intervention.

For neurological disease, CRISPR-Cas13d with SOD1-targeting crRNA reduced mRNA and protein levels, slowed overall disease progression, improved neuromuscular function, and decreased muscle atrophy in amyotrophic lateral sclerosis mice models. CRISPR-Cas13d targeting HTT in Huntington’s disease mice models also significantly reduced HTT protein levels, demonstrating that CRISPR-Cas13d could be employed as a versatile tool for treating different diseases of the CNS (Powell et al., 2022). These results offer promise for the safe and long-term treatment of CNS diseases through RNA silencing. Recombinant or novel Cas13 enzymes have also recently been described to improve the overall safety of RNA silencing. For instance, the new DjCas13d and hfCas13e enzymes have been shown to be more specific with comparable efficiency to wildtype Cas13d and Cas13e, respectively (Tong et al., 2022; Wei et al., 2022).

The compact CRISPR-Cas13 systems also allow translation blocking, base editing, and splice modulation within adeno-associated virus vectors for durable, single-administration therapies. An additional advantage is that these applications utilize dead Cas13 (dCas13), removing the catalytic properties of the enzyme, and thereby avoiding the collateral activity observed with active Cas13. For instance, recent work has demonstrated that dead CasRx (dCasRx) can repress mRNA translation when targeted to the ribosome binding site or open reading frame of fluorescent genes in Escherichia coli (Charles et al., 2021). Initiation factors can also be fused to dCasRx and targeted to the 5’ untranslated region of proteins to enhance gene expression (Otoupal et al., 2022).

Genes such as HEXA and SOD1, causative for Tay Sachs disease and spinal muscular atrophy respectively, also carry many disease-causing single-point mutations. In these cases, CRISPR-Cas13 RNA base editing can be employed for treatment through specific correction of the point mutations without affecting sequence integrity. Indeed, of 128 single-point mutations recorded for the HEXA gene, approximately 34% are editable by RNA base editors. For the SOD1 gene, approximately 25% are editable (https://www.hgmd.cf.ac.uk).

Current treatment strategies for neurodegenerative diseases may also be reimagined with Cas13 for safer and more durable therapies. For instance, ASOs targeting exon 7 of SMN2 for exon inclusion is a market-approved treatment for spinal muscular atrophy. Alternatively, Cas13 may be fused to splicing regulators like RBFOX1 for exon inclusion (Du et al., 2020). Exon skipping may also be employed in diseases where cryptic or mutant exons are included (Konermann et al., 2018).

Challenges of RNA-targeting therapeutics against neurological diseases: ASO-based therapeutics currently show tremendous potential in the clinical landscape, however, only offer short-lived therapeutic effects, necessitating a lifetime of drug administration. Due to the chemical modifications, a long-lasting delivery approach such as viral vector-based delivery is not feasible for ASOs. siRNA, when unmodified, also lead to massive off-target activities. Recent advancements through chemical modifications have led to the development of stable siRNAs that have greatly reduced off-target effects (Alterman et al., 2019). Similar to ASOs, chemically modified siRNAs are also not feasible for viral delivery. Therefore, with ASOs and siRNA-based therapies, repeated dosing is required for management of chronic neurological diseases. For this reason, CRISPR-Cas technology has been taken up globally by researchers recently for developing novel therapeutics.

Optimizing safety for clinical application of CRISPR-Cas13 RNA-targeting therapy remains a significant obstacle, particularly in light of off-targets by collateral activity and immune responses to the Cas enzyme in humans. A recent study elucidated a significant concern for the clinical use of CRISPR-CasRx, as the nuclease was found to be fatal in mice when used to target an isoform of the SIK3 gene (Li et al., 2022). While specific knockdown of SIK1 was observed, mortality was found to be not due to loss of SIK1 expression or guide-dependent off-targets but rather the collateral activity from CRISPR-CasRx. This was found to be dependent on the abundance of the target gene, and in this case, cleaved the 28s rRNA, impairing protein translation and activating an apoptosis pathway. Furthermore, pre-existing adaptive immunity to CRISPR-CasRx has also been reported through antibody responses and T cell-mediated recognition leading to inflammation and tissue damage (Tang et al., 2022). These findings suggest that Cas13 expression levels must be carefully regulated for RNA silencing applications.

Endogenous ADAR-mediated RNA editing strategies are considered safer alternatives to CRISPR-Cas13-based methods for RNA base editing due to the lack of foreign genetic material being introduced into the host. However, the expression of ADAR enzymes varies between tissues and editing efficiency is often not comparable to CRISPR-Cas13-based methods due to poor recruitment by the gRNA. Strategies to enhance the expression and stability of gRNAs without chemical modifications, through circular RNAs, have recently been explored and shown promise, allowing for safe and long-term RNA base editing (Katrekar et al., 2022). Nevertheless, ADAR-mediated RNA editing is limited to A-to-I(G) and C-to-U base editing applications, hindering widespread use.

Conclusions and future directions: RNA-targeting therapy has repeatedly demonstrated clinical potential in the form of ASO and siRNA strategies. Undoubtedly, these technologies will continue to support gene therapy development against neurological diseases. Over the last half-decade, CRISPR-Cas13 and ADAR-mediated RNA editing strategies are proving to be valuable additions to the RNA-targeting toolbox and strong candidates for future RNA-targeting therapies. Especially, with the recently described compact CRISPR-Cas13 enzymes, a multitude of applications are possible and the field is ripe for therapeutic development against various genetic disorders, including neurological diseases. This allows current therapies for neurodegenerative disease to be reimagined for more efficient, safer, and longer-lasting therapies. Given the safety and myriad of applications now achievable, RNA-targeting therapy might forge the way for treating neurological disease in the future.

This work was funded by grants from the National Health and Medical Research Council of Australia (1185600; to GSL) and Retina Australia (to GSL). The Center for Eye Research Australia receives Operational Infrastructure Support from the Victorian Government.

Additional file:

Additional Table 1: List of ongoing clinical trials for RNA-targeting therapies against neurological diseases.