Junctional epidermolysis bullosa (JEB) caused by COL17A1 pathogenic variants (JEB-C17) is characterized by skin fragility, chronic wounds, and an increased risk of squamous cell carcinoma. Approximately 20% of cases are caused by nonsense mutations, which induce premature termination codons (PTC) resulting in truncated protein production and/or trigger mRNA degradation through nonsense-mediated decay (NMD) pathway. Different approaches have been suggested for nonsense mutation suppression including translational readthrough inducing drugs (TRIDs) therapy, which is extensively examined in hereditary skin diseases due to relatively low costs for translation into the clinic. We evaluated a panel of TRIDs in patient-derived JEB-C17 keratinocytes carrying different COL17A1 nonsense variants. We observed variable responses to TRIDs across mutations. N-oxalylglycine (NOG) has been shown to reduce eRF1 hydroxylation and consequently inhibit NMD. We investigated NOG as an enhancer of aminoglycoside mediated readthrough for JEB-C17. We previously developed a cocktail consisting of aminoglycosides, NMD inhibitors as enhancers of read through activity and antioxidants that act as anti-inflammatory agents. In this study, we explored the effect of NOG as a readthrough enhancer in combination with TRIDs or TRID-cocktail. Our results support mutation-dependent TRID selection for further preclinical scientific investigations and highlight personalized therapeutic approaches as a key strategy for JEB-C17.
The regeneration of skeletal muscle involves precisely regulated intercellular communication, including extracellular vesicle (EV)-mediated processes. In this study, we show that during in vitro myogenic differentiation of human skeletal muscle progenitor cells (myoblasts), miR-193b-3p accumulates within small extracellular vesicles (sEVs) in a temporally regulated manner. Both sEV-mediated and synthetic delivery of miR-193b-3p enhanced myogenic differentiation in vitro and promoted histological recovery in a murine volumetric muscle loss model. Mechanistically, we demonstrate that miR-193b-3p directly suppresses adaptor-related protein complex 2 subunit mu 1 (AP2M1), a clathrin-associated adaptor protein that has not been well characterized in muscle biology, and identify it as a negative regulator of myogenic progression. AP2M1 knockdown phenocopied the effects of miR-193b-3p, suggesting that this regulatory axis may contribute to muscle regeneration. In addition, transcriptomic analyses of aged rodent and sarcopenic human datasets revealed conserved upregulation of AP2M1 in degenerative muscle states, supporting its potential clinical relevance. Collectively, these findings reveal a myogenic differentiation-dependent, sEV-mediated miRNA regulatory mechanism that contributes to muscle regeneration. Based on these results, we propose the miR-193b-3p–AP2M1 axis as a potential therapeutic target for muscle-wasting disorders.
A platform process underpins the manufacturing of RNA-based vaccines and therapeutics. However, it remains constrained by high operational expenditures from costly reagents, inefficient raw-material utilization, and extensive purification. We report an integrated sequential-batch in vitro transcription (IVT)-oligo-dT chromatography process that links RNA synthesis and purification via a shared buffer, enabling reagent recycling. Across five cycles, 5′ cap analog utilization improved 3.72× and calculated raw-material cost efficiency improved 2.21× for the NaCl workflow at the demonstrated 8 mL scale, while stable RNA production was maintained across recycling cycles. Product quality was assessed across seven critical quality attributes (CQAs), including RNA integrity, 5′ capping, poly(A)-tail heterogeneity, sequence identity, residual nucleotides, dsRNA content, and cell-based functional activity, measured by protein expression and cytokine responses. Purified recycling-process RNA contained ≈70-77% less double-stranded RNA (dsRNA) than purified standard-process RNA, purified RNA integrity exceeded 90%, and five-cycle mean 5′ capping exceeded 80% with NaCl; 3′ polyadenylate (poly(A)) tail length and heterogeneity remained stable across recycling cycles. THP-1 cell assays showed no progressive cycle-dependent loss of protein expression or increase in cytokine secretion across recycling cycles. This integrated framework advances cost-efficient, resource-efficient mRNA production while identifying candidate control points for extended reagent recycling.
CRISPR hybrid guides containing a combination of RNA and DNA nucleotides (CRISPR hybrid RNA DNA or chRDNA) enhance both Cas9 and Cas12a nuclease specificity and reduce off-target editing in vitro. CRISPR-Cas9 with all-RNA crRNA guides has been implemented for in vivo intervention of familial hypercholesterolemia, an inherited autosomal dominant disorder exhibiting high circulating low-density lipoprotein cholesterol, and transthyretin amyloidosis, a progressive life-threatening disease characterized by accumulation of misfolded transthyretin protein leading to neurodegeneration or cardiomyopathy. We targeted three clinically validated genes, Pcsk9, Angptl3, and Ttr, that address these indications using our Cas12a chRDNA genome-editing technology delivered in lipid nanoparticles. A single intravenous dose of LNP encapsulated Cas12a mRNA with a chRDNA guide led to high editing efficiencies without detectable off-target editing, corresponding with near complete reduction of all three targeted plasma proteins in wild-type mice. Additionally, Pcsk9 and Angptl3 knockouts were accompanied by reductions of plasma cholesterol. No liver toxicity, chronic cytokine release, off-target editing, or on-target editing in non-hepatic tissue was observed in rodents administered high doses. We propose that LNP-delivered Cas12a mRNA and chRDNA is a safe, highly specific, and powerful tool for genome editing in vivo.
Plasmodium vivax (Pv) remains the primary cause of malaria outside of Africa, yet no licensed vaccine is available against this pathogen. Here, we evaluate the immunogenicity and protective efficacy of novel vaccines targeting E140, a recently identified multi-stage antigen conserved across Plasmodium species. Lipid nanoparticle (LNP)-formulated nucleoside-modified mRNA vaccines against P. berghei E140 (PbE140) and P. vivax E140 (PvE140) induced robust antigen-specific IgG antibody responses, germinal center B cell and long-lived plasma cell responses. Vaccination with PbE140 mRNA-LNP resulted in reduced parasitemia and improved survival in pathogen-challenged mice. Importantly, antibodies elicited by PvE140 mRNA-LNP in mice reduced invasion of primary human reticulocytes by P. vivax merozoites by 56-78% in ex vivo functional assays. These findings suggest that E140 may be a promising antigen candidate for next-generation vaccines against P. vivax.
Human T cells modified with nucleic acids constitute a powerful and emerging therapeutic modality for cancer, autoimmune disorders, and aging related diseases. However, delivering nucleic acids, such as mRNA encoding synthetic receptors, transcription factors, cytokines, or genome editors to T cells can be challenging, as nucleic acids can unintentionally reduce viable T cell yield and function, particularly when delivered sequentially ex vivo. To address this challenge, we evaluated the efficiency of serial non-viral delivery of synthetic mRNA encapsulated within lipid nanoparticles (LNPs) incorporating cationic lipid, 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP), and fusogenic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). DOTAP/DOPE-containing LNPs delivered mRNA more efficiently than a clinically benchmarked 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-containing LNP formulation, resulting in significantly higher protein expression in primary human T cells. Furthermore, DOTAP/DOPE-LNP delivery resulted in 100% higher yield of live cells compared to electroporation, an advantage that compounded over serial rounds, with LNP-treated cultures maintaining substantially more viable cells through all four transfections. Additionally, serial transfection of mRNAs encoding a therapeutically relevant chimeric antigen receptor (CAR) payload produced functional CAR T cells. These results demonstrate that LNPs can be a viable platform for serial and iterative T cell engineering using multiple mRNA payloads, providing an alternative to electroporation for the engineering of therapeutic T cells.
Messenger RNA (mRNA) vaccines are a versatile platform for rapid vaccine development; however, nucleoside modifications must be optimized to balance translational efficiency and innate immune activation. Here, we evaluated N1-ethylpseudouridine (Et1Ψ) as an alternative to the widely used N1-methylpseudouridine (m1Ψ). Et1Ψ-modified mRNA exhibited a distinct codon-dependent translational sensitivity, with increased UUU codon content leading to reduced protein expression. Importantly, synonymous codon optimization that eliminated UUU codons restored translation efficiency to levels comparable to those of m1Ψ-modified mRNA across multiple cell types. Despite this constraint, Et1Ψ consistently reduced innate immune activation in vitro and in vivo. Following codon optimization, Et1Ψ-modified mRNA supported robust protein expression comparable to that from m1Ψ-modified mRNA. When applied to vaccine antigens, including SARS-CoV-2 spike and tetravalent dengue envelope domain III constructs, Et1Ψ-modified mRNA elicited strong humoral and cellular immune responses, with neutralizing activity comparable to that of m1Ψ-modified mRNA. Collectively, these findings identify Et1Ψ as an alternative nucleoside modification that combines reduced innate immunogenicity with codon-aware sequence optimization, highlighting codon–modification interactions as a key design principle for mRNA therapeutics.
The 7-methylguanosine (m7GpppN) cap at the 5′ end of mRNA is an important regulatory feature that contributes to transcript stability, translation initiation, and innate immune recognition. Synthetic biology harnesses these properties through engineered cap chemistries (e.g., anti-reverse cap analogs, enzymatic capping) and refined in vitro transcription to control therapeutic mRNA yield, durability, and immunogenicity. This review integrates current structural and mechanistic insights into m7G cap function with its application in vaccine design, emphasizing how cap chemistry affects eIF4E binding, decapping rates, and IFIT-mediated discrimination. We further discuss how cap chemistry interacts with other mRNA design features, including nucleoside modification, untranslated region architecture, poly(A) tail design, impurity control, and lipid nanoparticle formulation. As an illustrative example, we consider the potential relevance of cap-guided design principles to mRNA vaccine strategies against intracellular bacterial pathogens such as Brucella spp., where antigen expression and Th1-oriented cellular immunity are both important. Rather than presenting original experimental data, this review aims to synthesize current mechanistic and translational knowledge and to identify practical considerations for cap selection in next-generation mRNA vaccine design.
Phosphorodiamidate morpholino oligonucleotides (PMOs) are a class of antisense oligonucleotides. While PMOs have enabled several nucleic acid therapeutics, their structural and energetic solution properties remain poorly understood. Using solution viscosity measurements combined with computational molecular modeling, we explored interactions of therapeutic 22-mer, 25-mer, and 30-mer PMOs in concentrated solutions. Self-association of PMO monomers involves non-specific interactions, is energetically favorable (with estimates of -32 to -67 kcal/mol interaction energies), and exhibits biphasic kinetics involving a fast phase of hydrophobic anchoring followed by a slower phase associated with optimization of intermolecular base pairing and stacking interactions. The final complexes possess broad self-association interfaces of ∼700-1,600 Å2. Accurate interpretation of the viscosity vs. concentration data for concentrated PMO solutions must account for PMO dimer formation, as supported by the molecular dynamics simulations. Formation of higher-order PMO species was inferred from viscosity-concentration profiles based on models 1-4: for the 25-mer at concentrations above 160 mg/mL (dimerization) and 240 mg/mL (trimerization), and for the 30-mer above 190 mg/mL (dimerization) and 270 mg/mL (trimerization), respectively. The results provide atomic-level details on PMO structure, molecular properties, and interaction energies in concentrated environments, identifying weak preferential functional group interaction patterns that underlie thermodynamic stability.
Antisense oligonucleotide (ASO) therapeutics silence gene expression through RNase H-mediated mRNA degradation or steric blockade, yet their clinical efficacy is limited by inefficient intracellular trafficking, with <1% of internalized ASOs escaping endosomes to access target RNA. A deeper understanding of the cellular mechanisms governing ASO trafficking and activity is therefore critical. Here, we identify the endosomal sorting complex required for transport-I (ESCRT-I) as a previously unrecognized regulator of ASO pharmacological activity. Using human hepatocyte-derived cell models and FDA-approved ASO drugs inotersen and mipomersen, we demonstrate that suppression of specific ESCRT-I subunits, VPS23 and VPS28, markedly enhances ASO-mediated target silencing, whereas depletion of other subunits has minimal effect. Mechanistically, VPS23 functions as a central ESCRT-I component linking endosomal trafficking to intracellular glucose homeostasis. Loss of VPS23 reduces expression of the glucose transporter GLUT2, lowers intracellular glucose levels, and possibly enables ASO endosomal escape without altering cellular uptake or RNase H1-dependent activity. VPS23 suppression disrupts endosomal morphology, decreases ASO retention in late endosomes, and enhances cytosolic availability of ASOs. Notably, this regulatory effect extends beyond ASOs to small interfering RNA (siRNA) therapeutics, indicating a broader role for ESCRT-I in RNA drug biology. Together, these findings uncover a metabolic-endosomal axis controlling nucleic acid drug efficacy and provide new mechanistic insight into intracellular determinants of RNA-based therapeutics.
The lncRNA CyKILR produces two splice variants with antagonistic roles in lung tumorigenesis. While its nuclear counterpart functions as a tumor suppressor, we revealed that the cytoplasmic variant, CyKILRb, is a potent oncogenic driver that activates the PI3K/AKT signaling axis. Specifically, downregulation of CyKILRb induced the loss of the PI3K activator PIK3R2 and the tumor promoter RPS6KB2, while simultaneously increasing the tumor suppressors, p21 and p27. CyKILRb ectopic expression produced the contrasting effect, and suppression of either PIK3R2, PI3K, or AKT attenuated CyKILRb-induced cell proliferation and clonogenicity. Combinatorial expression studies oriented the pathway from CyKILRb→↑PIK3R2→PI3K→AKT→enhanced oncogenicity. An in silico analysis comparing predicted microRNA response elements (MREs) across the CyKILRb and PIK3R2 transcripts identified shared contiguous complementary miRNA binding sequences. Of these miRs, miR-3151-5p, a tumor suppressor and known PIK3R2 regulator, was identified. This miR associated with both RNAs in cells, and treatment of NSCLC cells with a miR-3151-5p mimic reduced PIK3R2 and clonogenicity. Ectopic expression of CyKILRb containing a mutated MRE for miR-3151-5p showed an inability to upregulate PIK3R2 and tumor-promoting phenotypes in contrast to WT CyKILRb. These findings define a novel role for CyKILRb as a competing endogenous RNA for tumor suppressive miRs, which induces the PI3K/AKT axis.
Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-β superfamily, functions in skeletal muscle and neuronal regeneration and has been implicated in tumor suppression. In hepatocellular carcinoma (HCC), GDF11 expression is markedly downregulated, but the mechanisms responsible for this repression remain unclear. In this study, we examined whether the oncogenic miR-106b-25 cluster contributes to GDF11 suppression in HCC. We found that this cluster decreases GDF11 expression at both the mRNA and protein levels, with miR-93-5p acting as the predominant regulator. Inhibition of miR-93-5p with antisense oligonucleotides restored GDF11 expression and reduced HCC cell proliferation, migration, and invasion. Mechanistically, we identified the RNA-binding protein (RBP) PCBP2 as a key facilitator of miR-93-5p targeting of GDF11. PCBP2 binds a C-rich element adjacent to the miR-93-5p target site in the GDF11 3′ UTR, thereby enhancing miR-93-5p-mediated repression. PCBP2 knockout attenuated miR-93-5p-mediated repression, whereas re-expression of PCBP2 restored it, supporting its modulatory role. Collectively, these findings identify PCBP2 as a modulator of miR-93-5p-mediated GDF11 repression and suggest that this regulatory interaction contributes to HCC cell proliferation, migration, and invasion. This work provides insights into the post-transcriptional control of the tumor suppressor and highlights the therapeutic potential of targeting miRNA–RBP interactions.
Targeting angiotensinogen (AGT) with small interfering RNAs (siRNAs) is a promising strategy to suppress renin–angiotensin system (RAS) activity and to potentially mitigate progression of chronic kidney disease (CKD). Although AGT siRNAs have been shown to exert kidney-protective effects in experimental models of kidney injury, the underlying mechanisms remain unclear. Juxtaglomerular renin-lineage cells (CoRL) have recently been recognized as progenitors capable of replenishing several glomerular cell types during injury. Here, we investigated whether AGT siRNA alters CoRL-driven glomerular cell repopulation in the 5/6 nephrectomy (5/6NX) model using renin lineage-tracing mice treated with either AGT siRNA or luciferase-targeting control. AGT silencing achieved ∼85% reduction in plasma AGT and induced a marked compensatory rise in plasma renin concentration. Despite these systemic effects, AGT knockdown did not alter kidney function, as reflected by unaffected blood urea levels, urinary albumin-to-creatinine ratios, glomerular hypertrophy, or kidney fibrosis. While the number of CoRL-derived mesangial cells remained unchanged, AGT inhibition resulted in a modest but significant increase in CoRL-derived parietal epithelial cells and a pronounced increase in CoRL-derived podocytes. These findings indicate that liver-directed AGT silencing does not confer short-term functional benefit but enhances CoRL-mediated podocyte regeneration, identifying a potential mechanism that may contribute to delayed renal repair.
Chemical modification is essential for the clinical application of small interfering RNAs (siRNAs), as it improves their stability and specificity. However, predicting the activity of chemically modified siRNAs remains challenging owing to the scarcity of high-quality datasets and the computational expense of molecular dynamics (MD) simulations. In this study, we propose fast and robust activity prediction of chemically modified siRNAs via structure-based energy (FRAMEs), a novel framework that combines rapid structural prediction via deep learning with physics-based energy calculations for feature engineering of siRNA modifications. To address data scarcity, FRAMEs employs inference-augmented tabular deep learning to achieve robust activity prediction. The total energy score correlates strongly with experimental IC50 and melting temperature, achieving performance comparable to MD-based metrics. Under both leave-one-out and stratified 5-fold cross-validation, inference-augmented TabPFN consistently outperformed all classical machine learning baselines, with the two evaluation schemes yielding mutually reinforcing conclusions. Furthermore, by exploiting physically meaningful stochasticity, FRAMEs stabilizes predictions on small datasets and exhibits strong generalization to an independent real-world dataset, outperforming existing methods. Guided by FRAMEs, several fully modified siRNA candidates targeting oncogenes relevant to cancer therapy were designed and experimentally verified. Cell-based gene-silencing assays confirmed their potent knockdown activity, validating the practical utility of FRAMEs for the rational design of therapeutic modified siRNAs.
