Kenneweg F, Hempel K, Höhne LPJ
… +15 more, Dräger G, Blume J, Viereck T, Stohwasser A, Groß S, Büchler G, Jansen K, Juchem M, Bär C, Pfanne A, Just A, Thum S, Gietz A, Kirschning A, Thum T
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41971067
·
Full text
Targeted RNA delivery with precise spatial and temporal control marks a significant advancement in therapeutic development, offering the potential to reduce drug dosages while minimizing off-target effects. In this study...Targeted RNA delivery with precise spatial and temporal control marks a significant advancement in therapeutic development, offering the potential to reduce drug dosages while minimizing off-target effects. In this study, we present a novel platform that employs superparamagnetic iron oxide nanoparticles (SPIONs) for externally controlled, thermally triggered, organ-specific locked nucleic acid (LNA) release. Our platform technology leverages a newly designed thermosensitive conjugate, based on a thermosensitive linker system that utilizes the thermal sensitivity of the -butyloxycarbonyl (Boc) group. This tool ensures stability during systemic circulation while enabling traceless, on-demand drug release at the target site. As a proof of concept, we applied this technology in a disease model of cardiac fibrosis, conjugating SPIONs with an inhibitor of microRNA (miRNA)-21, a key pro-fibrotic regulator. The nanoparticle system was thoroughly characterized for its stability, biocompatibility, and heat-induced release properties and subsequently validated for biodistribution, toxicology, and therapeutic potential in preclinical models. This innovative SPION-based delivery platform provides a versatile and precise framework for RNA-based therapeutics, with broad translational potential across various disease applications.
Sokulska M, Nalewaj M, Czapik T
… +2 more, Rachwalak M, Szabat M
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41971066
·
Full text
G-quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine-rich DNA or RNA sequences. The G4s are formed by planar G-tetrads and stabilized by monovalent metal cations such as potassium or sodium. T...G-quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine-rich DNA or RNA sequences. The G4s are formed by planar G-tetrads and stabilized by monovalent metal cations such as potassium or sodium. The structural diversity of G4s arises from differences in strand orientation, loop arrangement, and molecularity, leading to multiple topologies. The length of guanine tracts (G-tracts) and the number of strands involved further influence G4 folding, stability, and biological function. G-quadruplexes are commonly found in key genomic regions such as telomeres, promoters, and untranslated regions, where they play important roles in regulating fundamental biological processes, including replication, transcription, and translation. In this review, we summarize the molecular factors influencing G4 formation and stability. We also discuss recent advances in therapeutic strategies for targeting G-quadruplexes. Particular attention is given to antisense oligonucleotides (ASOs), ASO-ligand conjugates, peptide nucleic acids (PNAs), and PNA-conjugates as chemical tools for selective G4 recognition and modulation. We highlight the potential of nucleic-acid-based approaches for molecular therapeutics, including applications in anticancer and antiviral treatments.
Stavrou M, Wallace LM, Thangaraj MP
… +15 more, Taylor NK, Kagiava A, Papacharalambous R, McAllister C, Zender G, Saad NY, Bayazit MB, Heslegrave A, Tryfonos C, Richter J, Zetterberg H, Price B, Salzman R, Kleopa KA, Harper SQ
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41948127
·
Full text
Charcot-Marie-Tooth (CMT) type 1A, the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication, leading to overproduction of PMP22 protein in Schwann cells. To treat CMT1A, we devel...Charcot-Marie-Tooth (CMT) type 1A, the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication, leading to overproduction of PMP22 protein in Schwann cells. To treat CMT1A, we developed a PMP22 gene silencing therapy using adeno-associated viral vectors (AAV9) to deliver a therapeutic miRNA expression cassette (U6.miR871) via lumbar intrathecal administration. A single injection produced long-term miR871 expression, triggered selective RNA interference against the PMP22 mRNA, and subsequently lowered protein levels and improved disease manifestations in a humanized CMT1A model. To support clinical translation, we confirmed on-target specificity of miR871 for PMP22 , identified a safe and effective dosing range in mice, demonstrated absence of significant toxicity in rodents and non-human primates (NHPs), and performed a detailed AAV biodistribution study in a large animal model. We found vector biodistribution and miR871 expression in distal peripheral nerves, PMP22 target engagement in mice and NHPs, and silencing to levels expected to support normal myelination in humans. We identified the minimally efficacious to maximum tolerated dose range of AAV9.U6.miR871 in mice and confirmed safety range in NHPs for extrapolation to anticipated clinical trials. Our study supports the scale-up potential of gene therapy to treat CMT1A and other demyelinating peripheral neuropathies.
Anwar I, Wang X, Pratt RE
… +2 more, Dzau VJ, Hodgkinson CP
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41940076
·
Full text
Fibroblast-to-cardiomyocyte reprogramming is influenced by signals present in the injured heart. However, the nature of those signals is unclear. The efficacy of fibroblast-to-cardiomyocyte reprogramming was found to be...Fibroblast-to-cardiomyocyte reprogramming is influenced by signals present in the injured heart. However, the nature of those signals is unclear. The efficacy of fibroblast-to-cardiomyocyte reprogramming was found to be negatively impacted by extracts derived from the freeze-thawing of normal cardiac tissue. Mass spectrometry identified the adult minor beta chain of hemoglobin (Hbb-b2) as the most abundant protein in the extract, prompting investigation of hemoglobin (Hb) as a candidate inhibitory factor. Indeed, Hb was found to suppress fibroblast-to-cardiomyocyte reprogramming by inhibiting the expression of various cardiomyocyte-specific markers and cardiomyocyte formation. Hb is known to interact with TLR2 and TLR4. While inhibition of either receptor was unable to reverse the effects of Hb, inhibition of both TLR2 and TLR4 reversed the suppressive effects of Hb. To clarify the mechanism, assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed. ATAC-seq revealed that Hb induced widespread transcription factor network remodeling, whereby stress-associated factor networks were induced and latent CEBP/PAR-bZIP networks were suppressed. These effects were localized to a region 500-750 bp upstream of the transcription start site in cardiomyocyte-specific genes. These findings identify Hb as a potential negative regulator of fibroblast-to-cardiomyocyte reprogramming, with implications for cardiac regeneration strategies.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41940074
·
Full text
Nucleic acid-based therapeutics, which involve the manipulation of genetic materials to treat or prevent diseases, have gained considerable attention, leading to the approval of medicines such as COVID-19 vaccines, patis...Nucleic acid-based therapeutics, which involve the manipulation of genetic materials to treat or prevent diseases, have gained considerable attention, leading to the approval of medicines such as COVID-19 vaccines, patisiran (Onpattro), and nusinersen (Spinraza). However, their clinical application is hindered by challenges such as nuclease degradation, poor biodistribution, limited cellular uptake, and inefficient endosomal escape. Extracellular vesicles (EVs), which are natural nanoscale drug delivery systems derived from various eukaryotic and prokaryotic cells, offer a safe, efficient, specifically targeted, and non-pathogenic method for nucleic acid delivery. In this review, we summarize the classical methods and the latest research advances in EV preparation and nucleic acid loading. Additionally, we review the primary administration routes for nucleic acid-loaded EVs, such as intravenous, local, oral, intranasal, and inhalation delivery. By addressing these aspects, this review aims to guide the optimal design and clinical application of nucleic acid-loaded EVs.
Llinares C, Namkung S, Yip M
… +8 more, Vales Á, Olagüe C, Mauleón I, Isola S, Moeini P, González-Aseguinolaza G, Tai PWL, Unzu C
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41940073
·
Full text
Recombinant adeno-associated virus (rAAVs) vectors are the flagship vehicles for delivering DNA payloads for human gene therapy. However, only a few outstanding therapies have reached the market in the past decade. One r...Recombinant adeno-associated virus (rAAVs) vectors are the flagship vehicles for delivering DNA payloads for human gene therapy. However, only a few outstanding therapies have reached the market in the past decade. One reason for the slow development of rAAV-based gene therapies has been our limited knowledge of basic AAV biology. Therefore, the goal of this work was to investigate the influence of rAAV inverted terminal repeat (ITR) design, promoter use, and serotype selection on vector genome heterogeneity and transduction efficiency. Our analyses revealed additional, larger and smaller, vector genome species that were identified as unresolved and bound by the ITRs. Alkaline gel analysis and long-read single-molecule sequencing confirmed the presence of double-stranded genomes with self-complementary (sc) configurations for most of the serotypes and ITR designs tested. sc genome formation and ITR repair were favored under specific capsid-genome feature combinations, resulting in genomes larger than 5 kb in some cases. Furthermore, we found that vector genome length had an impact on ITR repair. Finally, transduction studies confirmed that the AAV capsid plays a role in functional ITR repair and potency. In conclusion, sc genome packaging is favored under specific ITR designs, and their stability is influenced by the AAV capsid.
Jamil M, Zahra A, Maqbool R
… +2 more, Arshad MI, Khan SH
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41940072
·
Full text
Prime editing is a precise "search and replace" genome editing tool that enables programmed genome modifications without the need for double-stranded breaks or additional repair templates. However, the retention of the u...Prime editing is a precise "search and replace" genome editing tool that enables programmed genome modifications without the need for double-stranded breaks or additional repair templates. However, the retention of the unedited 5' strand hinders the incorporation of stable edits into the genome, and the rate of indel errors is also high. Recently, et al. reported an engineered prime editor (PE), i.e., a very precise PE (vPE), that introduces nick-relaxing mutations, which can favor stable genome edits by destabilizing the 5' end and degrading the nicked end, thereby minimizing indel errors. This vPE holds promising potential as a transformative force in advancing human disease therapeutics and enabling innovative breakthroughs in crop improvement.
Puzzo F, Bayram B, Macaubas C
… +5 more, Lin A, Jang H, Zhang F, Mellins E, Kay MA
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41909468
·
Full text
Recombinant adeno-associated virus (rAAV) vectors and CRISPR-Cas9 are widely used in gene therapy. However, how endogenous DNA secondary structures may potentially affect genome editing outcomes is not fully understood....Recombinant adeno-associated virus (rAAV) vectors and CRISPR-Cas9 are widely used in gene therapy. However, how endogenous DNA secondary structures may potentially affect genome editing outcomes is not fully understood. RNA/DNA hybrids (R-loops), which form mostly during transcription, are dynamically regulated in cells and have been implicated in influencing DNA repair pathway choice. Here, we investigated whether genomic R-loops are associated with differences in Cas9-mediated genome editing outcomes and . By targeting regions with relatively low or high R-loop levels within the murine albumin () and actin () loci, we observed comparable insertion/deletion (indel) frequencies across sites with different R-loop abundance. In contrast, homology-directed repair (HDR) efficiency appeared reduced at R-loop-enriched sites in proliferating hepatocyte-derived cells (HEPA1-6) but not in quiescent hepatocytes . Manipulations resulting in reducing R-loop levels, including RNaseH1 overexpression or pharmacological induction of G1 arrest were associated with increased HDR at these genomic sites. In addition, T cell activation correlated with elevated R-loop accumulation suggesting they might influence genome editing. Together, these observations suggest that endogenous R-loop levels might influence HDR efficiency during Cas9-mediated editing and is a parameter to consider when designing genome editing strategies.
Nagamatsu Y, Umezu T, Hong T
… +6 more, Niijima T, Ohno SI, Harada Y, Kanekura K, Ochiya T, Kuroda M
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41909467
·
Full text
An aberrant six-base repeat in intron 1 of is the most frequent cause of solitary and familial amyotrophic lateral sclerosis and frontotemporal dementia. This mutation is a potential target for CRISPR/Cas9-based genome...An aberrant six-base repeat in intron 1 of is the most frequent cause of solitary and familial amyotrophic lateral sclerosis and frontotemporal dementia. This mutation is a potential target for CRISPR/Cas9-based genome editing. However, the blood-brain barrier and limitations of current viral or nanoparticle-based delivery systems to neurons significantly restrict the clinical application of CRISPR-Cas9 in the brain. To address these challenges, we developed a drug delivery system using acerola-derived exosome-like nanoparticles (AELNs), which may overcome several limitations associated with human exosomes. AELNs stably form complexes with ribonucleoproteins (RNPs) comprised of Cas9 proteins and guide RNAs (gRNAs). We improved the delivery efficiency and selectivity of AELN/RNP complexes in GLP2-receptor-expressing neurons by incorporating GLP2 peptides into the AELN/RNP complexes. Intranasal administration of peptide-tagged AELN/RNP complexes confirmed the successful genome editing of , demonstrating the potential of this system for treating neurodegenerative diseases. This study presents a potentially innovative approach for genome editing using a noninvasive delivery system.
Tang Y, Li P, Li M
… +9 more, Zhang P, Lu Z, Qiao H, Wei J, Xu Z, Wan X, Xu S, Chen YH, Zhang G
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41909465
·
Full text
Inflammatory bowel disease (IBD) presents significant therapeutic challenges. The immune regulatory protein TIPE2 is a risk factor for inflammatory diseases including IBD, but its potential as a therapeutic target remain...Inflammatory bowel disease (IBD) presents significant therapeutic challenges. The immune regulatory protein TIPE2 is a risk factor for inflammatory diseases including IBD, but its potential as a therapeutic target remains undefined. Here, we developed a replication-defective adenovirus encoding TIPE2-specific short hairpin RNA (shRNA) (AdV-shTIPE2) to knock down TIPE2 gene expression in mice and tested its therapeutic potential for treating dextran sulfate sodium (DSS)-induced colitis. Intravenous AdV-shTIPE2 administration conferred profound protection against colitis as judged by body weight loss, fecal bleeding, and colon length changes. Histological analyses also revealed significant preservation of epithelial integrity and diminished pathological damage in AdV-shTIPE2-treated mice. Mechanistically, TIPE2 knockdown limited infiltration of CD45 leukocytes, particularly CD11bLy6G myeloid cells, and reduced colonic inflammatory cytokine gene expression. Analysis of human IBD bulk and single-cell transcriptomic data confirmed TIPE2 upregulation and identified a distinct TIPE2-expressing monocyte subpopulation highly enriched in human intestinal lesions of IBD patients, demonstrating a conserved pathogenic TIPE2-myeloid axis across species. Together, these results validate TIPE2-driven inflammatory cell infiltration as a key driver of IBD and establish targeted TIPE2 silencing as a promising therapeutic strategy worthy of future clinical investigation.
Cheng J, Lin Z, Wu L
… +6 more, Li Q, Yin H, Wang H, Chen H, Chen X, Ji ZL
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41883585
·
Full text
Tissue-specific methylation sites (TSMs) are important epigenetic features associated with gene regulation, tissue development, and disease pathogenesis. However, the lack of comprehensive and reliable resources for TSMs...Tissue-specific methylation sites (TSMs) are important epigenetic features associated with gene regulation, tissue development, and disease pathogenesis. However, the lack of comprehensive and reliable resources for TSMs restricts advancements in epigenetic and translational research. We present TiSMeD (http://www.bio-add.org/TiSMeD/), a multi-omics database integrating 6,782 DNA methylation, 16,894 transcriptome, and 241 proteome profiles across 48 normal human tissues. Using a scoring framework based on SPM and Tscore, we identified 67,427 high-confidence TSMs, 4,607 tissue-specific genes, and 2,833 tissue-specific proteins, along with over 11 million housekeeping methylation sites. TiSMeD enables interactive exploration and data retrieval, supporting biomarker discovery and disease research. We demonstrate its utility in tracing the tissue-of-origin of cell-free DNA (cfDNA), prioritizing 1,849 cancer biomarkers from The Cancer Genome Atlas (TCGA), and constructing a multi-cancer tracing and diagnostic model achieving 95.7% accuracy. TiSMeD serves as a robust, user-friendly platform integrating multi-omics data to advance epigenetic research and biomarker translation.
Kobel L, Van de Venn L, Schröder M
… +7 more, Bechter LV, Huang D, Abdolazimi Y, Pertel T, Gopalakrishnan S, Corn JE, Kontarakis Z
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41883584
·
Full text
Programmable guided nucleases have revolutionized genome editing and biomedical research, with transformative potential for gene and cell therapy. Although the widespread adoption of the CRISPR-Cas system has provided de...Programmable guided nucleases have revolutionized genome editing and biomedical research, with transformative potential for gene and cell therapy. Although the widespread adoption of the CRISPR-Cas system has provided deep insights into target recognition and specificity, the behavior of clinically relevant tools like transcription activator-like effector nucleases (TALENs) remains poorly characterized in human cells. To address this gap, we implemented DisTAL-Seq, a TALEN-specific adaptation of the DISCOVER-Seq pipeline, which detects MRE11 recruitment to double-strand breaks (DSBs). Based on the DISCOVER-Seq principle, DisTAL-Seq incorporates alignment logic tailored to TALEN-binding properties, including variable RVD specificity, cleavage offset, and dimerization behavior. Using DisTAL-Seq, we identified and validated on- and off-target sites across diverse TALENs and T cell donors. This unbiased approach revealed key features of TALEN activity in human cells, including number of tolerated mismatches to a target site and relative location of the induced DSB. DisTAL-Seq thus extends DISCOVER-Seq to the TALEN family and provides a robust platform for assessing modifications in enzyme architecture and application contexts on a genome-wide scale, supporting the development of safer and more effective genome editing tools.
Seo HY, Jung H, Lee SY
… +9 more, Jung HG, Cho H, Son YM, Bak Y, Hwang SY, Kim JH, Park IH, Shin JS, Oh JW
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41883582
·
Full text
mRNA vaccines are a versatile platform for infectious disease prevention and therapeutic applications, yet their performance is limited by exclusive reliance on cap-dependent translation, which is markedly suppressed und...mRNA vaccines are a versatile platform for infectious disease prevention and therapeutic applications, yet their performance is limited by exclusive reliance on cap-dependent translation, which is markedly suppressed under hypoxia and cellular stress. Here, we report a hybrid 5' untranslated region (5'UTR) that enables dual translation initiation via both cap-dependent and internal ribosome entry site (IRES) mechanisms. This element integrates a minimal stem-loop 4.5-5 module (SL4.5-5) from the SARS-CoV-2 genomic 5'UTR, in which a conserved 5'-UUUCGU-3' motif within the SL5 loops is essential for function. Incorporating the SL4.5-5 module downstream of conventional 5'UTRs confers cap-independent translation capacity and enhances overall translation efficiency under translation-restrictive conditions such as hypoxia. When applied to the 5'UTRs of clinically validated COVID-19 vaccines, this module improves antigen expression in both modified and unmodified mRNAs. Notably, unmodified Omicron BA.5 and XBB.1.5 mRNA vaccines containing this element elicited potent humoral and cellular immune responses at sub-microgram doses, comparable to those induced by the approved N1-methylpseudouridine-incorporated mRNA vaccine, raxtozinameran. These findings identify SL4.5-5 as a modular IRES element that enables dual translation initiation, promoting efficient protein synthesis under cap-dependent translation-restrictive conditions and expanding the functional landscape of mRNA vaccines and therapeutics beyond cap-dependent limitations.
Cheng SY, Giguere D, Kim S
… +17 more, Seddon JM, Caiazzi J, Gross K, McHugh N, Echeverria D, Alterman JF, Gray-Edwards H, Benatti HR, Renner L, Woolard H, Stoddard J, McGill TJ, Neuringer M, Brush RS, Agbaga MP, Khvorova A, Punzo C
Mol Ther Nucleic Acids
· 2026 Jun · PMID 41858838
·
Full text
Age-related macular degeneration (AMD) remains a leading cause for visual impairment in the elderly. We recently showed that activated mammalian target of rapamycin complex 1 (mTORC1) in photoreceptor cells causes AMD-li...Age-related macular degeneration (AMD) remains a leading cause for visual impairment in the elderly. We recently showed that activated mammalian target of rapamycin complex 1 (mTORC1) in photoreceptor cells causes AMD-like pathologies in mouse. Employing mouse genetics, we dissect the mTORC1 pathway and identify ribosomal protein S6 kinase beta-1 () as a key component required for disease onset in our mouse model. Using a previously identified fully chemically modified tetravalent small interefing RNA (siRNA) that enriches in photoreceptors, we target in mouse, pigs, and non-human primates (NHP) by intravitreal injection. We find that silencing in diseased mice reverses phospholipid changes induced by activated mTORC1, restores lysosomal activity of retinal-pigmented epithelium cells, and reduces lipoprotein buildup at Bruch's membrane (BM). In pigs, which do not develop disease, we find a similar shift in phospholipids as in mouse, indicating a conserved role for in photoreceptor phospholipid metabolism. In aged NHPs with macular drusen, the lipoprotein-rich BM deposits that are a hallmark of human AMD, silencing slows drusen growth over a 6-month period. These findings establish as modifier of lipoprotein buildup at the BM and support our siRNA platform as a potential treatment modality for AMD and other retinal diseases.