Molecular Therapy. Nucleic Acids最新文献

Despite the proven safety of dystrophin-targeting phosphorodiamidate morpholino oligomer (PMO) therapy, poor delivery of the PMOs limit the efficacy of this dystrophin restoring gene therapy for Duchenne muscular dystrophy (DMD). Limited myogenesis and excessive fibrosis in DMD are pathological features that contribute to the poor efficacy of PMOs. We show that the severe DMD mouse model (D2-mdx) not only replicates these pathological features of DMD but also mirrors the resulting PMO-mediated dystrophin restoration deficit. High transforming growth factor β (TGF-β) activity, which is a common feature of DMD patient and D2-mdx muscles, limits myogenesis and causes fibrosis. We developed a TGF-β-targeting PO (TPMO), which when used acutely, lowered macrophage TGF-β activity and signaling in the dystrophic muscle, enhanced muscle regeneration, and enhanced dystrophin restoration when used in combination with dystrophin exon skipping PMO (DPMO). Chronic use of this combination PMO therapy in D2-mdx mice reduced muscle fibrosis and muscle loss, allowed dystrophin restoration in skeletal muscle and heart, and led to an overall enhancement of skeletal muscle function. This approach leverages the safety of PMO-based therapy and represents the first combination PMO treatment for DMD that simultaneously enhances dystrophin restoration, reduces fibrosis, and alleviates myogenic deficits to ultimately improve health and function of dystrophic muscles.

Heart failure (HF) remains a significant healthcare burden, with an unmet need for novel therapies to target the preceding pathological hypertrophy in HF patients. Here we report the development of novel conditional-siRNA (Cond-siRNA) constructs that are selectively activated by disease-specific RNA biomarkers to enable cell-specific inhibition of a target disease-causing RNA. We designed a Cond-siRNA that can be activated by Nppa mRNA, upregulated specifically in cardiomyocytes (CMs) under pathological stress, to silence the key pro-hypertrophic gene calcineurin (CaN) A-a by the effector small interfering RNA (siRNA). In both neonatal rat ventricular myocytes (NRVMs) and H9c2 CMs, Cond-siRNA showed minimal baseline activity but selectively silenced CaN upon Nppa mRNA induction by phenylephrine (PE) stress in cell culture models and pressure overload (PO) in a heart-on-a-chip model. In NRVMs, Cond-siRNA reduced CaN mRNA only after PE or PO, but not with vehicle, confirming Nppa-specific activation. This specificity was further validated as Cond-siRNA did not affect CaN in cardiac fibroblasts or T cells lacking Nppa. Reduced CaN protein levels and NFATc1 nuclear translocation correlated with decreased NRVM hypertrophy after PE treatment, confirming Cond-siRNA's efficacy. This study offers proof-of-concept for Cond-siRNA as a targeted therapy to mitigate hypertrophic progression, paving the way for novel HF treatments.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant cancer predisposition syndrome driven by the loss of fumarate hydratase (FH) activity. Recently, we identified a pathogenic variant in intron 9 of the FH gene that disrupts splicing by creating a novel splice acceptor site, resulting in the aberrant inclusion of a cryptic exon. Inclusion of the cryptic exon introduces a premature termination codon, leading to loss of FH activity. To restore FH expression, we sought to identify strategies to drive exclusion of the cryptic exon from the mature mRNA. To this end, we generated a minigene GFP reporter system that recapitulates the splicing defect observed in patients. We employed CRISPR-Cas9-mediated genome editing and antisense oligonucleotides (ASOs) to modulate splicing and demonstrated that both strategies can successfully promote skipping of the cryptic exon in a reporter cell line. Furthermore, we were able to show that ASOs can be used to shift the balance between the FH mRNA isoforms originated from the reference and the variant allele in patient-derived fibroblasts using ASOs. These findings support the potential for splicing modulation as a therapeutic approach for HLRCC-associated non-coding loss-of-function mutations in FH.

Gene editing is a groundbreaking therapeutic approach that can potentially treat a broad spectrum of genetic and acquired diseases. This review highlights recent clinical trials employing advanced gene editing technologies such as CRISPR-Cas9, zinc-finger nucleases (ZFNs), and base editors across multiple disease areas including metabolic disorders, autoimmune diseases, muscular dystrophies, and inherited eye disorders. Central to the success of these therapies is the development of efficient and safe delivery systems, including lipid nanoparticles (LNPs), viral vectors (adenoviral and lentiviral), electroporation techniques, and virus-like particles (VLPs), which facilitate precise editing of target cells in vivo or ex vivo. These delivery platforms have enabled promising early-phase clinical trials demonstrating feasibility, safety, and durable gene modification in patient populations. For example, LNPs have been pivotal in delivering mRNA editors for liver-targeted metabolic diseases. At the same time, viral vectors have been used for ex vivo modification of T cells and hematopoietic stem cells in autoimmune and infectious diseases. Despite encouraging results, challenges remain in optimizing delivery specificity, minimizing off-target effects, and ensuring long-term safety and efficacy. Ongoing and upcoming trials continue to refine these delivery technologies and expand the therapeutic reach of gene editing.

Small interfering RNA (siRNA) has emerged as a powerful tool for gene silencing, offering great potential for therapeutic applications. However, the clinical use of siRNA is limited by several challenges, including poor stability in biological fluids, off-target effects, and toxicity due to non-specific cellular uptake. To address these limitations, extracellular vesicles (EVs) derived from milk are being investigated as natural carriers to deliver siRNA and microRNA. These EVs offer advantages such as low immunogenicity, biocompatibility, and the ability to cross biological barriers. Here, we optimized methods for loading siRNA into milk-derived EVs (mEVS) and assessed their ability to protect siRNA from degradation while preserving its gene-silencing efficacy. We targeted a potential biomarker, Aurora kinase A (AURKA), known to be deregulated in many types of solid tumors, including colon cancer. Our results demonstrate that mEVs-loaded siRNA retains the stability and functionality of internalized siRNA, leading to efficient gene silencing in target cells. This approach highlights the potential of mEVs as a safe and valuable delivery system, overcoming key limitations of siRNA therapeutics and opening new avenues and opening new avenues for diagnostic and therapeutic strategies in colon cancer.

Plasmodium vivax poses significant challenges to malaria control due to its relapsing nature. This study explores the immunogenicity and efficacy of nucleoside-modified mRNA-lipid nanoparticle (LNP) vaccines targeting the P. vivax circumsporozoite protein (PvCSP). Two mRNA constructs encoding PvCSP were designed and tested in mice. Despite lower protein expression, the vaccine encoding the wild-type signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor of PvCSP induced significantly higher antibody titers against the PvCSP and its repeat region compared with the mRNA construct with SP but without GPI. The immunogenicity of PvCSP mRNA-LNP vaccines was evaluated using various administration routes and immunization schedules. Both intradermal and intramuscular delivery generated dose-dependent antibody responses, but the former demonstrated superior responses at a lower dose. Conversely, intravenous administration resulted in very poor responses. Notably, administering a delayed third dose intramuscularly 5 months after the second dose resulted in significantly higher levels of anti-repeat region antibodies and enhanced T cell responses in both the spleen and liver. This delayed regimen provided strong protection against sporozoite challenge, with the magnitude and avidity of anti-repeat region antibodies linked to this protection. These findings highlight the potential of the nucleoside-modified mRNA-LNP vaccine platform in combating P. vivax pre-erythrocytic stage infection.

Glioblastoma multiforme (GBM) is the most prevalent malignant brain tumor. Treating this type of cancer is challenging due to its high heterogeneity, rapid cell growth, and highly malignant nature, which results in a poor prognosis. A key feature of GBM's malignancy is that it resists drug treatments and evades cell death mechanisms. Ferroptosis is a promising therapeutic avenue for combating drug-resistant cancers because it is a recently discovered mechanism of programmed cell death that oxidizes membrane lipids and is triggered by an accumulation of reactive oxygen species. Recent findings suggest that ferroptosis is an innovative path for improving human GBM therapy. More exploration of the regulatory pathways and interactions of ferroptosis is essential to developing effective therapeutic strategies for this aggressive type of cancer. Inducing ferroptosis or integrating it with current treatments may present an opportunity to improve outcomes in GBM patients. This review investigates the role of ferroptosis in GBM and identifies its important molecular mediators. It also explores promising therapeutic strategies that target ferroptosis as a novel approach for GBM treatment.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the 3'-UTR of the DMPK gene that lead to nuclear foci accumulation and splicing defects. Circular RNAs (circRNAs) are emerging regulators of muscular disorders, but their role in DM1 remains largely unknown. By analyzing available RNA-sequencing datasets from DM1 patients, followed by validation in patients and matching control muscle biopsies, we identified seven circRNAs that were significantly increased in DM1 muscles and displayed high circular-to-linear isoform ratios. Among them, circARHGAP10 correlated positively with CTG repeat length and inversely with muscle strength, indicating its potential as a biomarker. Silencing of circARHGAP10 in DM1 myogenic cells reduced DMPK expression, decreased nuclear foci, and partially rescued normal splicing. Bioinformatics prediction and pull-down of circARHGAP10 indicated that circARHGAP10 binds miR-409-3p. circARHGAP10 and miR-409-3p were both found to be upregulated in DM1 muscle biopsies and silencing of circARHGAP10 led to the downregulation of miR-409-3p, indicating their co-regulation. Interestingly, miR-409-3p overexpression blocked the beneficial effects of circARHGAP10 silencing on DMPK levels, foci, and splicing. Thus, circARHGAP10-dependent regulation of DM1-associated mechanisms is mediated, at least in part, via interaction with miR-409-3p. In conclusion, circARHGAP10 exhibits promising potential as a biomarker and therapeutic target for DM1.

Messenger RNA (mRNA) has emerged as an attractive new technology of drugs. The efficacy of mRNA technology depends on both the efficiency of mRNA delivery and translation. Untranslated regions (UTRs) and the poly(A) tail play a crucial role in regulating mRNA intracellular kinetics. Intending to improve the therapeutic potential of synthetic mRNA, we evaluated various UTRs and tail designs, using Pfizer-BioNTech coronavirus disease 2019 (COVID-19) vaccine sequences as a reference. First, we screened six 5' UTRs (cap-dependent/-independent), evaluated nine 5' UTR-3' UTR combinations, and a novel heterologous A/G tail in cell models, and in vivo using luciferase as a reporter gene. Then, to decipher the translation mechanism of selected UTRs, we correlated mRNA expression with ribosome load, mRNA half-life, mRNA immunogenicity, and UTR structures. Our results showed that the heterologous tail we introduced is as potent as the Pfizer-BioNTech tail and confirmed the high potency of the human α-globin 5' UTR. They also revealed the potential of the VP6 and SOD 3' UTRs. We validated our results using mRNA encoding the SARS-CoV-2 spike protein formulated as lipid nanoparticles (LNPs) for mouse immunization. Overall, the selected 3' UTRs and heterologous A/G tail have great potential as new elements for therapeutic mRNA design.

Nucleic acid molecules are emerging as potential therapeutic tools, as evidenced by the transfection of small interfering RNA (siRNA) molecules in therapeutic applications and messenger RNAs in immunotherapeutic vaccination. In most cases, these nucleic acids are conditioned as lipid nanoparticles made with different lipid moieties to promote their intracellular delivery. Over the past few years, we have documented the delivery of siRNAs using a single short (15 amino acids) peptide called WRAP5, which follows an extremely simplified formulation phase that enables the formation of nanoparticles with a diameter of 60-80 nm. We indeed demonstrated the expected dose-response reduction in the levels of the targeted proteins. To apply this technology to the cellular delivery of mRNAs, we investigated the ability of the WRAP5 peptide to transfect mRNAs of different sizes and promote the expression of their proteins. These peptide-based nanoparticles, which also have diameters ranging from 60 to 80 nm, showed remarkable stability over time when simply stored at 4°C and fully retained their transfection properties in vitro for up to several months post-formulation. Interestingly, we demonstrated in vivo that these nanoparticles were able to induce an immune response against the protein synthesized from the vectorized mRNA.