Molecular Therapy. Nucleic Acids最新文献

[This retracts the article DOI: 10.1016/j.omtn.2020.04.018.].

The pro-inflammatory cytokine interleukin-6 (IL-6) via its IL-6 signal transducer (IL6ST/gp130) plays an important role in neuronal survival, neuro-regeneration, and pathological pain. While its critical importance in the nervous system is well established, the underlying molecular mechanisms and the involvement of microRNAs (miRNAs) as critical regulators of biological processes in health and disease are not sufficiently understood. We identified miR-486-5p as the single significantly deregulated miRNA in sensory neurons with a conditional depletion of gp130. In situ hybridization and immunofluorescence in dorsal root ganglia (DRG) localized miR-486 to small diameter neurons, including peptidergic nociceptors. miR-486^-/- mice exhibited normal baseline and neuropathic pain-like behaviors and recovered similarly to wild-type (WT) littermate controls in response to sciatic crush injury. On the other hand, DRG neurons derived from mice with a conditional deletion of IL6ST/gp130 in Na_v1.8-expressing primary afferent nociceptors (SNS-gp130^-/-) show strongly compromised neuro-regeneration, which was significantly rescued by overexpressing miR-486, indicative of a specific role of miR-486 in IL-6/gp130-dependent neuro-regenerative processes. Our findings highlight context-dependent differential expression and roles of miRNAs after nerve injury driving nerve regeneration versus neuropathic pain.

Potential strategies to develop new treatments for Parkinson's disease (PD) aim at targeting disease-associated proteins like alpha-synuclein (aSyn), which accumulates in neurons of PD patients and contributes to neuronal degeneration. A promising new approach is the therapeutic use of small interfering RNAs (siRNAs) for aSyn knockdown, but is challenging due to siRNA instability, poor delivery, and inefficient uptake. Therefore, we developed a nanoparticle-based approach for intranasal delivery of siRNAs, circumventing the blood-brain barrier and enhancing the potential of siRNAs for clinical application. Tyrosine-modified polyethylenimines (PEIs), or polypropylenimine dendrimers (PPIs), were complexed with siRNA targeting the aSyn-encoding gene SNCA (siSNCA) and combined with liposomes. Nanoparticles efficiently transfected SH-SY5Y cells with low cytotoxicity and significantly reduced SNCA mRNA levels. In Thy1-aSyn mice, intranasally administered labeled nanoparticles distributed extensively across the brain, including the olfactory bulb, substantia nigra, and prefrontal cortex. After only 4 days of treatment, siSNCA-loaded nanoparticles significantly reduced aSyn protein and SNCA mRNA levels in the brain. Mice showed neither overt adverse behavioral effects nor increased reactive microglia. These findings highlight the potential of nanoparticle-mediated intranasal siRNA delivery as a promising, non-invasive approach to reduce aSyn levels in the brain, offering a novel therapeutic strategy for Parkinson's disease.

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.