Knop O, Ben Haim N, Kalter N
… +9 more, Rosenberg M, Breier D, Baider K, Lee YN, Adam E, Somekh I, Nagler A, Somech R, Hendel A
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42181695
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X-linked severe combined immunodeficiency (SCID-X1) is a severe primary immunodeficiency caused by mutations in the gene, a shared subunit of cytokine receptors critical for the development and function of T and natural...X-linked severe combined immunodeficiency (SCID-X1) is a severe primary immunodeficiency caused by mutations in the gene, a shared subunit of cytokine receptors critical for the development and function of T and natural killer (NK) cells. The standard treatment, allogeneic hematopoietic stem cell transplantation (HSCT), requires a compatible donor and is often associated with significant transplant-related complications. We aimed to develop a more robust and universal gene therapy by modification of patient hematopoietic stem and progenitor cells (HSPCs) using the CRISPR-Cas9/rAAV6 gene-editing platform. To evaluate efficiency, we utilized a feeder-free, platform enabling T and NK cell differentiation from modified HSPCs. We investigated two approaches: the cut-site method (inserting a corrective cassette downstream of the start codon within the locus) and a replacement method (replacing the entire gene under endogenous regulation). We demonstrated that the cut-site insertion strategy is preferable for SCID-X1 correction, achieving superior homology-directed repair rates, lower toxicity, and significantly improved T and NK cell differentiation, based on phenotypic marker expression. Importantly, corrected patient-derived HSPCs from two SCID-X1 patients, modified using the cut-site approach, successfully demonstrated phenotypic evidence of T cell differentiation. Therefore, our findings firmly establish the feasibility of the cut-site strategy as a universal, high-efficiency, therapeutic solution for SCID-X1.
Labonia MCI, Martinez de Castilla PE, van der Kraak PH
… +8 more, Brans MAD, Yang Q, Lei Z, de Jager SCA, de Voogt WS, Schiffelers RM, Sluijter JPG, Vader P
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42181694
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The efficiency and safety of modified messenger RNA (modRNA) delivery into the heart using lipid nanoparticles (LNPs) remain undetermined. We previously demonstrated that modRNA encapsulated in C12-200 LNPs outperforms c...The efficiency and safety of modified messenger RNA (modRNA) delivery into the heart using lipid nanoparticles (LNPs) remain undetermined. We previously demonstrated that modRNA encapsulated in C12-200 LNPs outperforms current state-of-the-art intramyocardial modRNA delivery methods. Surprisingly, C12-200 LNPs triggered robust local immune cell activation 5 days post-injection, which was not evident on day 1. To investigate whether this immune response is driven by LNP composition or modRNA transfection efficiency, we systematically compared cardiac transfection efficiency, off-target biodistribution, and immunogenicity of modRNA formulated with clinically validated LNPs from Onpattro, BNT162b2/Comirnaty, and mRNA-1273/Spikevax. All tested formulations outperformed C12-200 in cardiac delivery, with mRNA-1273 and Onpattro showing markedly reduced off-target accumulation in the liver and spleen. Histopathological analysis revealed formulation-dependent immune cell infiltration, most pronounced with C12-200. C12-200 significantly elevated cytokine levels in both serum and heart tissue, whereas Onpattro increased cytokine levels mainly locally. In contrast, cytokine levels in animals treated with BNT162b2 and mRNA-1273 were comparable to those in PBS-treated controls. Importantly, cardiac transfection efficiency did not correlate with cytokine induction or histopathological changes, indicating that LNP-driven immunogenicity is independent of transfection efficacy. These findings provide a foundation for refining LNP formulations to optimize cardiac modRNA delivery while minimizing immune-related adverse effects.
Keshta A, Hashimoto R, Kitai Y
… +5 more, Nakata Y, Sakamoto A, Gotoh S, Takeda M, Takayama K
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42112103
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Respiratory syncytial virus (RSV) is one of the major causes of lower respiratory tract infections, particularly in infants and older adults. However, the host factors mediating infection remain poorly defined. It has be...Respiratory syncytial virus (RSV) is one of the major causes of lower respiratory tract infections, particularly in infants and older adults. However, the host factors mediating infection remain poorly defined. It has been suggested that four host surface proteins, namely intercellular adhesion molecule-1 (ICAM-1), epidermal growth factor receptor (EGFR), nucleolin (NCL), and insulin-like growth factor 1 receptor (IGF1R), may interact with the RSV fusion (F) protein. To investigate these roles under physiologically relevant conditions, we employed human induced pluripotent stem cell (iPSC)-derived respiratory organoids as a model for RSV infection. In this model, ICAM-1 and EGFR were genetically depleted using the CRISPR-Cas9 genome editing technique, while NCL and IGF1R were inhibited with neutralizing antibodies. Suppression of ICAM-1 or NCL significantly reduced nucleoprotein gene expression, whereas inhibition of EGFR or IGF1R had no observable effect on viral gene expression. Notably, simultaneous suppression of ICAM-1 and NCL resulted in a more substantial reduction in infectious viral titers and RSV F protein expression than inhibition of either protein alone. Our results suggest that both ICAM-1 and NCL may play important roles during RSV infection in human iPSC-derived respiratory organoids.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42112102
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N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) therapeutics have emerged as a groundbreaking modality with unparalleled efficacy for battling previously "undruggable" diseases. The unique pharmacok...N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) therapeutics have emerged as a groundbreaking modality with unparalleled efficacy for battling previously "undruggable" diseases. The unique pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of GalNAc-siRNA therapeutics provide an opportunity to leverage PK/PD modeling strategies for drug development. By utilizing the wealth of literature data, we developed and validated a mechanistic computational model-driven platform to guide the development of new GalNAc-siRNA therapeutics, optimizing their clinical translation. This platform integrates preclinical and clinical data from all seven FDA-approved GalNAc-siRNA drugs-fitusiran, givosiran, inclisiran, lumasiran, vutrisiran, nedosiran, and plozasiran-spanning multiple species (mouse, rat, monkey, and human). To enhance user accessibility, we further implemented a web-based Shiny application. The platform was used to inform the development of an investigational new angiotensinogen-silencing GalNAc-siRNA (SAL0132). Multiple PK/PD datasets from rats and monkeys were satisfactorily fitted, and extrapolated to humans. The platform successfully predicted the PK and simulated the PD profiles of SAL0132 in humans, which demonstrated model-informed strategies to support efficient drug development of this modality. In conclusion, this platform enables users to predict GalNAc-siRNA PK/PD profiles across species by inputting specific model parameters, providing a powerful resource to guide the development of next-generation GalNAc-siRNA therapeutics.
Chen S, Liu H, Cong D
… +11 more, Dong A, Wang Y, Bi J, Guo S, Yang J, Wang X, Ren G, Zhang K, Wang H, Lai F, Dang Y
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42112101
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Gapmer antisense oligonucleotides (ASOs) enable sequence-specific degradation of target mRNAs, offering therapeutic access to previously "undruggable" genes and holding great promise for treating chronic and genetic dise...Gapmer antisense oligonucleotides (ASOs) enable sequence-specific degradation of target mRNAs, offering therapeutic access to previously "undruggable" genes and holding great promise for treating chronic and genetic diseases. However, the rapid development of ASO therapeutics remains limited by challenges in rational sequence design and translational validation. Here, we present ClinASO, an integrated computational-experimental platform that unifies key determinants of ASO efficacy-including RNase H1 cleavage preference, SNP avoidance, off-target filtering, and cross-species conservation-into a single, data-driven workflow. This system enables rapid identification of potent ASO leads and direct validation in wild-type animal disease models. Using ClinASO, we efficiently identified potent ASOs against and , both exhibiting superior silencing activity compared to their clinical counterparts. Furthermore, ClinASO generated a potent ASO targeting genes implicated in metabolic dysfunction-associated liver disease (MASLD). In multiple human cells, the ASO achieved robust silencing effect. Notably, by conjugating GalNAc, this ASO demonstrated durable, liver-specific knockdown, significantly ameliorating hepatic steatosis and normalizing systemic lipid profiles in MASLD mouse model. Together, these findings establish ClinASO as an efficient, experimentally validated online tool for the rational design and rapid development of translatable ASO therapeutics.
Shcherbinina E, Fong M, Biscans A
… +1 more, Sarshad AA
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42112100
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RNA molecules are dynamic regulators of gene expression, with duplex RNAs such as siRNAs and saRNAs functioning as versatile effectors that can both silence and activate gene expression through Argonaute (AGO) proteins....RNA molecules are dynamic regulators of gene expression, with duplex RNAs such as siRNAs and saRNAs functioning as versatile effectors that can both silence and activate gene expression through Argonaute (AGO) proteins. While cytoplasmic RNA interference (RNAi) is well established, the mechanisms and outcomes of nuclear RNAi and RNA activation (RNAa) are only emerging. This review integrates recent discoveries on nuclear RNAi and RNAa, outlining how siRNAs can target nuclear long noncoding RNAs, promoter-associated transcripts, and pre-mRNAs to mediate silencing, transcriptional repression, or alternative splicing. Conversely, saRNAs can recruit AGO2 and transcriptional cofactors to activate gene expression through chromatin remodeling and RNA polymerase II engagement. Beyond their mechanistic roles, we highlight the growing therapeutic potential of duplex RNAs, which can be harnessed to selectively silence or activate gene expression, offering new strategies for RNA-based precision medicine.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42100560
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Long interspersed nuclear element-1 (LINE-1), a prominent class of retrotransposons, is abundantly distributed throughout mammalian genomes and plays critical roles in development, aging, and disease. In this review, we...Long interspersed nuclear element-1 (LINE-1), a prominent class of retrotransposons, is abundantly distributed throughout mammalian genomes and plays critical roles in development, aging, and disease. In this review, we provide a comprehensive overview of the multifaceted functions of LINE-1, with a focus on its regulatory impact on gene transcription and cellular responses across the DNA, RNA, and protein levels. We highlight recent advances in understanding the transcriptional and epigenetic mechanisms controlling LINE-1 transcription. Furthermore, we explore the current challenges and future opportunities in LINE-1 research, emphasizing its potential as a novel therapeutic target. Continued investigation into LINE-1 biology holds promise for deepening our understanding of human health and disease.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42100557
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Autologous cell therapies for knee osteoarthritis, including bone marrow aspirate concentrate (BMAC) and stromal vascular fraction (SVF), have advanced clinically despite an incomplete understanding of their mechanisms....Autologous cell therapies for knee osteoarthritis, including bone marrow aspirate concentrate (BMAC) and stromal vascular fraction (SVF), have advanced clinically despite an incomplete understanding of their mechanisms. Chatterjee et al. apply single-cell transcriptomics to multicenter trial of stem cell therapy for osteoarthritis (MILES) trial samples, revealing that site-to-site technical variability in BMAC composition overwhelms biological signals associated with treatment response. This finding mandates urgent standardization of harvest protocols before personalized genomic profiling can be meaningfully implemented. While responder/non-responder differences were subtle, pathway enrichment analyses and cell-cell communication inferences suggest that therapeutic outcomes may depend more on dynamic post-injection interactions than on baseline cellular states. The study establishes a foundational framework for evidence-based quality control in cellular orthopedics.
Man Z, Asano S, Kakutani T
… +3 more, Kiyoshi A, Hino K, Ikeda A
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42095135
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Werner syndrome (WS) is a rare autosomal recessive progeroid disorder caused by biallelic mutations in and is frequently complicated by refractory skin ulcers for which no effective therapy exists. We developed WRN-108,...Werner syndrome (WS) is a rare autosomal recessive progeroid disorder caused by biallelic mutations in and is frequently complicated by refractory skin ulcers for which no effective therapy exists. We developed WRN-108, a splice-switching antisense oligonucleotide (ASO) designed to induce exon 27 skipping and restore the open reading frame (ORF) disrupted by the most common mutation in Japanese WS patients, c.3139-1G>C, which leads to exon 26 skipping. In WS patient-derived fibroblasts, WRN-108 efficiently induced exon 27 skipping, restored WRN protein expression, and re-established its nuclear localization. Treatment improved cell proliferation and reduced senescence-associated markers, G-quadruplex accumulation, and γH2AX signaling, consistent with partial restoration of WRN-dependent genome maintenance functions. Topical administration in a rat skin wound model resulted in effective dermal penetration and sustained tissue retention. In a cynomolgus monkey wound model, WRN-108 induced exon 27 skipping in the skin and achieved dermal exposure without local toxicity. Short-term toxicity studies in mice and miniature pigs further confirmed its favorable tolerability. These findings provide preclinical evidence that ASO-mediated exon skipping can restore WRN function, highlighting the translational potential of WRN-108 as a therapeutic approach for refractory skin ulcers in WS patients harboring the c.3139-1G>C mutation.
Picht S, Farrera-Sal M, Hiller AL
… +14 more, Brumhard S, Klaas AM, Schulenberg S, Friedrich R, Scholz C, Hemmerling L, Simon DN, Krönke G, Pichon C, Sander LE, Volk HD, Gossen M, Schmueck-Henneresse M, Drzeniek NM
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42095134
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Chimeric antigen receptor (CAR) T cells offer a promising strategy for the treatment of autoimmune diseases. However, clinical translation is limited by the high cost, complexity, and poor scalability of current manufact...Chimeric antigen receptor (CAR) T cells offer a promising strategy for the treatment of autoimmune diseases. However, clinical translation is limited by the high cost, complexity, and poor scalability of current manufacturing, restricting broad patient access and persisting safety concerns including insertional mutagenesis risk from integrating vectors and uncontrolled long-term CAR T cell persistence. -transcribed (IVT) mRNA enables transient, non-integrating CAR expression with improved safety and scalability, making it particularly suited for non-malignant indications where prolonged persistence may not be required. Here, we systematically compare two IVT mRNA delivery platforms, electroporation and lipid nanoparticles (LNPs), for transient CAR T cell engineering in primary human T cells using single-cell transcriptomics and functional cell assays. We show that electroporation yields higher transfection efficiency and more sustained CAR surface expression, whereas LNP delivery reduces stress- and senescence-related transcriptional signatures as well as exhaustion marker expression, while enhancing antigen-driven activation, chemotactic responses, and cytotoxic function. Our comparative analysis highlights that the mode of mRNA delivery is associated with distinct transcriptional signatures and functional properties of CAR T cells, providing a framework to guide future development of mRNA-based approaches. These insights support LNP-mediated delivery as a functionally favorable strategy for transient CAR T cell engineering in autoimmune disease and beyond.
Elliot SJ, Grimaldo S, Catanuto P
… +7 more, Pereira-Simon S, Xia X, Civettini G, Shahzeidi S, Pastar I, Tomic-Canic M, Glassberg MK
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42088451
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Mesenchymal stem cells (MSCs) deliver their effects via paracrine signaling, including the release of extracellular vesicles (EVs), which transfer microRNAs (miRNAs) and mRNAs to recipient cells. Aging alters exosome com...Mesenchymal stem cells (MSCs) deliver their effects via paracrine signaling, including the release of extracellular vesicles (EVs), which transfer microRNAs (miRNAs) and mRNAs to recipient cells. Aging alters exosome composition and function, raising questions about donor age, the use of autologous vs. allogeneic donors, and exosome dose and dosing frequency for exosome-based therapies in fibrotic lung disease. To address these gaps, we investigated how exosomes from younger (28-39 years, adult-exo) and older (58-66 years) donors influence fibrosis and tissue repair. Exosomes were delivered intravenously to 18-months-old male C57BL/6 mice on day 12 post-bleomycin (BLM), with lung injury evaluated on day 21 by histologic, molecular, mitochondrial, and telomere analyses. A human skin injury model was used to quantitate exosome-mediated wound healing. Adult-exo reduced lung injury and accelerated skin wound closure. RNA sequencing (RNA-seq) revealed that adult-exo carried elevated antifibrotic miRNAs, such as let-7. Pathway enrichment linked this cargo to extracellular matrix (ECM) remodeling, mitochondrial function, and senescence. Adult-exo treatment also downregulated fibrosis- and senescence-associated miRNAs, such as miR-34, in lung tissue. experiments confirmed alterations in multiple downstream pathways, mediating reparative effects. These findings underscore age-dependent differences in stem cell-derived exosomes and demonstrate that synergistic miRNA cargo drives maximal efficacy.
Yu Z, Hussein M, Bao Y
… +8 more, Alcalá-Lalinde A, Kroon PZ, Thuillier E, Zhang Z, Enjuanes L, Zuñiga S, Berkhout B, Herrera-Carrillo E
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42065115
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RNA viruses evolve rapidly, enabling them to evade host immunity and antiviral therapies and complicating durable diagnostics strategies. There is a pressing need for approaches that provide broad-spectrum viral suppress...RNA viruses evolve rapidly, enabling them to evade host immunity and antiviral therapies and complicating durable diagnostics strategies. There is a pressing need for approaches that provide broad-spectrum viral suppression and detection. We designed four Cas13d crRNAs targeting a conserved 26-nucleotide sequence in coronavirus (CoV) nsp12. Their antiviral efficacy was evaluated against the target sequences of all seven human CoVs, showing potent activity across the tested samples. The same crRNAs were adapted for a Cas13d-based specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay to detect multiple human CoVs, demonstrating high sensitivity, with the ability to detect as few as a single copy of SARS-CoV-2 RNA, while showing no detectable signal for other seasonal respiratory viruses, such as influenza. This dual-function approach underlines the versatility and potential of CRISPR technologies in both managing and detecting viral infections. Additionally, bioinformatic analysis revealed that the crRNA targets are highly conserved across animal coronaviruses, suggesting that targeting of this sequence could facilitate the rapid development of treatment options and diagnostics during a new pandemic of an emerging coronavirus. This could significantly aid in pandemic preparedness and response efforts.
Eaton EJ, Wick BJ, Chacón JS
… +9 more, Wang AJ, Kluesner M, Barnes JT, Kar B, Wang M, Johnson MJ, Skeate JG, Webber BR, Moriarity BS
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42028578
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Adenine base editors (ABEs) are a promising yet underutilized tool for inducing protein knockout compared to Cas9 nuclease, owing in part to a lack of user-friendly platforms for reagent design and implementation. Here,...Adenine base editors (ABEs) are a promising yet underutilized tool for inducing protein knockout compared to Cas9 nuclease, owing in part to a lack of user-friendly platforms for reagent design and implementation. Here, we present a comprehensive workflow to achieve high-efficiency gene knockouts with ABE as an alternative to Cas9 nuclease-based approaches. This includes optimized guide RNA (gRNA) design using SpliceR, a web-based application, followed by genomic and functional validation of ABE-mediated knockouts for several target genes. We validated gRNAs for 14 immunologically relevant targets, using both NGG and NG PAM compatible ABE8e-variants in primary immune cells. To facilitate data analysis, we developed MultiEditRbatch, an updated version of MultiEditR as a user-friendly analysis tool with addition of batch mode for high-throughput analysis of Sanger sequencing data. MultiEditRbatch is available as a web-based application and an R package, enabling robust assessment of base editing outcomes.
Gibor G, Tzvi N, Meir A
… +10 more, Abu-Hariri H, Shemer A, Kilim S, Abelian S, Harush O, Itzhaki O, Shapira-Frommer R, Jacoby E, Cafri G, Wolf Y
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42028577
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Current mRNA approaches in immuno-oncology lack specificity for optimal T cell mRNA expression, necessitating tailored mRNA expression systems. In this study, we developed novel mRNA constructs in which the standard α-gl...Current mRNA approaches in immuno-oncology lack specificity for optimal T cell mRNA expression, necessitating tailored mRNA expression systems. In this study, we developed novel mRNA constructs in which the standard α-globin (HBA1) 5' UTR is replaced with sequences derived from genes highly expressed in effector T cells. Using primary human T cells, expression levels of UTR-modified reporter genes were evaluated, revealing significant variability based on the substituted UTR. For instance, interferon gamma (IFN-γ) UTRs facilitated enhanced and sustained protein expression, whereas TNF UTRs showed diminished expression. Unexpectedly, the -predicted RNA stability of the various UTR-modified constructs did not correlate with the altered expression. These UTR-mediated differences in protein expression were unique to T cells, as HEK cells introduced with the same constructs showed distinct expression profiles. CD19-CAR constructs expressed in T cells using various 5' UTRs demonstrated different protein expression and function toward antigen-positive target cells, as well as tonic signaling, manifested by the immune output in the absence of antigen. Specifically, for CD19-CAR, using the TIGIT 5' UTR proved optimal for achieving maximal reactivity while minimizing tonic signaling. These findings provide proof of concept for the pivotal role of T cell-specific UTRs in optimizing CAR-T cell functionality by fine-tuning expression, reducing tonic signaling, and minimizing off-target effects, thus emphasizing their potential in advancing the therapeutic potential of mRNA-based CAR-T cell therapies.
Totsune E, Wada Y, Mikami-Saito Y
… +8 more, Arai-Ichinoi N, Saijo N, Takayama J, Maeda Y, Nakajima Y, Ohara O, Kure S, Kikuchi A
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42028575
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Propionic acidemia is a rare autosomal recessive disorder caused by mutations in the or gene, resulting in deficient propionyl-CoA carboxylase activity. We identified a unique homozygous deep-intronic variant, NM_0002...Propionic acidemia is a rare autosomal recessive disorder caused by mutations in the or gene, resulting in deficient propionyl-CoA carboxylase activity. We identified a unique homozygous deep-intronic variant, NM_000282.4:c.1285-1358C>G, in an individual with neonate-onset propionic acidemia. Fibroblasts from this individual expressed only mRNA containing an 84-bp pseudoexon, which is present at low levels in healthy controls, leading to the loss of PCCA and PCCB proteins and severely reduced propionyl-CoA carboxylase activity. Transfection of fibroblasts with chemically synthesized antisense oligonucleotides (ASOs) designed to skip the pseudoexon restored productive splicing, rescued PCCA protein expression, and markedly increased propionyl-CoA carboxylase activity above wild-type levels. The efficacy of the ASOs was further evaluated in fibroblasts from 7 additional individuals with propionic acidemia carrying mutations in or . ASO treatment successfully restored enzymatic activity, particularly in fibroblast lines, with residual activity exceeding 1% of normal. These findings suggest that ASO-mediated splicing correction targeting the 84-bp pseudoexon can restore mRNA, protein, and enzymatic function in individuals with deep intronic mutations, as well as in other individuals with propionic acidemia, indicating the feasibility of ASO therapy as a molecular treatment strategy for a subset of individuals with propionic acidemia.