Wu JR, Hernandez Y, Canjels A
… +2 more, Bailey KL, Kwon EJ
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41583553
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The consequence of traumatic brain injury (TBI) is significant loss of nervous tissue that leads to long-term neurological deficits. Nucleic acid payloads offer potential treatments that can address the complex pathophys...The consequence of traumatic brain injury (TBI) is significant loss of nervous tissue that leads to long-term neurological deficits. Nucleic acid payloads offer potential treatments that can address the complex pathophysiology that unfolds after injury. TBI causes a transient disruption of the blood-brain barrier, allowing access of systemically administered nanoparticles to the affected nervous tissue. In this work, we evaluated the dosing window post-injury in which lipid nanoparticles (LNPs) carrying mRNA can access and transfect the injured brain after systemic administration and identified 24 h post-injury as a delivery time that achieves both LNP access and transfection activity. We observed that transfected cell types were majorly astrocytes and endothelial cells with no appreciable transfection of neurons. To increase neuronal transfection, we functionalized LNPs with the peptide RVG and were able to increase the proportion of neurons transfected 6.4-fold over untargeted LNPs. These results identify timelines in which LNPs can access the injured brain parenchyma to mediate gene expression and strategies to achieve neuron-specific gene delivery.
Zhang W, Wang H, Liu D
… +7 more, Mao X, Zhang Y, Yang Y, Liu Z, Pan T, Liu Y, Zhang Q
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41568169
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The CRISPR-Cas13 system exhibits potent RNA cleavage activity and has been widely utilized for RNA-targeting applications. However, its collateral cleavage of bystander RNAs limits therapeutic applications. In this stud...The CRISPR-Cas13 system exhibits potent RNA cleavage activity and has been widely utilized for RNA-targeting applications. However, its collateral cleavage of bystander RNAs limits therapeutic applications. In this study, we generated a series of LwaCas13a mutants through structure-based design and site-directed mutagenesis strategies. A triple mutant enCas13a (Q521R/E796A/E810A) was obtained with significantly enhanced target RNA cleavage activity along with only slightly increased collateral activity. To reduce the collateral activity, we optimized crRNA terminal extensions and obtained M1crRNA and M3crRNA variants that, in combination with enCas13a, maintained or reduced collateral activity while preserving enhanced targeted cleavage activity. Thus, by optimizing the Cas protein and crRNA, we have created an improved CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity. This system demonstrated superior performance in targeting endogenous genes and antiviral applications. Mechanistic studies revealed that enhanced protein-crRNA interactions and altered complex conformations underlie the improved cleavage activity. This engineering approach provides a generalizable strategy for developing CRISPR-Cas systems with enhanced therapeutic potential.
Martinez MA, Richards DY, Winn SR
… +4 more, Baris AM, Vonada A, Harper L, Harding CO
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41568168
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Low transgene integration frequency limits the therapeutic efficacy of homology-directed repair (HDR) as a treatment for genetic disorders. This study demonstrates improved efficacy of HDR-mediated gene insertion for th...Low transgene integration frequency limits the therapeutic efficacy of homology-directed repair (HDR) as a treatment for genetic disorders. This study demonstrates improved efficacy of HDR-mediated gene insertion for the treatment of murine phenylalanine hydroxylase (PAH) deficiency, a model of human phenylketonuria (PKU), through pharmacologic inhibition of competing DNA repair pathways. The targeted integration of a expressing transgene was enhanced with vanillin, a potent inhibitor of non-homologous end joining (NHEJ), reducing mean serum phenylalanine concentrations by 56.8% in treated mice. This was further improved following co-inhibition of NHEJ and microhomology-mediated end joining (MMEJ), yielding transgene insertions in approximately 10% of genomes with an associated 70.6% decrease in serum phenylalanine. Phenylalanine concentrations were further reduced to 392 μM after oral administration of sapropterin dihydrochloride, a pharmacologic cofactor of PAH. Separately, we demonstrate that rare hepatocytes harboring transgene insertions were successfully expanded to a therapeutically relevant population through selection based upon resistance to acetaminophen toxicity, but this method was hampered by adverse effects upon AAV vector production and the neurologic function of treated neonatal mice related to the presence of shRNA sequences in the AAV vector. These results demonstrate that pharmacologic inhibition of alternative DNA repair pathways can significantly enhance HDR-mediated transgene insertion .
Anderson AM, Tjärnhage E, Hinke DM
… +3 more, Braathen R, Grodeland G, Bogen B
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41568167
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Novel vaccines that confer broad protection against influenza A viruses (IAVs) are urgently needed. Hemagglutinin (HA) is the major influenza antigen targeted by protective immune responses. We have here developed a DNA...Novel vaccines that confer broad protection against influenza A viruses (IAVs) are urgently needed. Hemagglutinin (HA) is the major influenza antigen targeted by protective immune responses. We have here developed a DNA vaccine that simultaneously presents HA from 18 subtypes of IAV to the immune system. The vaccine consists of a DNA plasmid mixture that encodes a variety of dimeric vaccine proteins. Each dimer expresses two different HAs, as well as a targeting moiety directing the vaccine protein to antigen-presenting cells (APCs). When the vaccine proteins were targeted toward chemokine receptors 1, 3, and 5 (CCR1/3/5) on APC by means of macrophage inflammatory protein 1-alpha (MIP1α) (CCL3), vaccinated mice were broadly protected against infection with H1N1, H3N2, H5N1, and H7N1 influenza viruses. Furthermore, antibody-mediated protection against H1N1 was maintained when the H1 antigen was removed from the plasmid mixture, indicating that the diversity of HAs in the mixture promoted formation of antibodies specific for shared, conservative epitopes. The results may guide the development of a broadly protective influenza A vaccine for humans.
Zhong J, Zhang Z, Xiao L
… +4 more, Wang C, Yang Y, Zhang Q, Wang Z
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41552388
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Circular RNAs (circRNAs) have recently emerged as a promising new drug modality with significant therapeutic potential due to their higher stability and lower immunogenicity. Here, we report the development of a circRNA...Circular RNAs (circRNAs) have recently emerged as a promising new drug modality with significant therapeutic potential due to their higher stability and lower immunogenicity. Here, we report the development of a circRNA encoding human relaxin-2 (cRLN2), a short peptide hormone with well-established therapeutic potential, to treat liver fibrosis in a mouse model. Compared to the modified linear mRNA, cRLN2 mediated stronger and more prolonged expression of relaxin . In addition, the nanoparticle-mediated delivery of cRLN2 achieved a sustained translation into active relaxin in healthy mice with low immunogenicity. In a mouse model of liver fibrosis, cRLN2 treatment significantly decreased hepatic stellate cell activation and consequently reduced collagen deposition in fibrotic mice, while the treatment by relaxin protein showed limited anti-fibrosis effects. Toxicity evaluation confirmed that cRLN2 exhibits excellent safety and tolerability in mice. Collectively, our findings demonstrate that cRLN2 can efficiently express therapeutic proteins and alleviate liver fibrosis without obvious toxic effects, highlighting the potential of circRNAs as a novel therapeutic platform to treat fibrotic diseases.
Biswas DD, Rodriguez MYH, El Haddad L
… +11 more, Hoffmann G, Copson E, Mayman S, Jose J, Lang S, Chen O, Dela Pena A, Levin E, Abdelbarr M, Slyne A, ElMallah MK
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41552386
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Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder caused by a deficiency of dystrophin, leading to progressive muscle degeneration and eventually cardiorespiratory failure. Exon-skipping therapies...Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder caused by a deficiency of dystrophin, leading to progressive muscle degeneration and eventually cardiorespiratory failure. Exon-skipping therapies using cell-penetrating peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) restore production of a shortened but functional dystrophin protein. Since respiratory insufficiency is the leading cause of morbidity and mortality in DMD, we sought to examine the impact of PPMO on respiratory pathology. We evaluated the effects of RC-1001, a PPMO targeting a dystrophin mutation, in mice, a preclinical model of DMD. These mice received monthly intravenous doses of RC-1001 (30, 50, or 100 mg/kg), starting at 2 months of age, and were monitored until the study endpoint at 12 months of age. Respiratory function was evaluated using whole-body plethysmography and the forced oscillometry technique, followed by histological and molecular analysis of respiratory muscles. PPMO-treated mice showed dose-independent improvements in respiratory function, with postmortem studies revealing significant dystrophin restoration, reduced inflammation, and decreased fibrosis in respiratory muscles. Additionally, dystrophin restoration and strength improvements were observed in limb muscles. Overall, PPMO-mediated exon skipping effectively targets respiratory pathology and is a promising therapy for respiratory insufficiency in patients with DMD.
Maeda K, Hirunagi T, Sahashi K
… +9 more, Kamiya Y, Iida M, Sakakibara K, Onodera K, Ohyama M, Okada Y, Okano H, Asanuma H, Katsuno M
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41552385
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Polyglutamine (polyQ) diseases are inherited neurological disorders caused by an expansion of the cytosine-adenine-guanine (CAG) repeat in the causative genes. These include Huntington's disease, spinal and bulbar muscul...Polyglutamine (polyQ) diseases are inherited neurological disorders caused by an expansion of the cytosine-adenine-guanine (CAG) repeat in the causative genes. These include Huntington's disease, spinal and bulbar muscular atrophy (SBMA), and spinocerebellar ataxias (SCAs). Clinical trials have been conducted using nucleic acid therapeutics to silence the causative gene for these diseases, but none have been approved for use. Furthermore, while oligonucleotides targeting the CAG repeats are an attractive therapeutic option, concomitant silencing of the wild-type allele with normal CAG repeats can result in neuronal dysfunction. In this study, we developed an acyclic serinol nucleic acid (SNA)-modified small interfering RNA (siRNA) targeting CAG repeats. We also evaluated the safety and efficacy of the siRNA in different mouse models of polyQ diseases. Intracerebroventricularly administered siRNA was widely distributed throughout the central nervous system, where it selectively silenced the alleles encoding polyQ proteins without affecting their wild-type counterparts. Consequently, the intranuclear aggregation of polyQ proteins was reduced in mouse models of SBMA and SCA type 3. The siRNA attenuated neuromuscular degeneration and improved the lifespan and motor function of the SBMA mice. These findings suggest that SNA-modified siRNAs targeting CAG repeats represent a promising approach for treating polyQ diseases.
Chen H, Qin W, Bao H
… +10 more, Chen X, Ye X, Wang Y, Liu L, Zhang Y, Sun Y, Zhang T, Dong Y, Cen S, Zhang W
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41552384
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Poly(A) tail is crucial in regulating mRNA stability and protein translation. Thus, it is an essential element of mRNAs transcribed for mRNA medicines. However, the repetitive nature of the poly(A) tail can lead to sign...Poly(A) tail is crucial in regulating mRNA stability and protein translation. Thus, it is an essential element of mRNAs transcribed for mRNA medicines. However, the repetitive nature of the poly(A) tail can lead to significant poly(A) length variation and compromise the quality of the mRNA drug substance. Previous studies improved poly(A) transmission stability by inserting a non-adenosine spacer. Here, we designed new segmented poly(A) variants and evaluated their transmission stability in bacteria transformation and their ability in supporting protein expression in animals. We identified specific variants with multiple non-adenosine insertions that can maintain high transmission stability with robustness in and facilitate high protein expression in animals. Among the newly designed poly(A) variants, RG2 showed high and consistent transmission stability comparable to the A30-70 variant that is the industry gold standard but higher protein expression in animals than A30-70. We also isolate new factors that can influence the stable transmission of poly(A), such as poly(A) surrounding sequences and bacteria culture temperature. Thus, our work offers new tools valuable for rapidly developing mRNA vaccines and therapeutics.
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41541271
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Lipid nanoparticles (LNPs) are pivotal for mRNA delivery, yet predominant hepatic accumulation limits their therapeutic application in immunologically active sites like the spleen. Engineering LNPs for splenic mRNA deliv...Lipid nanoparticles (LNPs) are pivotal for mRNA delivery, yet predominant hepatic accumulation limits their therapeutic application in immunologically active sites like the spleen. Engineering LNPs for splenic mRNA delivery is therefore essential for advancing mRNA-based immunotherapies. Here, we rationally designed a series of LNPs by modulating the alkyl chain length of PEGylated lipids (fast-shedding C14 DMG-PEG vs. slow-shedding C18 DSG-PEG) and their molar ratios (0.75% and 1.5%). This systematic variation precisely controlled nanoparticle physicochemical properties and protein corona composition. Among these formulations, DSG-PEG LNPs at 0.75% mol exhibited the largest size (∼170 nm by DLS; ∼60 nm core by cryo-EM) and a distinct protein corona. Notably, this DSG-PEG 0.75% mol formulation demonstrated significantly enhanced selective spleen accumulation and efficient mRNA delivery . Furthermore, the optimized LNP potently activated dendritic cells, expanded antigen-specific CD4 T cell populations, and significantly inhibited tumor growth in a B16-ovalbumin (OVA) melanoma model. Collectively, our results establish a rational strategy via precise PEGylated lipid engineering to redirect LNP biodistribution from the liver to the spleen. These findings validate the spleen as a critical target for mRNA vaccines and provide a versatile platform for next-generation cancer immunotherapies.
Huang ZA, Li ZM, Wang J
… +6 more, Ji GS, Gao ZC, Chen AF, Tian ZK, Yu F, Song GS
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41541269
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The -acetylgalactosamine (GalNAc)-conjugate delivery platform has emerged as a pivotal enabling technology for the clinical translation of oligonucleotide therapeutics. The development of GalNAc-conjugated small interfer...The -acetylgalactosamine (GalNAc)-conjugate delivery platform has emerged as a pivotal enabling technology for the clinical translation of oligonucleotide therapeutics. The development of GalNAc-conjugated small interfering RNA (siRNA) therapeutics has been significantly advanced through structural optimization of the delivery platform. Here, we report that a series of ribofuranose- and deoxyribofuranose-incorporated GalNAc-siRNA conjugates targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA were synthesized and evaluated for their bioactivities and tissue distribution . When compared in detail with inclisiran, an approved siRNA drug that incorporates a triantennary GalNAc construct (designated L96) for targeted liver delivery, inc-G5, which contains ribofuranose ring within its GalNAc cluster, exhibits superior efficacy. Additionally, pharmacokinetic (PK) analysis in rats revealed that G5-conjugated siRNAs preferentially accumulate in the liver, have an extended elimination half-life, and exhibit the highest liver-to-kidney ratio, which indicated excellent liver delivery specificity. Moreover, the synthesis of G5 can be readily scaled up to the kilogram level, thereby providing robust support for the production of oligonucleotide drugs. These results underscore the critical application of ribofuranose integration in GalNAc-siRNA drugs, establishing G5 as a promising liver-targeted delivery moiety for siRNAs to achieve sustained gene silencing with improved hepatic specificity and therapeutic durability.
Fiduccia I, Vitale E, Varrica R
… +11 more, Ricci D, Marino S, Zito A, Pace A, Colige A, Moutschen M, Borutzki Y, Bileck A, Meier-Menches SM, Lentini L, Pibiri I
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41541268
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Nonsense mutations are among the genetic causes of LRBA (lipopolysaccharide-responsive beige-like anchor) deficiency, a rare autosomal-recessive immunodeficiency disorder. These mutations introduce premature stop codons,...Nonsense mutations are among the genetic causes of LRBA (lipopolysaccharide-responsive beige-like anchor) deficiency, a rare autosomal-recessive immunodeficiency disorder. These mutations introduce premature stop codons, leading to the loss of LRBA protein expression. Following the recent market withdrawal of ataluren, the only approved translational readthrough-inducing drug (TRID), there is an urgent need for alternative therapeutic options. In this study, we investigated the efficacy of three 1,2,4-oxadiazole-based TRIDs-NV848, NV914, and NV930-using primary fibroblasts from a patient homozygous for the R1683X nonsense mutation. All compounds restored full-length LRBA protein with correct cytoplasmic localization, as confirmed by western blot and immunofluorescence, outperforming ataluren in readthrough efficiency. NV848 exhibited the strongest activity and uniquely increased LRBA mRNA levels, suggesting transcript stabilization. In contrast, NV930 and NV914 induced readthrough without stabilizing mRNA. Global proteomic profiling revealed minimal off-target effects for NV848, limited protein modulation by NV914, and widespread variations of 828 proteins by NV930, affecting pathways related to vesicular transport and mRNA splicing. However, network analysis revealed poor connectivity among differentially expressed proteins, with LRBA unrelated to any regulated cluster. These findings highlight the reported molecules as promising candidates for precision therapy in LRBA deficiency and shed light on the broader cellular impact of TRIDs.
Poolsup S, Yaghoobi E, Radchanka A
… +18 more, Mulloo N, Uguccioni S, Pezacki JP, Khraibah A, Jawad A, Uppal GK, Gu Y, Lapointe BP, Hüttmann N, Minic Z, Artyushenko PV, Shchugoreva IA, Rogova AV, Tomilin FN, Morozov D, Kichkailo AS, Kolovskaya OS, Berezovski MV
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41541267
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The SARS-CoV-2 spike (S) protein, crucial for viral entry, remains a key target for diagnostics and therapeutics amid evolving variants. Here, we describe the selection and characterization of novel DNA aptamers targetin...The SARS-CoV-2 spike (S) protein, crucial for viral entry, remains a key target for diagnostics and therapeutics amid evolving variants. Here, we describe the selection and characterization of novel DNA aptamers targeting the S1 subunit, including the Omicron strain, via systematic evolution of ligands by exponential enrichment (SELEX) and biolayer interferometry (BLI). Three aptamers-AptS1-tSP4, AptS1-tSP10, and AptS1-tSP11-exhibited nanomolar dissociation constants (14-59 nM), with AptS1-tSP10 demonstrating good selectivity over MERS-CoV and robust binding in human saliva and pseudovirus samples. Integration with proximity ligation assay and qPCR (PLA-qPCR) achieved a detection limit of 3 pM, surpassing many antibody-based methods. Mass spectrometry-based epitope mapping identified the receptor-binding domain (RBD) peptide VGGNYNYLYR as the primary binding site for AptS1-tSP10. Molecular dynamics and quantum mechanics simulations revealed stable interactions through hydrogen bonding and π-π stacking with neutral residues in both open and closed spike conformations, independent of variant mutations. These multifunctional aptamers offer a versatile platform for ultrasensitive, epitope-specific SARS-CoV-2 detection and pave the way for nucleic acid-based therapeutics to combat viral infections.
Kim YJ, Bang JY, Yu HW
… +5 more, Lim Y, Lee J, Park HJ, Seo YJ, Hong SH
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41541266
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Optimizing co-stimulatory signaling to enhance T cell responses is central to effective antitumor immunity. In this study, we developed single-stranded RNAs (ssRNAs) utilizing the internal ribosome entry site (IRES) of e...Optimizing co-stimulatory signaling to enhance T cell responses is central to effective antitumor immunity. In this study, we developed single-stranded RNAs (ssRNAs) utilizing the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV) to express OX40L, 4-1BBL, and ICOSL and evaluated their efficacy. Co-culture of splenocytes with tumor cells transfected with these ssRNAs resulted in increased cytokine production and proliferation, along with altered T helper (Th) subsets. , intramuscular delivery of ssRNAs expressing co-stimulatory molecules expanded antigen-specific CD8 T cells. Furthermore, intratumoral delivery of these ssRNAs significantly suppressed tumor growth and induced complete tumor regression in a subset of melanoma-bearing mice. Mechanistically, ssRNAs expressing co-stimulatory molecules promoted immune cell infiltration into the tumor site and increased the cytotoxic CD8 T cells while reducing regulatory T cells (Tregs) in secondary lymphoid organs. These findings suggest that IRES-based ssRNAs expressing co-stimulatory molecules represent a promising platform for the development of effective cancer immunotherapies.
Pradeep SP, DiVasto BE, Glazer PM
… +1 more, Bahal R
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41536812
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Targeting genomic DNA to silence oncogenes is a promising strategy for cancer therapy. Gamma peptide nucleic acid (γPNA) targeting genomic DNA has shown established efficacy and in multiple preclinical models. In this...Targeting genomic DNA to silence oncogenes is a promising strategy for cancer therapy. Gamma peptide nucleic acid (γPNA) targeting genomic DNA has shown established efficacy and in multiple preclinical models. In this study, we investigated a combination treatment by integrating γPNAs with direct or indirect inhibitors to further enhance the anti-tumor effect. Antigene γPNAs treatment combined with histone deacetylase inhibitors (HDACis) resulted in a synergistic reduction in mRNA expression, protein levels, and cell viability . Additionally, the combination of γPNAs with MYC/MAX inhibitors reduced the IC, thereby enhancing their anti-tumor efficacy. We also examined the co-delivery of γPNAs with small interfering RNA (siRNA), which significantly downregulated mRNA and protein expression. Furthermore, small molecules targeting indirectly exhibited high synergy scores. This study demonstrates that combining anti-gene γPNAs with small molecules or antisense approaches creates a potent and synergistic strategy for targeting -driven cancers, presenting a promising therapeutic avenue for cancer treatment.
Zhang A, Lim KRQ, Chen Z
… +2 more, Yokota T, Chen YW
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41536811
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Facioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of double homeodomain protein 4 (). The disease has no effective treatment. Previously we demonstrated effective knockdown and using 2'-O-...Facioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of double homeodomain protein 4 (). The disease has no effective treatment. Previously we demonstrated effective knockdown and using 2'-O-methoxyethyl (2'MOE) and locked nucleic acid (LNA) gapmer antisense oligonucleotides delivered via intramuscular injections. This study aimed to evaluate efficacy of the gapmers via systemic delivery using mouse models expressing at different levels. First, we injected the gapmers subcutaneously to mice at 20 mg/kg twice a week for 10 weeks. Results showed significant reduction in mRNA and improved muscle function, assessed by grip strength. Muscle fibrosis and circulating TGFβ1 levels were significantly reduced, approaching baseline level. A dose-dependent reduction was observed in 2'MOE gapmer treated. In the model, where DUX4 expression was induced by tamoxifen (5 mg/kg), treatment with 2'MOE gapmers effectively reduced , improved muscle function, and decreased inflammation. These findings highlight the therapeutic potential of gapmer-based reduction, leading to phenotypic improvement and restoration of muscle function in FSHD mouse models.
Chekuri A, Kondabolu K, Kirchner EG
… +10 more, Koli S, Chagnon M, Krasniqi-Vanmeter D, Stern ME, Bolduc J, Romano G, Pagani F, Vandenberghe LH, Morini E, Slaugenhaupt SA
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41536810
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Familial dysautonomia (FD) is a rare autosomal recessive neurodegenerative disorder caused by a splicing mutation in the gene. It predominantly affects the sensory and autonomic nervous systems, with progressive vision...Familial dysautonomia (FD) is a rare autosomal recessive neurodegenerative disorder caused by a splicing mutation in the gene. It predominantly affects the sensory and autonomic nervous systems, with progressive vision loss due to optic neuropathy being a universal and debilitating symptom. Retinal pathology in FD involves progressive thinning of the retinal nerve fiber layer (RNFL), resulting from the degeneration of retinal ganglion cells (RGCs). Notably, FD-associated vision loss has a postnatal onset, offering a critical window for therapeutic intervention before severe visual impairment develops in adolescence. Currently, no approved treatments exist to prevent or reverse vision loss in FD. In this study, we present a novel RNA-based therapeutic approach targeting pre-mRNA splicing in the retina. We engineered exon-specific U1 small nuclear RNAs (ExSpeU1s) to enhance inclusion of exon 20 in the mutant transcripts in the retina, thereby restoring full-length ELP1 expression. Delivery of ExSpeU1 via adeno-associated virus serotype 2 (AAV2) to the retina improved splicing, rescued RGC loss, and visual function in an FD mouse model. These findings highlight ExSpeU1-mediated splicing correction as a promising therapeutic approach for treating optic neuropathy in FD, offering potential to preserve vision and improve quality of life for patients.
Liou RH, Urrutia-Cabrera D, Liu CF
… +13 more, Wu S, Westin IM, Golovleva I, Liu GS, Kumar S, McLenachan S, Chen FK, Hsu FT, Huang CL, Edwards T, Martin KR, Cheng AW, Wong RCB
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41536809
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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 therap...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 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in 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 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 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 c.5461-10T>C variants and up to 78% reduction of the c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced 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 , providing an important step to advance the development of gene therapy to treat STGD1.