Antisense Oligonucleotides (ASOs) in Motor Neuron Diseases: A Road to Cure in Light and Shade.

Antisense oligonucleotides (ASOs) are short oligodeoxynucleotides designed to bind to specific regions of target mRNA. ASOs can modulate pre-mRNA splicing, increase levels of functional proteins, and decrease levels of toxic proteins. ASOs are being developed for the treatment of motor neuron diseases (MNDs), including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA). The biggest success has been the ASO known as nusinersen, the first effective therapy for SMA, able to improve symptoms and slow disease progression. Another success is tofersen, an ASO designed to treat ALS patients with SOD1 gene mutations. Both ASOs have been approved by the FDA and EMA. On the other hand, ASO treatment in ALS patients with the C9orf72 gene mutation did not show any improvement in disease progression. The aim of this review is to provide an up-to-date overview of ASO research in MNDs, from preclinical studies to clinical trials and, where available, regulatory approval. We highlight the successes and failures, underline the strengths and limitations of the current ASO research, and suggest possible approaches that could lead to more effective treatments.

Profiling tofersen as a treatment of superoxide dismutase 1 amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive motor neuron disorder with a fatal outcome 3-5 years after disease onset due to respiratory complications. Superoxide dismutase 1 (SOD1) mutations are found in about 2% of all patients. Tofersen is a novel oligonucleotide antisense drug specifically developed to treat SOD1-ALS patients. AREAS COVERED: Our review covers and discusses tofersen pharmacological properties and its phase I/II and III clinical trials results. Other available drugs and their limitations are also addressed. EXPERT OPINION: VALOR study failed to meet the primary endpoint (change in the revised Amyotrophic Lateral Sclerosis Functional Rating Scale score from baseline to week 28, tofersen arm vs. placebo), but a significant reduction in plasma neurofilament light chain (NfL) levels was observed in tofersen arm (60% vs. 20%). PrefALS study has proposed plasma NfL has a potential biomarker for presymptomatic treatment, since it increases 6-12 months before phenoconversion. There is probably a delay between plasma NfL reduction and the clinical benefit. ATLAS study will allow more insights regarding tofersen clinical efficacy in disease progression rate, survival, and even disease onset delay in presymptomatic SOD1 carriers.

Quantifying the activity profile of ASO and siRNA conjugates in glioblastoma xenograft tumors in vivo.

Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma.

Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

[SOD1 gene therapy delays ALS disease progression]. / Precisionsmedicinsk genterapi har bromsat utveckling av ALS.

We present a patient with familial amyotrophic lateral sclerosis caused by an aggressive A4S mutation in the SOD1 gene. In 2020, the patient was enrolled in the VALOR SOD1 gene therapy phase-3 trial. At screening, the ALSFRS-R score was 41 (48 is normal) and the level of CSF-neurofilament L (an indicator of ongoing neuronal damage) was 11 000 ng/L (ref <650 ng/L). In the four years following enrollment, the patient received monthly intrathecal treatment with tofersen, an antisense oligonucleotide compound that inhibits SOD1 protein expression and hence lowers the synthesis of toxic SOD1 protein species. Side effects have been minimal and mostly attributed to the spinal taps. The patient remains ambulatory with an active social lifestyle. The ALSFRS-R score has in the past 18 months stabilized around 35-37, CSF-NfL is 1 290 ng/L and plasma-NfL is 12 (reference <13). This is the first documented arresting intervention in a patient with ALS in Sweden.

[Experience of the treatment of spinal muscular atrophy type 3 Kugelberg-Welander with Nusinersen]. / Opyt lecheniya nusinersenom spinal'noi myshechnoi atrofii 3-go tipa Kugel'berga­Velander.

Before the advent of pathogenetic therapy, the diagnosis of spinal muscular atrophy (SMA) meant the loss of all hopes for recovery and the patient's setting on the path of a steady decline in motor functions, a deterioration in the quality of life and, ultimately, inevitable early death. Currently, new methods of pathogenetic therapy with nusinersen and risdiplam, as well as etiological therapy with onasemnogene abeparvovec, are available in the Russia. Nusinersen is an antisense oligonucleotide that modifies splicing of the SMN2 gene to increase production of normal full-length motor neuron survival protein, which is deficient in SMA. The mechanism of action of Nusinersen is based on the activation of the disabled exon 7 of the SMN2 gene. The article describes an example of long-term effective treatment using pathogenetic therapy of a patient diagnosed with SMA type 3.

Olezarsen for Hypertriglyceridemia in Patients at High Cardiovascular Risk.

BACKGROUND: Reducing the levels of triglycerides and triglyceride-rich lipoproteins remains an unmet clinical need. Olezarsen is an antisense oligonucleotide targeting messenger RNA for apolipoprotein C-III (APOC3), a genetically validated target for triglyceride lowering. METHODS: In this phase 2b, randomized, controlled trial, we assigned adults either with moderate hypertriglyceridemia (triglyceride level, 150 to 499 mg per deciliter) and elevated cardiovascular risk or with severe hypertriglyceridemia (triglyceride level, ≥500 mg per deciliter) in a 1:1 ratio to either a 50-mg or 80-mg cohort. Patients were then assigned in a 3:1 ratio to receive monthly subcutaneous olezarsen or matching placebo within each cohort. The primary outcome was the percent change in the triglyceride level from baseline to 6 months, reported as the difference between each olezarsen group and placebo. Key secondary outcomes were changes in levels of APOC3, apolipoprotein B, non-high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol. RESULTS: A total of 154 patients underwent randomization at 24 sites in North America. The median age of the patients was 62 years, and the median triglyceride level was 241.5 mg per deciliter. The 50-mg and 80-mg doses of olezarsen reduced triglyceride levels by 49.3 percentage points and 53.1 percentage points, respectively, as compared with placebo (P<0.001 for both comparisons). As compared with placebo, each dose of olezarsen also significantly reduced the levels of APOC3, apolipoprotein B, and non-HDL cholesterol, with no significant change in the LDL cholesterol level. The risks of adverse events and serious adverse events were similar in the three groups. Clinically meaningful hepatic, renal, or platelet abnormalities were uncommon, with similar risks in the three groups. CONCLUSIONS: In patients with predominantly moderate hypertriglyceridemia at elevated cardiovascular risk, olezarsen significantly reduced levels of triglycerides, apolipoprotein B, and non-HDL cholesterol, with no major safety concerns identified. (Funded by Ionis Pharmaceuticals; Bridge-TIMI 73a ClinicalTrials.gov number, NCT05355402.).

Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease.

Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient.

A Precision Therapy Approach for Retinitis Pigmentosa 11 Using Splice-Switching Antisense Oligonucleotides to Restore the Open Reading Frame of PRPF31.

Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31. The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.

Antisense oligonucleotide targeting hepatic Serum Amyloid A limits the progression of angiotensin II-induced abdominal aortic aneurysm formation.

BACKGROUND AND AIMS: Obesity increases the risk for abdominal aortic aneurysms (AAA) in humans and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. We reported that deficiency of Serum Amyloid A (SAA) significantly reduces AngII-induced inflammation and AAA in both hyperlipidemic apoE-deficient and obese C57BL/6 mice. The aim of this study is to investigate whether SAA plays a role in the progression of early AAA in obese C57BL/6 mice. METHODS: Male C57BL/6J mice were fed a high-fat diet (60% kcal as fat) throughout the study. After 4 months of diet, the mice were infused with AngII until the end of the study. Mice with at least a 25% increase in the luminal diameter of the abdominal aorta after 4 weeks of AngII infusion were stratified into 2 groups. The first group received a control antisense oligonucleotide (Ctr ASO), and the second group received ASO that suppresses SAA (SAA-ASO) until the end of the study. RESULTS: Plasma SAA levels were significantly reduced by the SAA ASO treatment. While mice that received the control ASO had continued aortic dilation throughout the AngII infusion periods, the mice that received SAA-ASO had a significant reduction in the progression of aortic dilation, which was associated with significant reductions in matrix metalloprotease activities, decreased macrophage infiltration and decreased elastin breaks in the abdominal aortas. CONCLUSIONS: We demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from the progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

APOC3 siRNA and ASO therapy for dyslipidemia.

PURPOSE OF REVIEW: The aim of this review is to present the clinical indications of apolipoprotein C-III (apoC3) inhibition in the therapeutic arsenal for the treatment of lipid disorders and associated risks and to compare the most advanced modalities of apoC3 inhibition currently available or in development, specifically APOC3 antisense oligonucleotides (ASO) and small interfering RNA (siRNA). RECENT FINDINGS: ApoC3 inhibition significantly decreases triglyceride levels by mechanisms coupling both lipoprotein lipase (LPL) upregulation and LPL-independent mechanisms. The main apoC3 inhibitors in advanced clinical development are the GalNAc-ASO olezarsen and the GalNAc-siRNA plozasiran. Clinical studies conducted with volanesorsen, the olezarsen precursor, showed a favorable effect on hepatic steatosis (nonalcoholic fatty liver disease, NAFLD). Olezarsen does not appear to be associated with the main side effects attributed to volanesorsen including thrombocytopenia. Plozasiran is in advanced clinical development and requires subcutaneous injection every 3 months and present to-date an efficacy and safety profile comparable to that of the monthly ASO. SUMMARY: Inhibition of apoC3 is effective across all the spectrum of hypertriglyceridemia, might have a favorable effect on hepatic steatosis (NAFLD) and the effect of apoC3 inhibition on cardiovascular risk is not limited to its effect on plasma triglycerides. APOC3 GalNAc-conjugated ASO and siRNA are both effective in decreasing plasma apoC3 and triglyceride levels.

RNA therapeutics for metabolic disorders.

The prevalence of metabolic disorders is increasing exponentially and has recently reached epidemic levels. Over the decades, a large number of therapeutic options have been proposed to manage these diseases but still show several limitations. In this circumstance, RNA therapeutics have rapidly emerged as a new hope for patients with metabolic diseases. 57 years have elapsed from the discovery of mRNA, a large number of RNA-based drug candidates have been evaluated for their therapeutic effectiveness and clinical safety under clinical studies. To date, there are seven RNA drugs for treating metabolic disorders receiving official approval and entering the global market. Their targets include hereditary transthyretin-mediated amyloidosis (hATTR), familial chylomicronemia syndrome, acute hepatic porphyria, primary hyperoxaluria type 1 and hypercholesterolemia, which are all related to liver proteins. All of these seven RNA drugs are antisense oligonucleotides (ASO) and small interfering RNA (siRNA). These two types of treatment are both based on oligonucleotides complementary to target RNA through Watson-Crick base-pairing, but their mechanisms of action include different nucleases. Such treatments show greatest potential among all types of RNA therapeutics due to consecutive achievements in chemical modifications. Another method, mRNA therapeutics also promise a brighter future for patients with a handful of drug candidates currently under development.

Types of RNA therapeutics.

RNA therapy is one of the new treatments using small RNA molecules to target and regulate gene expression. It involves the application of synthetic or modified RNA molecules to inhibit the expression of disease-causing genes specifically. In other words, it silences genes and suppresses the transcription process. The main theory behind RNA therapy is that RNA molecules can prevent the translation into proteins by binding to specific messenger RNA (mRNA) molecules. By targeting disease-related mRNA molecules, RNA therapy can effectively silence or reduce the development of harmful proteins. There are different types of RNA molecules used in therapy, including small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamer, ribozyme, and antisense oligonucleotides (ASOs). These molecules are designed to complement specific mRNA sequences, allowing them to bind and degrade the targeted mRNA or prevent its translation into protein. Nanotechnology is also highlighted to increase the efficacy of RNA-based drugs. In this chapter, while examining various methods of RNA therapy, we discuss the advantages and challenges of each.

RNA therapeutics history and future perspectives.

Ribonucleic acid (RNA) therapeutics have significantly used RNA-based drugs to the prevention and treatment of diseases as effective messenger RNA-based vaccines in response to the COVID-19 pandemic. The RNA therapeutics with five classes including antisense oligonucleotide, small interfering RNA, microRNA, APTAMER and messenger RNAs are being quickly developed to treat various human diseases as neurological disease, cardiovascular disease, genetic and rare disease, cancer disease, coronavirus disease… which cannot be treated by other conventional drugs as small molecule-based drugs and antibodies. Therefore, the discovery of these RNA therapeutics created a new avenue for treatment of various human diseases. This chapter demonstrates the history of important discoveries in RNA biology and their impact on key developments in RNA therapeutics as well as the advantages of RNA therapeutics; RNA therapeutics describes the action mechanisms and examples of RNA-based drugs approved for treatment of various disease; and RNA therapeutics discusses delivery methods for RNA therapeutics to target organs and cells. In conclusion, this chapter is designed to offer an updated important development and advance of RNA therapeutics for the prevention and treatment of various human diseases.

RNA therapeutics for disorders of excretory system.

The excretory system is responsible for removing wastes from the human body, which plays a crucial role in our lives. Current treatments for diseases related to this system have shown several limitations; therefore, there is a rising need for novel methods. In this circumstance, RNA-based therapeutics have rapidly emerged as new and promising candidates. In fact, to date, a handful of potential drugs have passed the development step and entered the clinical pipeline. Among them, one drug received FDA approval to enter the global market, which is Oxlumo (Lumasiran) for the treatment of primary hyperoxaluria type 1. For other excretory diseases, such as paroxysmal nocturnal hemoglobinuria, urothelial cancer or renal cancer, RNA-based candidates are also being tested under clinical trials. Currently, the most potential types of RNA therapeutics to treat disorders of the excretory system are those based on small interfering RNA (siRNA), antisense oligonucleotides (ASO) and messenger RNA (mRNA), Among them, siRNA therapeutics seem to be the most promising, including Oxlumo and two other developing drug candidates. This chapter will provide a general overview on the application of RNA therapeutics in disorders of the excretory system.

Metabolic disorders affecting the liver and heart: Therapeutic efficacy of miRNA-based therapies?

Liver and heart disease are major causes of death worldwide. It is known that metabolic alteration causing type 2 diabetes (T2D) and Nonalcoholic fatty liver (NAFLD) coupled with a derangement in lipid homeostasis, may exacerbate hepatic and cardiovascular diseases. Some pharmacological treatments can mitigate organ dysfunctions but the important side effects limit their efficacy leading often to deterioration of the tissues. It needs to develop new personalized treatment approaches and recent progresses of engineered RNA molecules are becoming increasingly viable as alternative treatments. This review outlines the current use of antisense oligonucleotides (ASOs), RNA interference (RNAi) and RNA genome editing as treatment for rare metabolic disorders. However, the potential for small non-coding RNAs to serve as therapeutic agents for liver and heart diseases is yet to be fully explored. Although miRNAs are recognized as biomarkers for many diseases, they are also capable of serving as drugs for medical intervention; several clinical trials are testing miRNAs as therapeutics for type 2 diabetes, nonalcoholic fatty liver as well as cardiac diseases. Recent advances in RNA-based therapeutics may potentially facilitate a novel application of miRNAs as agents and as druggable targets. In this work, we sought to summarize the advancement and advantages of miRNA selective therapy when compared to conventional drugs. In particular, we sought to emphasise druggable miRNAs, over ASOs or other RNA therapeutics or conventional drugs. Finally, we sought to address research questions related to efficacy, side-effects, and range of use of RNA therapeutics. Additionally, we covered hurdles and examined recent advances in the use of miRNA-based RNA therapy in metabolic disorders such as diabetes, liver, and heart diseases.

The therapeutically actionable long non-coding RNA 'T-RECS' is essential to cancer cells' survival in NRAS/MAPK-driven melanoma.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

Eplontersen: First Approval.

Eplontersen (Wainua™) is a ligand-conjugated antisense oligonucleotide directed to TTR, which is being developed by Ionis Pharmaceuticals and AstraZeneca for the treatment of TTR-mediated amyloidosis (ATTR). Eplontersen, which is targeted to the liver by a ligand containing three N-acetyl galactosamine residues, binds to wild-type and variant TTR mRNA, thus reducing the levels of circulating TTR protein and amyloid deposition. Subcutaneous eplontersen reduced serum TTR levels, inhibited neuropathy progression and improved health-related quality of life in patients with polyneuropathy of hereditary ATTR (ATTRv-PN; v for variant) in a phase III trial. Based on these results, eplontersen was approved in the USA for the treatment of ATTRv-PN on 21 December 2023 and is currently undergoing regulatory review for a similar indication in the EU, the UK, Switzerland and Canada. Eplontersen is also undergoing phase III development for ATTR cardiomyopathy. This article summarizes the milestones in the development of eplontersen leading to this first approval for ATTRv-PN.

Prenatal delivery of a therapeutic antisense oligonucleotide achieves broad biodistribution in the brain and ameliorates Angelman syndrome phenotype in mice.

Angelman syndrome (AS), an early-onset neurodevelopmental disorder characterized by abnormal gait, intellectual disabilities, and seizures, occurs when the maternal allele of the UBE3A gene is disrupted, since the paternal allele is silenced in neurons by the UBE3A antisense (UBE3A-AS) transcript. Given the importance of early treatment, we hypothesized that prenatal delivery of an antisense oligonucleotide (ASO) would downregulate the murine Ube3a-AS, resulting in increased UBE3A protein and functional rescue. Using a mouse model with a Ube3a-YFP allele that reports on-target ASO activity, we found that in utero, intracranial (IC) injection of the ASO resulted in dose-dependent activation of paternal Ube3a, with broad biodistribution. Accordingly, in utero injection of the ASO in a mouse model of AS also resulted in successful restoration of UBE3A and phenotypic improvements in treated mice on the accelerating rotarod and fear conditioning. Strikingly, even intra-amniotic (IA) injection resulted in systemic biodistribution and high levels of UBE3A reactivation throughout the brain. These findings offer a novel strategy for early treatment of AS using an ASO, with two potential routes of administration in the prenatal window. Beyond AS, successful delivery of a therapeutic ASO into neurons has implications for a clinically feasible prenatal treatment for numerous neurodevelopmental disorders.

Specificity, synergy, and mechanisms of splice-modifying drugs.

Drugs that target pre-mRNA splicing hold great therapeutic potential, but the quantitative understanding of how these drugs work is limited. Here we introduce mechanistically interpretable quantitative models for the sequence-specific and concentration-dependent behavior of splice-modifying drugs. Using massively parallel splicing assays, RNA-seq experiments, and precision dose-response curves, we obtain quantitative models for two small-molecule drugs, risdiplam and branaplam, developed for treating spinal muscular atrophy. The results quantitatively characterize the specificities of risdiplam and branaplam for 5' splice site sequences, suggest that branaplam recognizes 5' splice sites via two distinct interaction modes, and contradict the prevailing two-site hypothesis for risdiplam activity at SMN2 exon 7. The results also show that anomalous single-drug cooperativity, as well as multi-drug synergy, are widespread among small-molecule drugs and antisense-oligonucleotide drugs that promote exon inclusion. Our quantitative models thus clarify the mechanisms of existing treatments and provide a basis for the rational development of new therapies.