Mol Ther Nucleic Acids
· 2026 Jun · PMID 42028574
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Small molecules that induce DNA double-strand breaks (DSBs) are of significant interest for cancer therapy due to their ability to exploit repair vulnerabilities in tumor cells. Mimosine, a non-proteinogenic amino acid d...Small molecules that induce DNA double-strand breaks (DSBs) are of significant interest for cancer therapy due to their ability to exploit repair vulnerabilities in tumor cells. Mimosine, a non-proteinogenic amino acid derived from Leucaena species, is known to cause cell-cycle arrest and DNA strand breaks, yet its interaction with DNA repair pathways remains poorly understood. Here, we systematically evaluated the genotoxic effects of mimosine in a panel of DNA repair-efficient and deficient cells models. Using neutral comet and γH2Ax assay, we demonstrated that mimosine treatment induces DSBs that persist in cells lacking non-homologous end joining (NHEJ), homologous recombination (HR), and nucleotide excision repair (NER). We established that cells with efficient NHEJ repair pathways can resolve mimosine-induced DNA lesions. Cell cycle analysis shows that mimosine treatment stalls the G1 phase when the NHEJ pathway is active. Cell viability profiling across cancer cells shows greater sensitivity to mimosine in cells with DNA repair deficiency. These findings establish the repair pathway dependencies that drive the mechanism of action of mimosine and its use in cancer treatment therapy.
González-Domínguez I, Boza M, Abdeljawad A
… +13 more, Shea W, Puente-Massaguer E, Mishra M, Lemus N, Lai TY, Muramatsu H, Lam K, Heyes J, Martínez-Guevara JL, Krammer F, Palese P, Pardi N, Sun W
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42028573
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Influenza B viruses cause about one-third of annual influenza infections, leading to significant respiratory illness. Vaccination remains the most effective strategy to mitigate the health and socio-economic burdens of i...Influenza B viruses cause about one-third of annual influenza infections, leading to significant respiratory illness. Vaccination remains the most effective strategy to mitigate the health and socio-economic burdens of influenza. Recent advances focus on developing a universal influenza B vaccine using chimeric hemagglutinin (HA) (cHA) and mosaic HA (mHA) constructs. cHA vaccines are engineered by replacing the entire head domain of influenza B HA with those from exotic avian influenza viruses, while mHA vaccines involve substituting only the immunodominant epitopes in the HA head domain. In this study, we assessed the immunogenicity of cHA and mHA vaccines delivered via the nucleoside-modified mRNA-lipid nanoparticle platform. Sequential immunization with these constructs in murine models stimulated immune responses targeting conserved HA epitopes, enhancing cross-protection against diverse influenza B virus strains. Notably, mHA vaccination induced higher levels of cross-reactive antibodies and antigen-specific CD4 T cells compared to cHA, resulting in superior protection against the distantly related B/Lee/1940 influenza virus strain in both passive transfer and direct challenge mouse models. These findings highlight the potential of the mHA vaccination strategy and represent a significant step toward the clinical development of broadly protective influenza B vaccines.
Chakraborty C, Bhattacharya M, Das A
… +2 more, Agoramoorthy G, Lee SS
Mol Ther Nucleic Acids
· 2026 Mar · PMID 42028170
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The genome editing technology using CRISPR-Cas9 has created a new trajectory for treating diverse human diseases. The approval of CRISPR-Cas9 therapeutics was first given to Casgevy for treating transfusion-dependent β-t...The genome editing technology using CRISPR-Cas9 has created a new trajectory for treating diverse human diseases. The approval of CRISPR-Cas9 therapeutics was first given to Casgevy for treating transfusion-dependent β-thalassemia (TDT) by the UK Medicines and Healthcare Products Regulatory Agency (UKMHRA) on November 16, 2023. Subsequently, UKMHRA approved Casgevy for the treatment of sickle cell disease (SCD), which was approved by the US Food and Drug Administration (FDA) on December 8, 2023. The approval has created a new era of gene editing medicine. Numerous clinical trials have been initiated to treat different human diseases. In this comprehensive review, we present an overview of the therapeutic development that includes the earliest days of research using CRISPR-Cas9 technology, clinical trials, UKMHRA/FDA-approved therapeutics (Casgevy), and the CRISPR-Cas9 technology approved for the therapy of SCD and TDT. We also present details on the role of biopharmaceutical industry-academia collaboration and patent landscape of the CRISPR-Cas9 technology. Finally, the therapeutics' challenges, safety concerns, and cost-effectiveness are illustrated to provide a complete understanding on the current landscape of CRISPR-Cas9-mediated therapeutics for countries' policymakers, biopharmaceutical companies, and next-generation researchers to formulate the future strategies.
Carneiro AD, Nisanov AM, Lee H
… +1 more, Schaffer DV
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42023033
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Effective delivery to target cell types remains a major obstacle for gene therapy. Recombinant adeno-associated viruses (AAVs) have proven to be effective delivery vectors for the treatment of several diseases yet still...Effective delivery to target cell types remains a major obstacle for gene therapy. Recombinant adeno-associated viruses (AAVs) have proven to be effective delivery vectors for the treatment of several diseases yet still have to demonstrate efficient delivery to many cell types, including cells of the peripheral nervous system. Schwann cells-essential for myelination, nerve regeneration, and general nerve maintenance-are strategic gene therapy targets for treating peripheral neuropathies but present unique challenges for gene delivery due to their protective barriers and diffuse localization throughout the body. To address this issue, we engineered novel AAV variants with enhanced transduction efficiency toward Schwann cells. Using directed evolution, we identified two groups of capsids with significantly improved delivery to these cells in mice or human. In addition to higher transduction efficiencies, human-tropic capsids also demonstrated higher resistance to antibody neutralization compared to natural serotypes. studies of mouse-tropic capsids confirmed higher selectivity, improved packaging titers, and decreased localization to the liver. These engineered AAVs are valuable tools for studying Schwann cells and offer new avenues for the treatment of peripheral nerve disorders.
De Chiara A, Giachino C, Pirillo MF
… +16 more, Campanile A, Pellino E, Gallinaro A, Froechlich G, Falce C, Scognamiglio A, Totaro S, Liguori MV, Peltrini R, De Simone A, Capone S, Pietro Z, Negri D, Cara A, Nicosia A, Sasso E
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42023032
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The continuous evolution of SARS-CoV-2 variants, driven by mutations in the spike protein undermines viral recognition by antibodies elicited through prior infection or vaccination with the ancestral Wuhan strain. Origin...The continuous evolution of SARS-CoV-2 variants, driven by mutations in the spike protein undermines viral recognition by antibodies elicited through prior infection or vaccination with the ancestral Wuhan strain. Original antigenic sin of SARS-CoV-2 ancestral virus or vaccine led to a weakened neutralizing antibody response against successive variants upon administration of an updated vaccine. On the contrary, T cells retain cross-reactivity thanks to the high density of conserved epitopes. We designed mRNA vaccines encoding single-chain heterotrimers of the receptor-binding domain (RBD) natural variants of interest (VOI), (RBD-VOI) and of phylogenetically informed consensus representing the major variant lineages RBD-consensus (RBD-Cons). We demonstrate a broad neutralizing activity against omicron subvariants and mitigated immune imprinting when RBD-Cons was used as a booster after conventional Wuhan spike priming. To enhance cellular immunity, we designed a second mRNA vaccine component encoding the viral polymerase NSP12 able to induce a cross-reactive T cell response to be combined with the heterotrimeric RBD vaccine. Our results offer a rational strategy for next-generation, imprinting-resistant vaccines.
Kearney A, Lukomska A, Brady J
… +3 more, Damania A, Gupta M, Trakhtenberg EF
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42023031
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Central nervous system (CNS) projection neurons' failure to repair or regenerate injured axons has devastating consequences for those who have sustained CNS injuries. Thus, there is a need for translatable factors capabl...Central nervous system (CNS) projection neurons' failure to repair or regenerate injured axons has devastating consequences for those who have sustained CNS injuries. Thus, there is a need for translatable factors capable of promoting long-distance axon regeneration in the CNS. We hypothesized that supporting lysosomes in injured neurons by supplementing their structural factors through gene therapy may foster axon regeneration. To test our hypothesis, we selected Atp6v0c for experimental regulation because it plays roles in lysosomal acidification and the degradation of misfolded proteins in response to endoplasmic reticulum (ER) stress in injured neurons. We tested this in a rodent optic nerve crush (ONC) model of traumatic optic neuropathy (TON), in which injured prototypical CNS projection neurons, the retinal ganglion cells (RGCs), do not regenerate damaged axons and eventually degenerate. Atp6v0c transgene expression was achieved using intravitreally injected adeno-associated virus serotype 2 (AAV2), which transduces the RGCs. For benchmarking, we compared efficacy to AAV2 targeting of prominent regulators of axon regeneration, Pten, and Klf9. We found that Atp6v0c transgene promoted RGC survival and long-distance axon regeneration, comparable to targeting Pten and Klf9. Thus, Atp6v0c is an axon regeneration-promoting factor with potential for treating CNS injury and disease.
Melone G, Bayraktar R, Zhao H
… +19 more, Hynes SO, Kaar F, Freegrove A, Deng J, Baseviciene A, Uzair I, Martinez KO, Guzman-Rojas L, Chervo MF, Li M, Qian W, Zhou J, Phillips A, Zambo KDA, Nikolos F, Chan KS, Thomas C, Francis L, Chang JC
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42023030
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High-grade serous ovarian carcinoma (HGSC) is often diagnosed at an advanced stage and is characterized by an immunosuppressive tumor microenvironment that facilitates disease progression, therapeutic resistance, and poo...High-grade serous ovarian carcinoma (HGSC) is often diagnosed at an advanced stage and is characterized by an immunosuppressive tumor microenvironment that facilitates disease progression, therapeutic resistance, and poor survival. We recently demonstrated that nitric oxide (NO) blockade enhances the response of metaplastic breast cancer to PI3K inhibition and taxane therapy by reducing S-nitrosylation-mediated activation of the JNK/c-Jun pathway, thereby promoting tumor differentiation, suppressing stemness, and improving survival in patient-derived xenograft models. Here, we demonstrate that inducible nitric oxide synthase (iNOS) is constitutively expressed in HGSC and that pharmacologic inhibition of NO signaling with the pan-NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) enhances cisplatin efficacy by reprogramming the tumor microenvironment. By targeting NO signaling with either L-NMMA or through CRISPR-Cas9-mediated iNOS knockout and iNOS small interfering RNA (siRNA), we impaired epithelial-to-mesenchymal transition (EMT) by inhibiting NO-mediated stabilizing effects on vimentin in both and models. Mechanistically, L-NMMA-mediated inhibition of iNOS signaling reduced S-nitrosylation, accelerated vimentin ubiquitination, and promoted its proteasome-dependent degradation. These findings identify iNOS-mediated S-nitrosylation as a key regulator of vimentin stability and EMT and suggest that therapeutic inhibition of NO signaling may increase cisplatin sensitivity in HGSC and improve patient outcomes.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42023028
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Alternative splicing quantification is critical for understanding disease mechanisms and developing precision medicine approaches. Conventional gel-based methods suffer from poor resolution and limited quantitative preci...Alternative splicing quantification is critical for understanding disease mechanisms and developing precision medicine approaches. Conventional gel-based methods suffer from poor resolution and limited quantitative precision, especially for similar-sized exons. Here, we present multiplex alternative splicing quantification (MASQ), a dual TaqMan probe qPCR platform enabling precise quantification of alternative splicing events. MASQ employs FAM-labeled probes targeting the alternative exon and HEX-labeled probes for constitutive exon normalization, providing internal controls and eliminating variability. Using exon 27 as a model system, where inclusion promotes immune checkpoint resistance through enhanced PBAF complex recruitment to the promoter, we demonstrate superior analytical performance. MASQ exhibits exceptional linearity ( > 0.98), high precision (intra-assay CV < 9%), and femtogram-level sensitivity (LOD 195 fg). CRISPR-engineered cellular validation confirmed probe specificity, while analysis of uterine corpus endometrial carcinoma specimens revealed significantly elevated exon27 inclusion in cancer tissues (61.2% ± 14.5%) compared to normal endometrium (12.7% ± 4.0%, < 0.01). Platform generalizability was demonstrated through adaptation to exon 7 quantification and monitoring splice-switching oligonucleotide effects (44.6% reduction at 24 h, < 0.0001). MASQ represents a broadly applicable platform for alternative splicing analysis, enabling applications including biomarker discovery, diagnostic development, and monitoring.
Harikrishnan H, Lamsal M, Chan KK
… +9 more, Zhang J, Tian J, Fosu K, Nguyen HP, Clapp DW, Sierra Potchanant EA, Kapur R, Xiao W, Tran NT
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42003887
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Fanconi anemia (FA) is a rare recessive genetic disorder resulting from mutations in genes in the FA-DNA repair pathway. Among its subtypes, FA complementation group C () is associated with particularly severe hematologi...Fanconi anemia (FA) is a rare recessive genetic disorder resulting from mutations in genes in the FA-DNA repair pathway. Among its subtypes, FA complementation group C () is associated with particularly severe hematologic and developmental manifestations. Gene therapy targeting autologous hematopoietic stem/progenitor cells (HSPCs) from FA patients represents a promising curative strategy for FA-associated bone marrow failure (BMF), potentially circumventing the limitations of allogeneic transplantation. Despite substantial progress in developing gene therapy for FA group A ( mutations), therapeutic strategies for FA group C have received comparatively little attention. Although FA-deficient cells are believed to exhibit compromised homologous recombination (HDR), our reporter assay demonstrated that mouse HSPCs retain HDR activity, supporting the feasibility of precise gene editing. Building on this finding, we established a CRISPR-Cas9/AAV6-HDR platform to integrate Fancc cDNA into its endogenous locus while minimizing off-target effects and AAV integration. Correction of HSPCs restored expression, rescued colony-forming capacity, and improved cellular viability. Importantly, transplantation of corrected HSPCs into mice conferred resistance to mitomycin C-induced BMF, demonstrating durable functional correction. Collectively, these results establish CRISPR-Cas9-mediated HDR as a viable and potential therapeutic strategy for FA group C.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42003885
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Fibrosis is a common pathological endpoint of numerous chronic diseases and a major driver of progressive organ dysfunction and failure. It is characterized by persistent fibroblast activation, excessive extracellular ma...Fibrosis is a common pathological endpoint of numerous chronic diseases and a major driver of progressive organ dysfunction and failure. It is characterized by persistent fibroblast activation, excessive extracellular matrix deposition, and irreversible disruption of tissue architecture. Despite substantial clinical demand, current antifibrotic therapies largely rely on single-target interventions, often demonstrating limited efficacy. mRNA nanomedicine has emerged as a promising therapeutic paradigm due to its genomic safety, programmability, transient protein expression, and capacity for multipathway regulation. However, a unified framework integrating mRNA design, delivery technologies, and translational challenges in fibrosis remains lacking. This review systematically summarizes recent advances in mRNA-based nanotherapeutics for cardiac, renal, pulmonary, and hepatic fibrosis. We discuss the key principles of mRNA molecular engineering, including chemical modifications and sequence optimization, as well as innovations in lipid nanoparticles, polymeric carriers, and biomimetic nanoplatforms. These strategies enhance organ- and cell-specific targeting, improve pharmacokinetics, and mitigate immunogenicity. Importantly, mRNA nanomedicines enable coordinated modulation of profibrotic signaling networks, including the TGF-β/Smad, NF-κB, BMP, and PI3K/mTOR pathways, thereby promoting fibrosis resolution and functional recovery. Emerging delivery paradigms, such as inhalable formulations, hydrogel-based depots, and selective organ targeting (SORT) technologies, are also highlighted along with critical barriers, including endosomal escape, repeat-dosing immune responses, and manufacturing.
Owusu L, Kim S, Patel H
… +11 more, Foltz S, Chan GNY, Kim KH, Kopen A, Yang L, Lawlor MW, Buss N, Cunningham JJ, Liu Y, Danos O, Fiscella M
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42003884
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Use of adeno-associated virus (AAV)-mediated transfer of functional microdystrophins to address Duchenne muscular dystrophy (DMD) has been established. RGX-202, an AAV8 vector encoding a novel, optimized human microdystr...Use of adeno-associated virus (AAV)-mediated transfer of functional microdystrophins to address Duchenne muscular dystrophy (DMD) has been established. RGX-202, an AAV8 vector encoding a novel, optimized human microdystrophin with an extended C-terminal domain, expressed under the Spc5-12 muscle-specific promoter, was evaluated for tolerability and efficacy in dystrophin-deficient mouse in 12- and 26-week studies at vector doses ranging from 3 × 10 to 5 × 10 gc/kg. A single intravenous administration of RGX-202 was well tolerated and led to robust, dose-dependent expression of microdystrophin in skeletal and cardiac muscles at 12 weeks, which persisted to 26 weeks post-administration. Increased microdystrophin was associated with recruitment of the dystrophin-associated protein complex to the sarcolemma, particularly at doses ≥1 × 10 gc/kg. Histologic and magnetic resonance imaging examinations revealed marked and sustained suppression of dystrophic pathology in all RGX-202-treated mice. Functionally, gains were observed in the specific force of extensor digitorum longus muscle, grip strength, and the rescue of treadmill and gait deficits. These findings provide preclinical evidence for the therapeutic efficacy of RGX-202 at a minimum effective dose (MED) of 1 × 10 gc/kg in the murine DMD model. This MED served as the starting dose for the RGX-202 clinical study (NCT05693142), which has currently completed phase III enrollment.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42003883
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Viral vectors have transformed gene therapy by enabling efficient and targeted delivery of therapeutics. However, the efficacy and safety of viral vector-based therapies are frequently constrained by host innate immune r...Viral vectors have transformed gene therapy by enabling efficient and targeted delivery of therapeutics. However, the efficacy and safety of viral vector-based therapies are frequently constrained by host innate immune responses. Sensing of viral capsids, vector genomes, and transgene-derived nucleic acids activates inflammatory and type I interferon signaling, leading to transgene silencing, vector clearance, and immune-mediated loss of transduced cells, particularly at the high vector doses required for clinical efficacy. In contrast, natural viruses have evolved sophisticated mechanisms to evade or modulate these innate immune pathways, enabling sustained gene expression and persistence. This review examines the molecular basis of innate immune sensing of commonly used viral vectors and highlights viral immune evasion strategies as an underexplored resource for therapeutic design. We discuss how repurposing viral immunomodulatory elements-through vector-intrinsic engineering and transient co-delivery-can desensitize innate signaling pathways, thereby enhancing gene transfer while preserving host immune surveillance. Extending these principles beyond viral vectors, we consider their relevance to non-viral platforms, including lipid nanoparticles, polymeric carriers, and exosomes. By reframing viral immune evasion as a therapeutic opportunity rather than a liability, we outline new directions for safer and more effective gene delivery systems.
Barnes LF, Watt B, Jalal S
… +4 more, Karunanithy G, Ranaghan MJ, Rood J, Wang HL
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42003882
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Developing analytical methods for RNA-based products, such as mRNA vaccines, requires tools that support release testing, stability assessment, and characterization. Mass photometry (MP) is a label-free, single-molecule...Developing analytical methods for RNA-based products, such as mRNA vaccines, requires tools that support release testing, stability assessment, and characterization. Mass photometry (MP) is a label-free, single-molecule technique increasingly applied to biotherapeutics and vaccines. Here, we outline best practices and technical considerations for using MP to analyze vaccine mRNA constructs and other RNA samples. With appropriate calibration, MP provides mRNA molecular weight and quantifies heterogeneous species, including those difficult to resolve using traditional techniques. We systematically evaluated experimental parameters influencing RNA measurements, including surface chemistry and buffer composition, and show that cationic-coated sample carriers and salt-containing buffers are essential for reproducible detection and mass resolution. Low-mass species were assessed using multiple mRNA constructs, kinetic analysis, and buffer controls to distinguish genuine sample heterogeneity from method artifacts. We further examined the effects of ionic additives on oligomeric species. MP results were compared with charge detection mass spectrometry and fragment analyzer measurements, showing good agreement when accounting for differences in measurement principles. Overall, MP provides a rapid, low-sample consumption, and information-rich approach for mRNA characterization and quality assessment, either as a standalone method or as an orthogonal complement to established RNA analytical techniques.
Birkenshaw A, Thomson T, Truong MP
… +8 more, Komaki Y, Ramsden N, Timpano A, Huang C, Blakney AK, Kurek DZ, Kulkarni J, Ross CJD
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42003881
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Base editors have emerged as powerful tools for precise genome editing, offering significant therapeutic potential. A critical challenge lies in optimizing the delivery and dosage of single-guide RNA to maximize on-targe...Base editors have emerged as powerful tools for precise genome editing, offering significant therapeutic potential. A critical challenge lies in optimizing the delivery and dosage of single-guide RNA to maximize on-target editing efficiency while minimizing off-target and bystander effects. This study investigates the impact of guide RNA dosage on editing efficiency and tissue specificity using a reporter mouse model with a luciferase transgene correctable by adenine base editing. Mice were treated with lipid nanoparticles co-encapsulating a fixed dose of ABE8e RNA and varying doses of guide RNA. Editing outcomes were assessed through whole-body imaging, tissue analysis, and sequencing. Increasing the guide RNA dose to 4 mg/kg enhanced editing efficiency up to 3.3-fold in multiple tissues compared to the standard 1 mg/kg dose, with the liver exhibiting the highest on-target editing rates at 63%. Bystander editing increased significantly with higher guide RNA doses, particularly in highly edited tissues like the liver, where bystander edits showed a dose-dependent increase. These findings demonstrate the importance of careful guide RNA dose optimization to balance editing efficiency and tissue specificity with bystander effects in the therapeutic applications of ABE8e.
Zou M, Wang Z, Zhou W
… +9 more, Xue G, Hui Y, Pang F, Tan R, Xu Z, Jin X, Sun H, Wang P, Jiang Q
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41971068
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Colorectal cancer (CRC) exhibits substantial heterogeneity in response to immunotherapy, highlighting the critical need for improved stratification strategies. Here, we established a robust multi-omics framework by integ...Colorectal cancer (CRC) exhibits substantial heterogeneity in response to immunotherapy, highlighting the critical need for improved stratification strategies. Here, we established a robust multi-omics framework by integrating transcriptomic, epigenetic, and mutational profiles, identifying three cancer subtypes (CS1-CS3). Among these, the CS3 subtype emerged as a distinct immunogenic class characterized by genomic instability and robust immune activation, capturing a subset of immune-inflamed tumors classified as clinically microsatellite-stable (MSS) that may be overlooked by routine microsatellite instability (MSI) testing. Single-cell transcriptomics further supported this immune-active state by revealing enrichment of pro-inflammatory myeloid populations and specialized epithelial states. Longitudinal analysis of independent immunotherapy cohorts revealed that CS3 signature activity exhibits response-dependent dynamics. While elevated baseline CS3 activity was associated with favorable clinical outcomes, significant post-treatment score reductions were primarily enriched among patients achieving radiographic complete response (CR). To facilitate clinical translation, we derived an 11-gene prognostic model for risk stratification and leveraged pharmacogenomic screening to prioritize actionable combination candidates, including multi-kinase and epigenetic inhibitors. Collectively, these findings characterize a therapeutically relevant immune-active subtype and provide a comprehensive framework of biomarkers and actionable targets to advance precision immunotherapy in CRC.