Molecular Therapy Nucleic Acids

Exon-skipping therapy mediated by antisense oligonucleotides (ASOs) is expected to provide a therapeutic option for Duchenne muscular dystrophy (DMD). ASOs for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated ASOs for exon 44 skipping (applicable to approximately 6% of all DMD patients) to improve activity by using a novel ASO design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to simultaneously target two splicing regulators in exon 44, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a DMD patient to whom exon 44 skipping is applicable. We also assessed the in vivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an ASO with a novel connected-sequence design, showed both in vitro and in vivo exon-skipping activity.

Antisense oligonucleotides (ASOs) have emerged as one of the most innovative new genetic drug modalities, however, the high molecular weight limits their bioavailability for otherwise treatable neurological disorders. We investigated conjugation of ASOs to an antibody against the murine transferrin receptor (TfR), 8D3130, and evaluated it via systemic administration in mouse models of the neurodegenerative disease, spinal muscular atrophy (SMA). SMA like several other neurological and neuromuscular diseases, is treatable with single-stranded ASOs, inducing splice modulation of the survival motor neuron 2 (SMN2) gene. Administration of 8D3130-ASO conjugate resulted in bioavailability of 2.7% of the injected dose in brain. Additionally, 8D3130-ASO yielded therapeutically high levels of SMN2 splicing in the central nervous system of mildly affected adult SMA mice and resulted in extended survival of severe SMA mice. Systemic delivery of nucleic acid therapies with brain targeting antibodies offers powerful translational potential for future treatments of neuromuscular and neurodegenerative diseases.

AO_SCPLOWBSTRACTC_SCPLOWSmall interfering RNAs (siRNAs) exemplify the promise of genetic medicine in the discovery of novel therapeutic modalities. Their ability to selectively suppress gene expression makes them ideal candidates for development as oligonucleotide pharmaceuticals. Recent advancements in machine learning (ML) have facilitated unmodified siRNA design and efficacy prediction, but a model trained to predict the silencing activity of siRNAs with diverse chemical modification patterns has yet to be published, despite the importance of such chemical modifications in designing siRNAs with the potential to advance to the clinic. This study presents the first application of ML to classify efficient chemically modified siRNAs from sequence and chemical modification patterns alone. Three algorithms are evaluated at three classification thresholds and compared according to sensitivity, specificity, consistency of feature weights with empirical knowledge, and performance on an external validation dataset. Finally, possible directions for future research are proposed.

Human gremlin-1 is a physiologically versatile signaling molecule that has been associated with several human diseases including cancer. The ability of gremlin-1 to induce fibrosis in organs and transduce angiogenesis makes it a target for cancer therapy. RNAi-based therapy has proven to be very efficient and specific in tumor growth inhibition. The efficacy and specificity of siRNA-mediated gene silencing depends on the designing approaches. Here, empirical guidelines for siRNA design and comprehensive target site availability analysis were used to select effective siRNA from a plethora of potential candidates designed using several computation algorithms. Then, the selected siRNA candidates were subjected to stringent similarity searches in order to obtain siRNA candidates with reduced off-target effects (high specificity). The best candidates were compared to experimentally successful gremlin-1 siRNAs in order to predict the silencing potency of the selected siRNAs. siRNA-6 (sense strand: 5-CCAAGAAAUUCACUACCAU-3), siRNA-7 (sense strand: 5-CCAUGAUGGUCACACUCAA-3) and siRNA-47 (sense strand: 5-GGCCCAGCACAAUGACUCA-3) were predicted to be highly effective siRNA candidates for gremlin-1 silencing. These siRNAs can be considered for RNAi-based therapy because off-target effects are predicted to be minimal.

Guanidine- bridged nucleic acid (GuNA) is a bridged nucleic acid analog with high binding affinity towards complementary strands along with high nuclease resistance. GuNA has been developed to improve pharmacokinetics and safety profiles of phosphorothioate modified gapmers. Here, we evaluated antisense oligonucleotides (ASOs) modified with combination of GuNA and 2-O-methoxyethyl (MOE) could significantly improve KD activity in vitro and in vivo. Long-term efficacy evaluation showed that intracerebroventricularly administered GuNA modified gapmers stayed active for over 24 weeks in mouse brain. Furthermore, we found that GuNA-modified gapmers could evade lymphocyte-derived immune responses and kept ASO-induced toxicity in check. Taken together, the results of this study demonstrated that GuNA modification can improve the potential of ASOs, especially in the central nervous system.

siRNA delivery platforms capable of accessing both central and peripheral tissues are critically needed to expand the therapeutic potential of oligonucleotides. To address this, we developed a novel siRNA-antibody conjugate by attaching an Hprt-targeting siRNA to an engineered antibody shuttle. This shuttle targets the insulin-like growth factor 1 receptor (IGF1R) using a fused antibody fragment (Clone F) and utilizes an antibody backbone with no tissue-relevant binding in this study. The resulting conjugate, designated Clone F-Hprt, demonstrated robust in vivo knockdown across multiple tissues. Clone F-Hprt demonstrated enhanced penetration into central nervous system (CNS) tissues compared to unconjugated siRNA following intracerebroventricular (ICV) and intravenous (IV) administration. In peripheral tissues, Clone F-Hprt achieved widespread knockdown in muscle, heart, and lung, consistent with IGF1R expression. The conjugate was well tolerated across all routes, including with repeated dosing. Although several receptor-mediated approaches for CNS delivery are progressing to the clinic (e.g., targeting the transferrin receptor), clinical validation remains to be demonstrated. Our findings highlight IGF1R as an alternative receptor capable of supporting delivery across both central and peripheral tissues, offering a complementary strategy for expanding the therapeutic landscape of oligonucleotide delivery.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder with limited therapeutic options. Mutations in the gene encoding superoxide dismutase 1 (SOD1) represent a major genetic cause of familial ALS, driving motor neuron degeneration through toxic gain-of-function mechanisms. Although gene silencing approaches targeting SOD1 show substantial therapeutic potential, their clinical translation remains restricted by suboptimal delivery to the spinal motor neurons and safety concerns linked to conventional viral vectors. This study presents a minimally invasive gene therapy strategy that combines the retrograde transport capability of rAAV2-retro with the safety of an artificial microRNA (miRNA) to achieve pan-spinal SOD1 silencing. A single intramuscular injection of rAAV2-retro-miRNA into neonatal SOD1G93A mice resulted in widespread transduction of spinal motor neurons, significant reduction of mutant SOD1 protein, and multifaceted therapeutic benefits. Treated mice exhibited delayed disease onset, extended lifespan, preserved motor function, reduced neuroinflammation, and protection of neuromuscular junctions and spinal motor neurons. Importantly, the artificial miRNA construct demonstrated a superior safety profile relative to short hairpin RNA (shRNA)-based constructs, which induced marked toxicity and lethality in wild-type mice. These findings establish neonatal intramuscular delivery of rAAV2-retro-miRNA as a safe, efficient, and clinically translatable strategy for preemptive intervention in SOD1-mediated ALS, offering broader applicability to other motor neuron diseases.

The efficacy of adeno-associated virus (AAV)-mediated systemic gene therapy for central nervous system (CNS) diseases is often limited by the blood-brain barrier (BBB). This study systematically evaluated the tissue distribution of three BBB-crossing AAV capsid variants (PHP.eB, CNSRCV300, and BI-hTFR1) following intravenous injection in mice, using either a constitutive promoter (CAG) or a neuron-specific promoter (hSyn) to drive EGFP reporter expression. Compared with AAV9, both PHP.eB and CNSRCV300 demonstrated significantly enhanced BBB penetration and brain transduction efficiency. While the use of the hSyn promoter led to reduced transgene expression in the brain compared with the CAG promoter, and substantially decreased visible reporter expression in peripheral organs, viral deposition in the liver could still be detected via immunohistochemistry. Overall, CNSRCV300 exhibited the most favorable balance between brain-targeting efficiency and biosafety, highlighting its potential as a promising delivery vector. In summary, both the capsid and promoter jointly influence AAV-mediated expression in vivo, and although cell type-specific promoters can reduce off-target expression, residual viral deposition in non-target tissues remains a potential safety concern.

The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors such as NK cell activation, construct design, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped LVs for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with a NK cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and Retronectin, resulting in excellent transduction efficiency without compromising NK cell phenotype or growth. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the first exon of the huntingtin gene (HTT). Oligonucleotide therapeutics, such as short interfering RNA (siRNA), reduce levels of huntingtin mRNA and protein in vivo and are considered a viable therapeutic strategy. However, the extent to which they silence HTT mRNA in the nucleus is not established. We synthesized siRNA cross-reactive to mouse (wild-type) Htt and human (mutant) HTT in a di-valent scaffold and delivered to two mouse models of HD. In both models, di-valent siRNA sustained lowering of wild-type Htt, but not mutant HTT mRNA expression in striatum and cortex. Near-complete silencing of both mutant HTT protein and wild-type Htt protein was observed in both models. Subsequent fluorescent in situ hybridization (FISH) analysis shows that di-valent siRNA acts predominantly on cytoplasmic mutant HTT transcripts, leaving clustered mutant HTT transcripts in the nucleus largely intact in treated HD mouse brains. The observed differences between mRNA and protein levels, exaggerated in the case of extended repeats, might apply to other repeat-associated neurological disorders.

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominant neurodegenerative disorder caused by the expansion of CAG trinucleotide repeats in the ATXN3 gene. This mutation induces a toxic gain-of-function of the ATXN3 protein, leading to neurodegeneration, particularly in the cerebellum and brainstem. Despite extensive research, no disease-modifying treatments are available for SCA3 patients. In this study, we developed and tested a novel therapeutic strategy using recombinant adeno-associated virus (rAAV) to deliver bicistronic artificial microRNAs designed to selectively silence the mutant ATXN3 allele. Through in vitro screening, we identified a lead construct (miATXN3-10x2) that effectively and specifically silenced the mutant allele by targeting of a single nucleotide polymorphism (SNP) associated with the repeat expansion. This construct was packaged into rAAV9 and delivered via intra-cerebellar administration into two mouse models of SCA3, resulting in robust suppression of mutant ATXN3 in the cerebellum. To assess long-term efficacy, we performed intra-cisterna magna (ICM) injections of rAAV9-miATXN3-10x2 in a severe SCA3 transgenic mouse model. Widespread distribution of viral vectors and miATXN3 copies was observed in disease-relevant brain regions. Treated animals exhibited significant and sustained improvements in motor function at 5, 8, and 11 weeks post-injection. Histological analyses showed a reduction in mutant ATXN3 aggregates and a trend toward preventing shrinkage of cerebellar molecular layer. These findings were supported by dose-dependent reductions in mutant ATXN3 mRNA levels and decreased expression of neuroinflammatory markers in the cerebellum. Additionally, a significant increase of the neuronal marker NeuN was also observed in treated animals. Finally, transcriptomic profiling of the cerebellum demonstrated that treated transgenic animals exhibited an improved transcriptomic signature, shifting toward a wild-type profile. In conclusion, our findings highlight the therapeutic potential of a single administration of rAAVs encoding bicistronic artificial microRNAs for allele-specific gene silencing in SCA3. This study provides compelling preclinical evidence supporting the translation of this approach into clinical applications for SCA3 patients.

In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether such modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we provide a systematic comparison of genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiD, and Streptavidin-conjugated blocked-end linear DNA. All of these forms of linear DNA resulted in a high fraction of the cells being stably transfected - between 10% and 20% of the initially transfected cells, with CELiDs showing the highest rates of integration. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration. Moreover, our analysis suggest that conventional AAV-based gene therapy may be highly susceptible to integration, which is consistent with recent findings from long-term clinical studies.

BackgroundThe gene for apolipoprotein E4 (ApoE4 E4) confers an increased risk for development and lowers the age of onset of Alzheimers disease (AD), and is a highly suitable target for CRISPR-based editing because ApoE4 differs from ApoE3 by a single nucleotide polymorphism in the codon for residue 112 that codes for arginine (CGC) in E4 and cysteine (TGC) in E3. Editing of E4 to E3 could lower the risk of AD or ameliorate E4-related AD phenotypes. For AD, in order to deliver CRISPR components across the blood-brain barrier to the brain, we have developed a delivery platform termed Synthetic Exosomes (SEs) - microfluidically-synthesized deformable nanovesicles approximately the size of natural exosomes that have the ability to cross the BBB and deliver cargo to the brain. Here, we describe our use of SEs carrying CRISPR to successfully edit E4 to E3 in brain tissue of an E4-expressing mouse model. MethodsSeveral CRISPR guide RNAs (gRNA) and Cytosine Base Editor (CBE) mRNAs were synthesized by chemical and in vitro transcription syntheses, respectively. Four combinations of gRNA and CBE mRNA were tested in vitro for their relative activity to edit the E4 (cytosine) to E3 (thymine) in E4-expressing neuroblastoma (E4-N2A) and human Kelly neuroblastoma cells, to assess which combination produced the highest E4 to E3 base editing efficiency. The CRISPR RNA combination with the highest efficiency was encapsulated in SEs and injected intravenously (IV) via the tail vein into an AD model E4-expressing (E4-5XFAD) transgenic mouse; as a negative control, an E4-5XFAD mouse was injected with empty SEs. Five days after injection, mice were euthanized and brain, liver, and buffy coat (white blood cells (WBC)) collected to determine the editing of E4 to E3 measured by Next Generation Sequencing. In addition, E3 mRNA was measured in the brain and liver and compared to the %E3 gene editing. ResultsThe highest gRNA+CBE mRNA editing efficiency was [~]50% in E4-N2A cells and the same gRNA+CBE combination delivered in SEs to Kelly neuroblastoma cells showed 6.5% editing efficiency. In the E4-5XFAD mouse in vivo, five days after IV delivery of a single dose of the highest-activity SE-CRISPR gRNA+CBE mRNA, the percent of E4 edited to E3 was 0.14% in brain, 0.8% in liver, and 0.36% in WBCs. As evidence of functional editing, SE-CRISPR-treated mice had 0.03% E3 mRNA in brain and 0.09% E3 mRNA in liver. ConclusionsWhile this level of ApoE4 to E3 editing achieved five days after a single IV injection of SE-CRISPR is small, it provides initial in vivo proof-of-concept that the ApoE4 gene can be successfully edited, and editing results in functional expression of ApoE3 mRNA. The findings presented herein supports further optimization of the SE-CRISPR approach to increase the level of editing in brain as part of clinical development of SE-CRISPR as a powerful novel therapeutic approach for AD.

MicroRNAs (miRNAs) are implicated in the onset and progression of a variety of diseases. Modulating the expression of specific miRNAs is a possible option for therapeutic intervention. A promising strategy is the use of antisense oligonucleotides (ASOs) to inhibit miRNAs. Targeting ASOs to specific tissues can potentially lower the dosage and improve clinical outcomes by alleviating systemic toxicity. We leverage here automated peptide nucleic acid (PNA) synthesis technology to manufacture an anti-miRNA oligonucleotide (antagomir) covalently attached to a 12-mer peptide that binds to transferrin receptor 1. Our PNA-peptide conjugate is active in cells and animals, effectively inhibiting the expression of miRNA-21 both in cultured mouse cardiomyocytes and different mouse organs (heart, liver, kidney, lung, and spleen), while remaining well-tolerated in animals up to the highest tested dose of 30 mg/kg. Conjugating the targeting ligand to the PNA antagomir significantly improved inhibition of miRNA-21 in the heart by over 50% relative to the PNA alone. Given the modulation of biodistribution observed with our PNA-peptide conjugate, we anticipate this antagomir platform to serve as a starting point for pre-clinical development studies. Table of Contents Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/536802v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@645857org.highwire.dtl.DTLVardef@1e3be1corg.highwire.dtl.DTLVardef@d6286aorg.highwire.dtl.DTLVardef@1f76032_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisConjugating T12, a peptide targeting transferrin receptor 1 (TfR1), to a peptide nucleic acid (PNA) oligonucleotide targeting microRNA-21 increases delivery of the PNA-T12 conjugate to cardiac tissue relative to PNA alone.

Oligonucleotide (ON) therapeutics are promising as a disease-modifying therapy for central nervous system disorders. Intrathecal ON administration into the cerebral spinal fluid is a safe and effective delivery mode to the CNS. However, preclinical studies have shown acute toxicities following high-dose central ON delivery. Here we characterize a transient neurobehavioral change peaking 15 minutes after ON dosing and resolving after 120 minutes. Symptoms include shaking, muscle twitching, cramping, hyperactivity, stereotypic movements, hyperreactivity, vocalizations, tremors, convulsions, and seizures. These are collectively referred here as the acute neuronal activation response. Acute neuronal activation is observed in rats, mice, and non-human primates and is quantifiable using a simple scoring system. It is distinct from acute sedation seen with some phosphorothioate-modified antisense oligonucleotides, characterized by loss of spinal reflexes, ataxia, and sedation. The acute neuronal activation response is largely sequence-independent and is driven by ON chelation of divalent cations, particularly influenced by the divalent cations-to-ON ratio in the dosing solution. Acute neuronal activation can be safely mitigated by adjusting this ratio through magnesium supplementation in the ON formulation. We provide a comprehensive framework for quantifying and mitigating the acute neuronal activation response caused by high concentrations of centrally delivered ON therapeutics in preclinical species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/638138v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@b18796org.highwire.dtl.DTLVardef@13c9898org.highwire.dtl.DTLVardef@1463019org.highwire.dtl.DTLVardef@ffe719_HPS_FORMAT_FIGEXP M_FIG C_FIG

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated splicing factors or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids (PDOs) are a promising platform to recapitulate key aspects of disease states and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) transfection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma (PDAC) organoids. This optimized delivery method allows fast and efficient screening of ASOs that reverse oncogenic alternative splicing. In combination with advancements in chemical modifications and ASO-delivery strategies, this method has the potential to accelerate the discovery of anti-tumor ASO drugs that target pathological alternative splicing.

Therapeutic in vivo gene editing with highly specific nucleases has the potential to revolutionize treatment for a wide range of human diseases, including genetic disorders and latent viral infections like herpes simplex virus (HSV). However, challenges regarding specificity, efficiency, delivery, and safety must be addressed before its clinical application. A key concern is the risk of off-target effects, which can cause unintended and potentially harmful genetic changes. We previously developed a curative in vivo gene editing approach to eliminate latent HSV using HSV-specific meganuclease delivered by an AAV vector. In this study, we investigate off-target effects of meganuclease by identifying potential off-target sites through GUIDE-tag analysis and assessing genetic alterations using amplicon deep sequencing in tissues from meganuclease treated mice. Our results show that meganuclease expression driven by a ubiquitous promoter leads to high off-target gene editing in the mouse liver, a non-relevant target tissue. However, restricting the meganuclease expression with a neuron-specific promoter and/or a liver-specific miRNA target sequence efficiently reduces off-target effects in both liver and trigeminal ganglia. These findings suggest that incorporation of regulatory DNA elements for tissue-specific expression in viral vectors can reduce off-target effects and improve the safety of therapeutic in vivo gene editing.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by rapid progression and high mortality. With genetic mutations, particularly in the SOD1 gene, playing a significant role in ALS pathogenesis, targeted therapies have become a primary focus. This study introduces RD-12500 (RAG-17), a novel siRNA-ACO (Accessory Oligonucleotide) conjugate designed to address the challenges of delivering duplex RNAs to the central nervous system (CNS). RD-12500 exhibits remarkable in vitro stability and target specificity with minimal immunostimulation. In vivo studies demonstrate its extensive CNS biodistribution, sustained accumulation post-intrathecal administration, and a robust dose-exposure-activity correlation. Notably, RD-12500 significantly reduces cerebrospinal fluid (CSF) SOD1 protein levels, indicating potent SOD1 mRNA and protein knockdown in cynomolgus monkeys. Most notably, our study breaks new ground by demonstrating the effectiveness of RD-12500 in late-stage treatment scenarios. In SOD1G93A ALS mice, post-onset administration of RD-12500 significantly delayed disease progression, improved motor function, and extended survival, marking a significant advancement over other treatments which are typically initiated pre-symptomatically in the same model mice. These findings suggest RD-12500s potential to provide therapeutic benefits not only to pre-symptomatic but also to post-symptomatic and late-stage SOD1-ALS patients.

Loss-of-function mutations in the CTNNB1 gene cause {beta}-catenin deficiency, resulting in CTNNB1 syndrome--a rare neurodevelopmental disorder characterized by motor and cognitive impairments. Given the wide variety of mutations across CTNNB1 and its dosage sensitivity, a mutation-independent therapeutic approach that preserves endogenous gene regulation is critically needed. This study introduces spliceosome-mediated RNA trans-splicing as a novel approach to restore {beta}-catenin production. Precursor mRNA trans-splicing molecules (PTMs) targeting CTNNB1 introns 2, 5, and 6 were designed and evaluated using a split fluorescent YFP reporter system. Rationally designed short antisense RNAs, which mask splicing regulatory elements, significantly enhanced PTM-mediated trans-splicing at both RNA and protein levels. Additionally, introducing a self-cleaving ribozyme at the PTMs 5 end further improved trans-splicing efficiency, likely due to increased nuclear retention. CMV promoter-driven PTM expression yielded the highest efficiency. Importantly, successful trans-splicing of the endogenous CTNNB1 transcript confirmed the physiological relevance of this strategy. This study is the first to apply and optimize SMaRT for CTNNB1 correction, providing a promising, mutation-agnostic approach for treating CTNNB1 syndrome.