Molecular Therapy. Nucleic Acids最新文献

[This corrects the article DOI: 10.1016/j.omtn.2023.02.014.].

Messenger ribonucleic acid (mRNA), a promising tool in vaccine and therapeutic development, is reliant on intact mRNA delivery into target cells. Given its susceptibility to degradation, ensuring its stability is crucial, necessitating rigorous quality control throughout the product life cycle. This study presents an ion-pair reverse-phase liquid chromatography method that enables rapid and direct mRNA extraction from lipid nanoparticles, facilitated by using a surfactant in the sample preparation. This method, optimized using design of experiments (DoE), allows relative quantification of intact mRNA, mRNA fragments, and mRNA-lipid adducts. Forced degradation studies were used to investigate the impact of mRNA-lipid adducts on protein expression and to identify their chemical structures. The structures, identified by mass spectrometry, suggest reaction mechanisms that differ from those described in the literature so far. Further studies evaluated how formulation parameters such as pH, ionic strength, and buffering species affect mRNA-lipid adduct formation and mRNA fragmentation. A DoE assessed the impact of formulation parameters on mRNA integrity and mRNA-lipid adducts, showing that pH plays the major role. Overall, these findings have significant implications for the design and development of future mRNA-based biopharmaceuticals.

Adenine base editors (ABEs) enable efficient A-to-G base conversions in genomic DNA, serving as powerful tools for basic research and clinical disease treatment. TadA-8e with high processive and compatibility makes ABE8e to be the most widely used adenine base editor and has also facilitated the creation of more elegant base editors based on TadA-8e fusion, such as AYBE and eA&C-BEmax. However, ABE8e has more off-target events including DNA off-target and RNA off-target, which raises safety concerns for precision gene editing. Here, we split the TadA-8e of ABE8e (sABE8e) to enable controlled adenine base editing through rapamycin-induced dimerization between FRB and FKBP12. sABE8e has comparable on-target adenine editing activity to ABE8e while maintaining reduced DNA and RNA off-target effects. Harnessing this site of split TadA-8e, we have also developed controllable AYBE (sAYBE) and eA&C-BEmax (seA&C-BEmax), which both offer similar or slightly low base editing efficiency with decreased off-targets compared to AYBE or eA&C-BEmax. These precise and controllable base editing tools will advance the future application of base editors in basic research and clinical disease treatment.

Loss-of-function mutations in the CTNNB1 gene cause β-catenin deficiency, resulting in CTNNB1 syndrome, a rare neurodevelopmental disorder characterized by motor and cognitive impairments. Given the wide variety of mutations across CTNNB1 and its dosage sensitivity, a mutation-independent therapeutic approach that preserves endogenous gene regulation is critically needed. This study introduces spliceosome-mediated RNA trans-splicing as a novel approach to restore β-catenin production. Pre-trans-splicing RNA molecules (PTMs) targeting CTNNB1 introns 2, 5, and 6 were designed and evaluated using a split yellow fluorescent protein reporter system. Rationally designed short antisense RNAs, which mask splicing regulatory elements, significantly enhanced PTM-mediated trans-splicing at both mRNA and protein levels. Additionally, introducing a self-cleaving ribozyme at the PTM's 5' end further improved trans-splicing efficiency, likely due to increased nuclear retention. CMV promoter-driven PTM expression yielded the highest efficiency. Importantly, successful trans-splicing of the endogenous CTNNB1 transcript confirmed the physiological relevance of this strategy. This study is the first to apply and optimize spliceosome-mediated RNA trans-splicing (SMaRT) for CTNNB1 mRNA correction, providing a promising, mutation-agnostic approach for treating CTNNB1 syndrome.

Lung cancer is the leading cause of cancer-related death worldwide, and the complex molecular mechanisms underlying its development, particularly the role of alternative splicing (AS) in different subtypes, remain poorly understood. In this study, we performed RNA sequencing of 178 lung cancer patients and conducted a comprehensive analysis of the transcriptomic landscape with a focus on AS. We identified robust lung cancer- and subtype-specific AS biomarkers that were consistently effective in both tissue samples and cancer cell lines. Notably, several of these biomarkers also serve as critical regulators in lung cancer progression. Our regulatory network analysis, with a focus on RNA-binding proteins, revealed QKI and SR proteins as key splicing factors. Specifically, QKI was found to modulate the splicing of PLEKHA1 exon 15, a cancer-specific AS biomarker, while SRSF1 regulated the splicing of MKNK2 exon 14, a subtype-specific AS biomarker. Our study provides valuable insights into key AS events and their regulatory mechanisms in lung cancer, paving the way for potential therapeutic targets.

Parkinson's disease (PD) is a debilitating neurodegenerative condition. Synaptic dysfunctions are associated with the onset and progressive neurodegeneration exhibited in PD. Healthy, active synapses are a prerequisite for non-pathological neurotransmission. When neurotransmission becomes pathological, such as observed in neurodegenerative conditions like PD, the biomolecules found in and around such synapses need distinctive investigation. MicroRNAs (miRNAs) found in neuronal subcellular compartments, such as dendrites, pre-synaptic boutons, and synaptic vesicles, have been garnering attention in neurogenerative diseases. MiRNAs that modulate synaptic activity and synapse function are called synaptic miRNAs. Several miRNAs have been identified that regulate key synaptic proteins; however, information about synaptic miRNAs is largely unknown in PD. In this review, we focused on the most promising synaptic miRNAs, those that are critical for normal synapse function and play a crucial role in PD pathology. We also discussed the synaptic miRNA's interplay with PD-associated synaptic dysfunction. Investigating further how synaptic miRNAs impacts PD pathogenesis may uncover novel etiological information and potential pathways for treatments and a cure for PD.

Traditional viral-based chimeric antigen receptor (CAR) T cell therapies have vanquished multiple blood malignancies with decade-long remissions yet struggle against solid tumors. Nonviral engineering of CAR T cells via electroporation or lipid nanoparticle (LNP) delivery of CAR-encoding mRNA results in highly efficient yet transient CAR expression, challenging the adequacy of available preclinical models for mRNA-based CAR T cell evaluation. This study presents a unique three-pronged approach that combines mRNA-based CAR T cells, multi-targeting of glioblastoma (GBM)-associated receptors, and maximal surgical resection as a novel and readily translatable platform for preclinical evaluation of mRNA-based CAR T cells against solid tumors. We performed head-to-head in vitro and in vivo analyses of mRNA-based CAR T cells generated using different expansion conditions, mRNA delivery methods, or combination approaches. Besides potent in vitro cytotoxicity, our findings unveil a therapeutic window of anti-tumor efficacy, as well as robust and durable complete remissions in xenograft mouse models of GBM receiving maximal surgical resection and locoregional injections of multivalent CAR T cells (MVCAR). Such efficacies were significantly better in 5-day expanded versus quiescent T cells. Interestingly, MVCAR T cells were superior to pooled CAR T cells (CARPool) expressing the same CAR scFv combinations in an orthotopic resection model of GBM.