Antisense oligonucleotides

Antisense oligonucleotides (ASOs) are short, synthetic strands of nucleic acids aimed to bind to specific RNA sequences, leading to their degradation or preventing their translation into protein [4, 5]. ASOs have been developed for HD, targeting the mutant huntingtin (HTT) transcript to reduce its production. Studies have shown that reducing HTT levels in animal models leads to improvement in motor function and reduced neurodegeneration [6]. Some ASOs have been developed as splice-modulator like branaplam and PTC518 or as knockdown ASOs like tominersen, WVE-120101, and WVE-120102. In 2015, the first continuous clinical trial (NCT02519036) aimed at reducing HTT levels was launched. The trial utilized ASOs administered intrathecally to individuals in the early stages of HD, with the goal of lowering HTT alleles. Some ASOs, such as tominersen (Figure 1) entered clinical trials, showing encouraging initial results in HD patients. However, phase 3 clinical trials (NCT03761849) were halted due to off-target inflammatory effects. New phase 2 clinical trial is under process to mitigate these effects [7].

Display full size

Figure 1. 

Tominersen as an ASO. Tominersen binds to HTT mRNA and degrades them. As a result, mutant HTT protein production decreases. ASO: antisense oligonucleotide; HTT: huntingtin. Adapted from Van de Roovaart et al. 2023 [77]. © 2023 by the authors. CC BY 4.0

Branaplam initially in phase 2 clinical trial for spinal muscular atrophy (NCT02268552) showed promises against HTT protein and was in phase 2b trial (NCT05111249) until recently [8]. ASO therapy targeting the GGGGGCC repeat expansion in C9ORF72 gene shows promise for ALS/FTD treatment. ASOs effectively reduce GGGGGCC-containing transcripts and poly GP dipeptides in patient-derived cells and C9BAC mice. ASOs with reduced phosphorothioate content improve tolerability without compromising efficacy. Intrathecal delivery of optimal ASO in a patient with mutant C9ORF72 led to significant reductions in cerebrospinal fluid poly GP levels [9]. ASO therapy for SCA3 mice resulted in significant total choline rescue and partial reversals of taurine, glutamine, and total N-acetyl aspartate in the cerebellum and brainstem. ASO treatment fully or partially reversed neurochemical abnormalities in SCA3 mice, indicating the potential for these measures to serve as non-invasive treatment biomarkers in future SCA3 gene silencing trials [10]. As expansion in polyglutamine stretch in ataxin-3 gene is the main reason for SCA3, anti-ATXN3 ASO has been shown effective in several mouse and cell culture models [11–13]. It is also now being investigated in phase I clinical trial (identifier: NCT05160558) [14]. Preclinical studies using ASOs in SBMA mouse models showed improved muscle strength and extended lifespan by reducing toxic proteins [15]. Several ASOs are being evaluated for the treatment of DM1. These ASOs corrected splicing defects, reduced RNA foci, and improved muscle strength in mice [16–19]. Clinical trials: NCT02312011, DYNE-101, IONIS 486178 showed promise in correcting the DM1 phenotype in human. Challenges with ASOs in DM1 include poor blood-brain barrier (BBB) penetration and inadequate tissue uptake in skeletal muscle. Strategies to improve ASO delivery in DM1 include ligand-conjugated ASOs and cell-penetrating peptide conjugates [20]. As ASOs are not selective for the mutant allele, some strategies like mixmer design, nucleotide mismatch, and backbone modification can be used to improve specificity.

RNA interference by siRNA

RNA interference (RNAi) utilizes small interfering RNA (siRNA) molecules to target and degrade mRNA transcripts encoding toxic proteins. Like ASOs, siRNA-based therapies target mutant gene expression reduction. RNAi has been explored in diseases like DM1, where expanded CUG repeats in the DMPK gene cause toxic RNA foci that sequester proteins, disrupting normal cellular function [21]. A phase 1/2 clinical trial (NCT05027269) has been completed in 2023 for safety check. RNAi approaches targeting these repeats have demonstrated therapeutic potential in preclinical models. In several HD mouse models, RNAi was found beneficial [22–25]. Lipid nanoparticle delivered siRNA has been used to selectively silence polyQ-expanded androgen receptor, causative agent for in SBMA [26]. ASOs using single and double-stranded siRNAs proved promising to reduce gain-of-toxicity features caused by the GGGGCC repeat. Mice with C9ORF72 RNA repeats or inactivated C9orf72 alleles showed reduced RNA foci and dipeptide-repeat proteins using siRNA [27–29].

RNAi by microRNA

MicroRNA (miRNA) approach is a promising therapeutic strategy for HD by targeting the HTT protein. This approach involves delivering a therapeutic miRNA precursor using an adeno-associated viral (AAV) vector to reduce overall HTT translation in target cells. AAV5-miHTT intrastriatal injections were developed in 2019 and are in phase I/II clinical trial as of October 2024 (uniQure AMT-130 trial, NCT04120493). Another clinical trial (AMT-130, NCT05243017) using AAV5-miHTT was developed in 2021 and was in phase Ib/II stage as of October 2024. Using AAV-delivered anti-HTT RNAi technique, VY-HTT01 was developed for HD’s clinical trial that was discontinued later [30–32]. AAV-mediated miRNA targets AR gene expression via miRNAs like miR-196a, which enhances androgen receptor mRNA decay, showing promising results in SBMA mouse models [15]. Using antisense technology, antagomiR-23b has been tested against DM1 that upregulated MBLN1 protein, recovered mis-splicing and muscle strength. AntagomiR-23b is now in phase I/II clinical trial [33]. AAV5-miC (artificial anti-C9orf72-targeting miRNAs) in induced pluripotent stem cells (iPSCs) neurons and an ALS mouse model demonstrated successful silencing of C9orf72 and reduction of toxic transcripts and foci [34].

Direct repeat excision

CRISPR/Cas9 can be programmed to recognize and cut the expanded repeat sequences in affected genes. This approach has been applied in preclinical studies of SCA, and HD where the expanded CAG repeats excision reduced the toxic protein burden [35, 36]. The system has been effective in iPSCs as creating isogenic control iPSCs could help confirm precise correction of the mutations [37, 38]. In DM1, CRISPR/Cas9 has been implemented to delete large CTG repeats [39]. However, delivering CRISPR components efficiently and specifically to affected tissues remains a significant hurdle [40].

Base editing

Another approach involves base editing, where specific nucleotides in the expanded repeat sequence are altered without introducing double-stranded breaks. This method may offer a safer alternative to traditional CRISPR/Cas9 editing by minimizing off-target effects and limiting potential genomic instability [41]. A study demonstrated the effectiveness of a CRISPR-based editing approach by skipping exon 13 of the HTT gene. These editors reduced the formation of toxic N-terminal HTT fragments, reduced mutant HTT protein aggregation, and attenuated brain atrophy in HD rodent model. CRISPR base editing showed promising results in the rodent model by producing mutant HTT isoforms that are more resistant to toxic proteolysis, offering potential for long-term treatment [42, 43].

RNA repeats or transcription factor binding molecules

Small molecules that bind selectively to expanded RNA repeats can prevent the formation of toxic RNA foci. For example, compounds targeting the expanded CUG repeats in DM have shown promise in preventing the sequestration of essential RNA-binding proteins, thus restoring normal cellular function. This study explores the interaction of small molecules with trinucleotide repeat expansions in RNA, which are linked to diseases like DM1 and HD [44]. Using nuclear magnetic resonance (NMR) and molecular dynamics, researchers identified how three small molecules (a diguandinobenzoate, an imidazole derivative, and a quinoline) bind to the RNA’s hairpin structure. These findings enhance the understanding of ligand-RNA interactions and offer potential for designing new treatments targeting RNA-related disorders [45]. Another study reported SPI-24 and SPI-77 as Spt5-Pol II small molecule inhibitors for lowering mutant HTT mRNA and protein levels in HD mouse and cell lines [46].

Proteostasis regulators

Proteostasis regulators aim at the ubiquitin-proteasome system by enhancing the degradation of toxic proteins. These strategies target various components of the proteolytic machinery to address protein misfolding and aggregation in HD. Overexpression of proteasome activator PA28ɣ improved cell viability in HD neuronal models [47] and salvaged HD phenotypes in YAC128 mice model [48]. Elevated expression of the proteasome’s pbs-5 catalytic subunit improved resistance to proteotoxic stress and alleviated motor dysfunction in nematode HD models [49]. Enhanced expression of E3 ubiquitin ligases (such as CHIP, Ube3, Herp, and UBR5) may reduce mutant HTT aggregation by facilitating its ubiquitination and subsequent degradation via the proteasome [50, 51]. Overexpression of NUB1, which recruits ubiquitin ligases reduced mutant HTT neurotoxicity in fly models [52]. Ubiquilin-2 overexpression with the Hsp70-Hsp110 disaggregation system facilitates the transfer of Hsp70-bound mutant HTT for proteasome degradation [53].

Gene replacement

In cases where loss of function due to expanded repeats is the primary cause of disease, gene replacement therapy can be used to introduce functional copies of the affected gene. This approach has been tested for SBMA in mouse models, where delivery of wild-type androgen receptor genes reduced disease severity [15]. An AAV delivery system AT-466 has been developed for treating DM that inserts a functional version of the MBNL1 gene into cells impacted by DM1 [54–56].

Regulatory element modification

Another gene therapy approach involves modifying regulatory elements to increase the expression of non-expanded alleles or downregulate mutant alleles. This strategy is under investigation for FXS, where CGG repeats expansion in the FMR1 gene causing transcriptional silencing. This study emphasizes that directing transcriptional activators specifically to CGG repeats offers a promising approach for reactivating FMR1 expression in FXS patients. However, challenges remain, such as achieving sufficient FMRP protein production, which is necessary for therapeutic effects [57].

Heat shock proteins

Heat shock proteins (HSPs) known as molecular chaperones assist in protein folding and help clear misfolded proteins. Small molecules that enhance the expression or function of HSPs are being examined for their ability to reduce protein aggregation in diseases like HD [58].

Chemical chaperones

Chemical chaperones are small molecules that stabilize protein structures and prevent misfolding. Some research shows that chemical chaperones may have the ability to prevent the formation of toxic protein aggregates in various REDs [59–62]. A study showed molecular chaperones named Brichos alleviated the toxicity and aggregation processes by selectively engaging with amyloid fibrils in Alzheimer’s disease [63]. In addition to targeting the root cause of REDs, some therapeutic approaches focus on mitigating downstream cellular pathways that are disrupted by repeat expansions. As neurodegeneration is a hallmark of many REDs, some “neuroprotective agents” are being tested to slow disease progression. These agents aim to reduce oxidative stress, inflammation, and mitochondrial dysfunction, all of which contribute to neuronal death in REDs [64]. Besides, abnormal ion channel function has been implicated in certain repeat expansion disorders, such as SCAs. “Ion channel modulators” like riluzole may help to restore normal neuronal firing patterns and improve motor function in patients [65].