Gene therapy for targeting a prenatally enriched potassium channel associated with severe childhood epilepsy and premature death.

Dysfunction of the sodium-activated potassium channel KNa1.1 (encoded by KCNT1) is associated with a severe condition characterized by frequent seizures (up to hundreds per day) and is often fatal by age three years. We defined the early developmental onset of KNa1.1 channels in prenatal and neonatal brain tissue, establishing a timeline for pathophysiology and a window for therapeutic intervention. Using patch-clamp electrophysiology, we observed age-dependent increases in KNa1.1 K+ conductance. In neurons derived from a child with a gain-of-function KCNT1 pathogenic variant (p.R474H), we detected abnormal excitability and action potential afterhyperpolarization kinetics. In a clinical trial, two individuals with the p.R474H variant showed dramatic reductions in seizure occurrence and severity with a first-in-human antisense oligonucleotide (ASO) RNA therapy. ASO-treated p.R474H neurons in vitro exhibited normalized spiking and burst properties. Finally, we demonstrated the feasibility of ASO knockdown of KNa1.1 in mid-gestation human neurons, suggesting potential for early therapeutic intervention before the onset of epileptic encephalopathy.

Extracellular vesicle mimetics as delivery vehicles for oligonucleotide-based therapeutics and plasmid DNA.

Introduction: Small membrane particles called extracellular vesicles (EVs) transport biologically active cargo between cells, providing intercellular communication. The clinical application of EVs is limited due to the lack of scalable and cost-effective approaches for their production and purification, as well as effective loading strategies. Methods: Here we used EV mimetics produced by cell treatment with the actin-destabilizing agent cytochalasin B as an alternative to EVs for the delivery of therapeutic nucleic acids. Results: Cytochalasin-B-inducible nanovesicles (CINVs) delivered a fully modified N-(methanesulfonyl)- or mesyl (µ-) antisense oligonucleotide to B16 melanoma cells, selectively decreasing the level of target microRNA-21 with effectiveness comparable to that observed upon Lipofectamine 2000-mediated delivery. The efficiency of the CINV-mediated delivery of plasmid DNA encoding EGFP varied depending on the type of recipient cells. Surprisingly, under experimental conditions, CINVs were unable to deliver both modified and natural short RNA duplexes-small interfering RNA and immunostimulatory RNA-probably due to their poor loading into CINVs. Discussion: CINVs demonstrated unique properties for the delivery of therapeutic nucleic acids, especially for antisense oligonucleotide-based therapy.

l-Type amino acid transporter 1-targeting nanoparticles for antisense oligonucleotide delivery to the CNS.

l-Type amino acid transporter 1 (LAT1)-specific ligands and polyion complexes are used as brain-specific targets to deliver RNA-based drugs across the blood-brain barrier. We characterized an LAT1-targeting antisense oligonucleotide (ASO)-encapsulated nanoparticle, Phe-NPs/ASO. A 25% density of phenylalanine effectively binds to the surface of LAT1-targeting NPs in the GL261-Luc cells, and Phe-NPs/ASO shows higher binding affinity compared to that without phenylalanine by cellular binding assay. To further characterize the blood-brain barrier-targeting effect and tissue distribution following a single-dose intravenous injection in mice, we performed in vivo biodistribution studies using fluorescence imaging. The Phe-NPs/ASOs were detected in the brain tissue 1 h post-intravenous injection at an approximately 64-fold higher ratio than that of the same ASOs administered in the absence of any NP carrier. The brain tissue delivery of ASO-loaded Phe-NPs was also confirmed in a fluorescence imaging study performed in non-human primates. These results demonstrate that Phe-NPs may successfully deliver an ASO to the brain tissue across brain regions. Phe-NPs loaded with RNA-based drugs have the potential to treat diseases of the CNS, including all forms of neurodegenerative diseases.

Elevated plasma and CSF neurofilament light chain concentrations are stabilized in response to mutant huntingtin lowering in the brains of Huntington's disease mice.

BACKGROUND: Therapeutic approaches aimed at lowering toxic mutant huntingtin (mHTT) levels in the brain can reverse disease phenotypes in animal models of Huntington's disease (HD) and are currently being evaluated in clinical trials. Sensitive and dynamic response biomarkers are needed to assess the efficacy of such candidate therapies. Neurofilament light chain (NfL) is a biomarker of neurodegeneration that increases in cerebrospinal fluid (CSF) and blood with progression of HD. However, it remains unknown whether NfL in biofluids could serve as a response biomarker for assessing the efficacy of disease-modifying therapies for HD. METHODS: Longitudinal plasma and cross-sectional CSF samples were collected from the YAC128 transgenic mouse model of HD and wild-type (WT) littermate control mice throughout the natural history of disease. Additionally, biofluids were collected from YAC128 mice following intracerebroventricular administration of an antisense oligonucleotide (ASO) targeting the mutant HTT transgene (HTT ASO), at ages both before and after the onset of disease phenotypes. NfL concentrations in plasma and CSF were quantified using ultrasensitive single-molecule array technology. RESULTS: Plasma and CSF NfL concentrations were significantly elevated in YAC128 compared to WT littermate control mice from 9 months of age. Treatment of YAC128 mice with either 15 or 50 µg HTT ASO resulted in a dose-dependent, allele-selective reduction of mHTT throughout the brain at a 3-month interval, which was sustained with high-dose HTT ASO treatment for up to 6 months. Lowering of brain mHTT prior to the onset of regional brain atrophy and HD-like motor deficits in this model had minimal effect on plasma NfL at either dose, but led to a dose-dependent reduction of CSF NfL. In contrast, initiating mHTT lowering in the brain after the onset of neuropathological and behavioural phenotypes in YAC128 mice resulted in a dose-dependent stabilization of NfL increases in both plasma and CSF. CONCLUSIONS: Our data provide evidence that the response of NfL in biofluids is influenced by the magnitude of mHTT lowering in the brain and the timing of intervention, suggesting that NfL may serve as a promising exploratory response biomarker for HD.

Efficacy and safety of olezarsen in lowering apolipoprotein C-III and triglycerides in healthy Japanese Americans.

BACKGROUND: Olezarsen is a GalNAc3-conjugated, hepatic-targeted antisense oligonucleotide that lowers apolipoprotein C-III (apoC-III) and triglyceride levels. The efficacy and safety of olezarsen has not previously been studied in ethnically diverse American populations. The aim of this study is to assess the effect of olezarsen in healthy Japanese Americans. METHODS: A randomized, placebo-controlled, double-blind phase 1 study was performed in 28 healthy Japanese American participants treated with olezarsen in single-ascending doses (SAD; 30, 60, 90 mg) or multiple doses (MD; 60 mg every 4 weeks for 4 doses). The primary, secondary, and exploratory objectives were safety and tolerability, pharmacokinetics, and effects of olezarsen on fasting serum triglycerides and apoC-III, respectively. RESULTS: There were 20 participants (16 active:4 placebo) in the SAD part of the study, and 8 participants (6 active:2 placebo) in the MD part of the study. For the primary endpoint, no serious adverse events or clinically relevant laboratory abnormalities were reported. The majority of olezarsen plasma exposure occurred within 24 h post-dose. In the SAD cohorts at Day 15 the percentage reduction in apoC-III/TG was - 39.4%/ - 17.8%, - 60.8%/ - 52.7%, and - 68.1%/ - 39.2% in the 30, 60 and 90 mg doses, respectively, vs 2.3%/44.5% increases in placebo. In the MD cohort, at Day 92 the percentage reduction in apoC-III/TG was - 81.6/ - 73.8% vs - 17.2/ - 40.8% reduction in placebo. Favorable changes were also present in VLDL-C, apoB and HDL-C. CONCLUSIONS: Single- and multiple-dose administration of olezarsen was safe, was well tolerated, and significantly reduced apoC-III and triglyceride levels in healthy Japanese Americans.

Opportunities for Riboswitch Inhibition by Targeting Co-Transcriptional RNA Folding Events.

Antibiotic resistance is a critical global health concern, causing millions of prolonged bacterial infections every year and straining our healthcare systems. Novel antibiotic strategies are essential to combating this health crisis and bacterial non-coding RNAs are promising targets for new antibiotics. In particular, a class of bacterial non-coding RNAs called riboswitches has attracted significant interest as antibiotic targets. Riboswitches reside in the 5'-untranslated region of an mRNA transcript and tune gene expression levels in cis by binding to a small-molecule ligand. Riboswitches often control expression of essential genes for bacterial survival, making riboswitch inhibitors an exciting prospect for new antibacterials. Synthetic ligand mimics have predominated the search for new riboswitch inhibitors, which are designed based on static structures of a riboswitch's ligand-sensing aptamer domain or identified by screening a small-molecule library. However, many small-molecule inhibitors that bind an isolated riboswitch aptamer domain with high affinity in vitro lack potency in vivo. Importantly, riboswitches fold and respond to the ligand during active transcription in vivo. This co-transcriptional folding is often not considered during inhibitor design, and may explain the discrepancy between a low Kd in vitro and poor inhibition in vivo. In this review, we cover advances in riboswitch co-transcriptional folding and illustrate how intermediate structures can be targeted by antisense oligonucleotides-an exciting new strategy for riboswitch inhibitor design.

Uncovering ASO-Targetable Deep Intronic AIRE Variants: Insights and Therapeutic Implications.

High-throughput DNA sequencing has accelerated the discovery of disease-causing genetic variants, yet only in 10-40% of cases yield a genetic diagnosis. Increased implementation of genome sequencing has enabled a deeper exploration of the noncoding genome and recognition of noncoding variants as major contributors to disease. In a recent study, we identified a deep intronic variant in the AutoImmune REgulator (AIRE) gene (c.1504-818 G>A) as the cause of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a life-threatening monogenic autoimmune disorder most often caused by biallelic AIRE defects. This deep intronic variant disrupts normal splicing AIRE , causing pseudoexon inclusion and altered protein function. By developing an antisense oligonucleotide (ASO) targeting the pseudoexon sequence, we restored normal AIRE transcript in vitro, thereby revealing a potential genotype-specific candidate treatment. Our study illustrates key aspects of intronic variant detection, validation, and candidate ASO development. Herein, we briefly highlight the growing potential of ASO-based therapies for deep intronic variants, addressing the unmet need of personalized, genotype-specific therapies in diseases lacking curative options.

Messenger interference RNA therapies targeting apolipoprotein C-III and angiopoietin-like protein 3 for mixed hyperlipidemia: the future of plozasiran and zodasiran.

INTRODUCTION: Mixed hyperlipidemia represents a substantial public health issue and a considerable burden on healthcare systems. Although the introduction of statins and LDL-cholesterol lowering agents have significantly reduced the incidence of atherosclerotic cardiovascular diseases (ASCVD), a significant portion of the population continues to exhibit ASCVD progression due to elevated triglyceride-rich lipoprotein (TRL) levels. This persistent risk has catalyzed the development of novel pharmacological interventions targeting these lipoproteins. AREAS COVERED: Our special report commenced with a targeted PubMed search using keywords such as 'plozasiran,' 'zodasiran,' and terms related to APOC3 and ANGPTL3. As the review progressed, emergent research questions guided further searches, allowing for the inclusion of additional relevant articles to comprehensively illustrate the linkage between TRLs and cardiovascular disease, discuss the roles of APOC3, ANGPTL3, and the pharmaceutical agents that target these proteins, and provide a comparison on the ARCHES-2 and MUIR trials. EXPERT OPINION: The ARCHES-2 and MUIR trials demonstrated effective triglyceride reduction by these therapies, yet it is uncertain if this correlates with significant clinical benefits. Advances in antisense oligonucleotide technology, especially the GalNAc delivery platform, show promise for personalized lipid management, though challenges such as cost and safety concerns remain.

Recent Progress of Antisense Oligonucleotide Therapy for Superoxide-Dismutase-1-Mutated Amyotrophic Lateral Sclerosis: Focus on Tofersen.

Amyotrophic lateral sclerosis (ALS) is a refractory neurodegenerative disease characterized by the degeneration and loss of motor neurons, typically resulting in death within five years of onset. There have been few effective treatments, making the development of robust therapies an urgent challenge. Genetic mutations have been identified as contributors to ALS, with mutations in superoxide dismutase 1 (SOD1), which neutralizes the harmful reactive oxygen species superoxide, accounting for approximately 2% of all ALS cases. To counteract the toxic gain of function caused by SOD1 mutations, therapeutic strategies aimed at suppressing SOD1 gene expression have shown promise. Antisense oligonucleotide (ASO) is an artificially synthesized, short, single-stranded DNA/RNA molecule that binds to target RNA to alter gene expression, representing a next-generation therapeutic approach. In 2023, tofersen became the first ASO drug approved by the FDA for ALS. Administered intrathecally, tofersen specifically binds to SOD1 mRNA, inhibiting the production of toxic SOD1 protein, thereby improving biomarkers of ALS. The long-term efficacy and safety of tofersen require further validation, and the development of more optimized treatment protocols is essential. A series of studies and therapeutic developments related to SOD1 mutations have advanced the understanding of ALS pathophysiology and significantly contributed to treatment strategies for central nervous system disorders. This review focuses on an overview of SOD1 mutations and the development process of tofersen, aiming to deepen the understanding of advancements in ALS research and discuss future challenges and directions for ASO therapy.

Assessment of Platelet Aggregation and Thrombin Generation in Patients with Familial Chylomicronemia Syndrome Treated with Volanesorsen: A Cross-Sectional Study.

BACKGROUND: The antisense oligonucleotide against APOC3 mRNA volanesorsen was recently introduced to treat Familial Chylomicronemia Syndrome (FCS). Cases of decreased platelet count are reported among patients treated with volanesorsen. The aim of the study was to evaluate platelet function and thrombin generation (TG) assessment in FCS patients receiving volanesorsen. We performed a cross-sectional study on FCS patients treated with volanesorsen. METHODS: Changes in platelet count PLC were assessed from baseline to Tw12 and Tw36. To assess TG, samples were processed by CAT (with PPP-reagent LOW). The results were expressed by the thrombogram graphic (thrombin variation over time); LagTime; endogenous thrombin potential (ETP); peak; time to reach peak (ttpeak), StartTail and Velocity Index. Platelet aggregation was assessed by testing different agonists using the turbidimetry method. RESULTS: Four FCS patients and four matched healthy controls were included in the present study. Changes in PLC were 30% at Tw12 and 34% at Tw36. Thrombin generation results showed values in the normal range (for patients and controls, respectively, LagTime:10.42 ± 4.40 and 9.25 ± 0.99; ttPeak:14.33 ± 4.01 and 13.10 ± 0.67; StartTail: 32.13 ± 3.54 and 29.46 ± 1.69; Velocity Index: 20.21 ± 3.63 and 33.05 ± 13.21; ETP: 599.80 ± 73.47 and 900.2 ± 210.99; peak value: 76.84 ± 1.07 and 123.30 ± 39.45) and no significant difference between cases and controls. Platelet aggregation test showed values in range, with no significant difference compared to healthy controls. CONCLUSIONS: Our study showed for the first time that no significant changes in general hemostasis assessed by TG and in platelet function were observed in FCS patients receiving volanesorsen.

Joining forces to develop individualized antisense oligonucleotides for patients with brain or eye diseases: the example of the Dutch Center for RNA Therapeutics.

Antisense oligonucleotides (ASOs) offer versatile tools to modify the processing and expression levels of gene transcripts. As such, they have a high therapeutic potential for rare genetic diseases, where applicability of each ASO ranges from thousands of patients worldwide to single individuals based on the prevalence of the causative pathogenic variant. It was shown that development of individualized ASOs was feasible within an academic setting, starting with Milasen for the treatment of a patient with CLN7 Batten's disease in the USA. Inspired by this, the Dutch Center for RNA Therapeutics (DCRT) was established by three academic medical centers in the Netherlands with a track record in ASO development for progressive, genetic neurodegenerative, neurodevelopmental, and retinal disorders. The goal of the DCRT is to bundle expertise and address national ethical, regulatory, and financial issues related to ASO treatment, and ultimately to develop individualized ASOs for eligible patients with genetic diseases affecting the central nervous system in an academic, not-for-profit setting. In this perspective, we describe the establishment of the DCRT in 2020 and the achievements so far, with a specific focus on lessons learned: the need for processes and procedures, the need for global collaboration, the need to raise awareness, and the fact that N-of-1 is N-of-a-few.

Joining forces to develop individualized antisense oligonucleotides for patients with brain or eye diseases: the example of the Dutch Center for RNA Therapeutics Many rare diseases have a genetic cause. Antisense oligonucleotides (ASOs) are short pieces of modified DNA that have therapeutic potential for some patients with rare diseases. However, often this is in a patient-specific setting, meaning individualized therapy development is required, which has little commercial opportunity for pharmaceutical companies. It was shown however that individualized ASOs can be developed by academics, starting with Milasen, which was developed for a unique DNA variant found in a child with Batten's disease in the USA. Following in the footstep of these academic pioneers we established the Dutch Center for RNA Therapeutics (DCRT), which aims to develop individualized ASOs for eligible patients with eye or brain diseases in a not-for-profit setting. Our goal is to bundle expertise and address national, ethical, regulatory and financial issues related to individualized ASO development. In this perspective review we outline the achievements since establishing the DCRT in 2020, with a focus on lessons learnt along the way: the need for processes and procedures, the need for global collaboration, the need to raise awareness and the fact that very often ASOs developed for a single person, could be applied also to a few other patients with the same DNA variants.

Gene therapy for the leukodystrophies: From preclinical animal studies to clinical trials.

Leukodystrophies are progressive single gene disorders affecting the white matter of the brain. Several gene therapy trials are in progress to address the urgent unmet need for this patient population. We performed a comprehensive literature review of all gene therapy clinical trials listed in www.clinicaltrials.gov through August 2024, and the relevant preclinical studies that enabled clinical translation. Of the approximately 50 leukodystrophies described to date, only eight have existing gene therapy clinical trials: metachromatic leukodystrophy, X-linked adrenoleukodystrophy, globoid cell leukodystrophy, Canavan disease, giant axonal neuropathy, GM2 gangliosidoses, Alexander disease and Pelizaeus-Merzbacher disease. What led to the emergence of gene therapy trials for these specific disorders? What preclinical data or disease context was enabling? For each of these eight disorders, we first describe its pathophysiology and clinical presentation. We discuss the impact of gene therapy delivery route, targeted cell type, delivery modality, dosage, and timing on therapeutic efficacy. We note that use of allogeneic hematopoietic stem cell transplantation in some leukodystrophies allowed for an accelerated path to clinic even in the absence of available animal models. In other leukodystrophies, small and large animal model studies enabled clinical translation of experimental gene therapies. Human clinical trials for the leukodystrophies include ex vivo lentiviral gene delivery, in vivo AAV-mediated gene delivery, and intrathecal antisense oligonucleotide approaches. We outline adverse events associated with each modality focusing specifically on genotoxicity and immunotoxicity. We review monitoring and management of events related to insertional mutagenesis and immune responses. The data presented in this review show that gene therapy, while promising, requires systematic monitoring to account for the precarious disease biology and the adverse events associated with new technology.

An Evaluation of First-in-Human Studies for RNA Oligonucleotides.

Most oligonucleotide therapeutics use Watson-Crick-Franklin base-pairing hybridization to target RNA and mitigate disease-related protein production. Using targets that were previously inaccessible to small molecules and biologics, synthetic nucleotides have provided treatments for severely debilitating and life-threatening diseases. However, these therapeutics possess unique pharmacologies that require specific considerations for their distribution, clearance, and other clinical pharmacology characteristics. Namely, one hurdle in the drug development of these therapeutics remains the prediction of human dose that results in exposures comparable with or below those seen at no observed adverse effect level in animals. For first-in-human (FIH) clinical trials, this often involves allometric scaling based on body surface area (BSA) or body weight (BW). In this study, we reviewed the current literature and surveyed elements across 16 approved oligonucleotide therapeutic New Drug Applications approved by the U.S. Food and Drug Administration in the period from September 1998 to January 2024, and 89 Investigational New Drug (IND) programs with available FIH clinical trials conducted from January 2015 to January 2024, to understand dose selection in early-stage development of oligonucleotide therapeutics. The surveyed elements across these programs include study design, route of administration, dosing regimen, interspecies scaling approach, and the most sensitive species. Of 89 IND programs and 16 approved therapeutics, intravenous and subcutaneous were the most common route of administration, no observable adverse event levels were frequently derived from nonhuman primates, BSA and BW were adjusted for in similar frequencies, patients were predominantly enrolled in FIH trials, and the most common design was a single or multiple ascending dose trial.

A Phase 1, Single-Dose Study to Evaluate the Pharmacokinetics and Safety of Bepirovirsen in Adults with Hepatic Impairment and Healthy Participants (B-Assured).

Bepirovirsen is a developmental antisense oligonucleotide (ASO) for treatment of chronic hepatitis B virus infection. No pharmacokinetic (PK) studies comparing participants with hepatic impairment (HI) and healthy participants (HPs) have been conducted with ASOs. Given the target patient population, characterization of bepirovirsen PK in HI was imperative. This phase 1, nonrandomized, open-label study (NCT04971928) evaluated the PKs of a single 300-mg dose of bepirovirsen in participants with HI and matched HPs, enrolled in 2 parts (Part 1: moderate HI; Part 2: mild HI). If no predefined difference in the area under the concentration-time curve from time 0 (predose) to infinite time (AUC0-∞) and maximum observed concentration (Cmax; geometric mean ratio [GMR] 0.5-1.5) was identified in Part 1, findings were applied to mild HI, eliminating Part 2. Participants were monitored for 50 days post-treatment and noncompartmental analysis estimated PK parameters. Twenty-four participants (moderate HI, n = 12; HP, n = 12) received bepirovirsen and completed Part 1. AUC0-∞ and Cmax were lower in participants with moderate HI (GMR 0.69 and 0.67, respectively) than in HPs, while apparent clearance (CL/F) and apparent terminal phase volume of distribution (Vz/F) were higher (GMR 1.44 and 1.64, respectively), but fell within the predefined thresholds of difference for this study. Part 2 was omitted. Adverse events were mild. Moderate HI did not have a clinically relevant impact on bepirovirsen PK or safety.

Therapeutic Antisense Oligonucleotides in Oncology: From Bench to Bedside.

Advancements in our comprehension of tumor biology and chemoresistance have spurred the development of treatments that precisely target specific molecules within the body. Despite the expanding landscape of therapeutic options, there persists a demand for innovative approaches to address unmet clinical needs. RNA therapeutics have emerged as a promising frontier in this realm, offering novel avenues for intervention such as RNA interference and the utilization of antisense oligonucleotides (ASOs). ASOs represent a versatile class of therapeutics capable of selectively targeting messenger RNAs (mRNAs) and silencing disease-associated proteins, thereby disrupting pathogenic processes at the molecular level. Recent advancements in chemical modification and carrier molecule design have significantly enhanced the stability, biodistribution, and intracellular uptake of ASOs, thereby bolstering their therapeutic potential. While ASO therapy holds promise across various disease domains, including oncology, coronary angioplasty, neurological disorders, viral, and parasitic diseases, our review manuscript focuses specifically on the application of ASOs in targeted cancer therapies. Through a comprehensive examination of the latest research findings and clinical developments, we delve into the intricacies of ASO-based approaches to cancer treatment, shedding light on their mechanisms of action, therapeutic efficacy, and prospects.

Tofersen for SOD1 ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative condition affecting the motor system. The heterogenous nature of ALS complicates trial design. Genetic forms of ALS present an opportunity to intervene in a less heterogeneous population. ALS associated with gain of function mutations in SOD1 make 'knock-down' strategies an attractive therapeutic approach. Tofersen, an antisense oligonucleotide that reduces expression of SOD1 via RNAase mediated degradation of SOD1 mRNA, has shown robust effects on ALS biomarkers. While a Phase III trial of tofersen failed to meet its primary end point, open label extension data suggests that tofersen slows progression of SOD1 ALS.

What is this summary about? Amyotrophic lateral sclerosis is a fatal, progressive disease of the motor system. Most approved treatments have a modest effect on disease progression. Tofersen is a recently approved medication that acts by reducing a toxic protein found in a particular form of inherited amyotrophic lateral sclerosis. This is an overview of the published clinical data for tofersen as well as some background information about tofersen and other treatments for amyotrophic lateral sclerosis.What were the results? Treatment with tofersen reduced levels of the target protein and markers of nervous system damage. In longer follow-up, patients treated with tofersen showed less disease progression.What do the results of the study mean? Treatment with tofersen likely slows progression in this form of inherited ALS, though longer duration studies would likely be needed to demonstrate this effect.

Antisense Oligonucleotide Therapy for Calmodulinopathy.

BACKGROUND: Calmodulinopathies are rare inherited arrhythmia syndromes caused by dominant heterozygous variants in CALM1, CALM2, or CALM3, which each encode the identical CaM (calmodulin) protein. We hypothesized that antisense oligonucleotide (ASO)-mediated depletion of an affected calmodulin gene would ameliorate disease manifestations, whereas the other 2 calmodulin genes would preserve CaM level and function. METHODS: We tested this hypothesis using human induced pluripotent stem cell-derived cardiomyocyte and mouse models of CALM1 pathogenic variants. RESULTS: Human CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes exhibited prolonged action potentials, modeling congenital long QT syndrome. CALM1 knockout or CALM1-depleting ASOs did not alter CaM protein level and normalized repolarization duration of CALM1F142L/+ induced pluripotent stem cell-derived cardiomyocytes. Similarly, an ASO targeting murine Calm1 depleted Calm1 transcript without affecting CaM protein level. This ASO alleviated drug-induced bidirectional ventricular tachycardia in Calm1N98S/+ mice without a deleterious effect on cardiac electrical or contractile function. CONCLUSIONS: These results provide proof of concept that ASOs targeting individual calmodulin genes are potentially effective and safe therapies for calmodulinopathies.

Antisense oligonucleotides enhance SLC20A2 expression and suppress brain calcification in a humanized mouse model.

Primary familial brain calcification (PFBC) is a genetic neurological disease, yet no effective treatment is currently available. Here, we identified five novel intronic variants in SLC20A2 gene from six PFBC families. Three of these variants increased aberrant SLC20A2 pre-mRNA splicing by altering the binding affinity of splicing machineries to newly characterized cryptic exons, ultimately causing premature termination of SLC20A2 translation. Inhibiting the cryptic-exon incorporation with splice-switching ASOs increased the expression levels of functional SLC20A2 in cells carrying SLC20A2 mutations. Moreover, by knocking in a humanized SLC20A2 intron 2 sequence carrying a PFBC-associated intronic variant, the SLC20A2-KI mice exhibited increased inorganic phosphate (Pi) levels in cerebrospinal fluid (CSF) and progressive brain calcification. Intracerebroventricular administration of ASOs to these SLC20A2-KI mice reduced CSF Pi levels and suppressed brain calcification. Together, our findings expand the genetic etiology of PFBC and demonstrate ASO-mediated splice modulation as a potential therapy for PFBC patients with SLC20A2 haploinsufficiency.

Switching from inotersen to eplontersen in patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy: analysis from NEURO-TTRansform.

BACKGROUND: The phase 3 NEURO-TTRansform trial showed eplontersen treatment for 65 weeks reduced transthyretin (TTR), halted progression of neuropathy impairment, and improved quality of life (QoL) in adult patients with hereditary TTR-mediated amyloidosis with polyneuropathy (ATTRv-PN), vs. historical placebo. METHODS: NEURO-TTRansform enrolled patients with ATTRv-PN. A subset of patients were randomized to receive subcutaneous inotersen 300 mg weekly (Weeks 1-34) and subsequently switched to subcutaneous eplontersen 45 mg every 4 weeks (Weeks 37-81). Change in serum TTR and treatment-emergent adverse events (TEAEs) were evaluated through Week 85. Effects on neuropathy impairment, QoL, and nutritional status were also evaluated. RESULTS: Of 24 patients randomized to inotersen, 20 (83%) switched to eplontersen at Week 37 and four discontinued due to AEs/investigator decision. Absolute change in serum TTR was greater after switching from inotersen (-74.3%; Week 35) to eplontersen (-80.6%; Week 85). From the end of inotersen treatment, neuropathy impairment and QoL were stable (i.e., did not progress) while on eplontersen, and there was no deterioration in nutritional status. TEAEs were fewer with eplontersen (Weeks 37-85; 19/20 [95%] patients) compared with inotersen (up to Week 35; 24/24 [100%] patients). Mean platelet counts decreased during inotersen treatment (mean nadir reduction ‒40.7%) and returned to baseline during eplontersen treatment (mean nadir reduction, ‒3.2%). CONCLUSIONS: Switching from inotersen to eplontersen further reduced serum TTR, halted disease progression, stabilized QoL, restored platelet count, and improved tolerability, without deterioration in nutritional status. This supports a positive benefit-risk profile for patients with ATTRv-PN who switch from inotersen to eplontersen.

Recent Progress in Gene-Targeting Therapies for Spinal Muscular Atrophy: Promises and Challenges.

Spinal muscular atrophy (SMA) is a severe genetic disorder characterized by the loss of motor neurons, leading to progressive muscle weakness, loss of mobility, and respiratory complications. In its most severe forms, SMA can result in death within the first two years of life if untreated. The condition arises from mutations in the SMN1 (survival of motor neuron 1) gene, causing a deficiency in the survival motor neuron (SMN) protein. Humans possess a near-identical gene, SMN2, which modifies disease severity and is a primary target for therapies. Recent therapeutic advancements include antisense oligonucleotides (ASOs), small molecules targeting SMN2, and virus-mediated gene replacement therapy delivering a functional copy of SMN1. Additionally, recognizing SMA's broader phenotype involving multiple organs has led to the development of SMN-independent therapies. Evidence now indicates that SMA affects multiple organ systems, suggesting the need for SMN-independent treatments along with SMN-targeting therapies. No single therapy can cure SMA; thus, combination therapies may be essential for comprehensive treatment. This review addresses the SMA etiology, the role of SMN, and provides an overview of the rapidly evolving therapeutic landscape, highlighting current achievements and future directions.