Molecular Therapy. Nucleic Acids最新文献

Inherited retinal dystrophies (IRDs) are a group of incurable, genetically heterogeneous diseases that cause progressive degeneration of the retina, leading to the loss of vision. Genome editing technologies offer a powerful prospect for mutation correction and single-dose cures for these diseases. Here, we investigated the potential of adenine base editing (ABE) to correct a panel of causative genetic variations in patient-derived induced pluripotent stem cells (iPSCs) and identified parameters that can efficiently correct a pathogenic variation in the AIPL1 gene (c.665G>A, p.Trp222∗), which is associated with autosomal recessive Leber congenital amaurosis type 4. To investigate correction of the variant in a patient-relevant model, retinal organoids (ROs) were derived from corrected isogenic and patient-derived iPSCs. Adenine base editor components were delivered to ROs via lipofection as chemically modified RNA or via a split intein system following dual-AAV transduction. The data show AIPL1 rescue in photoreceptor cells with both delivery systems and restoration of the AIPL1 target protein, cyclic guanosine monophosphate phosphodiesterase 6-a critical component of the visual transduction system-in treated rod photoreceptors. These proof-of-principle experiments highlight the utility of ROs for investigating the potential of ABE technology as a means to treat IRDs.

We previously demonstrated that blocking tolerogenic signal transducer and activator of transcription 3 (STAT3) signaling in the tumor microenvironment can unleash Toll-like receptor 9 (TLR9)-mediated antitumor immunity. To enable systemic administration of minimally modified CpG-siSTAT3, we developed a panel of MC3-based lipid nanoparticle (LNP) formulations optimized for targeting immune cells and B cell lymphoma cells. The selected LNP2(CpG-siSTAT3) induced potent type I interferon (IFN) production in human peripheral blood mononuclear cells (PBMCs) and resulted in >50% STAT3 knockdown in human cancer cells at low oligonucleotide concentrations. In vivo, LNP2(CpG-siSTAT3) showed a 10-fold improvement in potency against B cell lymphoma xenotransplants compared to the naked oligonucleotide. Further changes in chemical composition yielded LNP2.1, which preferentially targeted human monocytes and dendritic cells (DCs). In A20 lymphoma-bearing mice, the fluorescently labeled LNP2.1(CpG-siSTAT3) quickly drained to local tumor-draining lymph nodes (TDLNs) after subcutaneous injection and was taken up by activated DCs and macrophages. Furthermore, LNP2.1(CpG-siSTAT3) administration significantly reduced A20 tumor growth by rapidly activating DCs and macrophages in TDLNs, thereby promoting T cell activation and specifically increasing tumor-infiltrating cytotoxic CD8 T cells secreting IFNγ and tumor necrosis factor alpha. The LNP2.1 formulation offers an effective vehicle for targeting tolerogenic myeloid cells in the B cell lymphoma microenvironment and potentially in solid tumors.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. Previously, we have shown correction of the most recurrent PKU variant using both base editing and prime editing. In this work, we set out to screen base editors and single guide RNAs (sgRNAs) in vitro to identify sgRNA-editor combinations that would provide sufficient correction for phenotypic rescue of the next four most recurrent PKU variants. For three candidates, we established efficient corrective editing in vitro. We then assessed the off-target editing profile of each sgRNA-editor combination. For two of these variants, we demonstrated efficient corrective editing in the livers of humanized mouse models via adeno-associated viral (AAV) delivery. This work identifies base editing strategies for in vivo correction of the second and third most common pathogenic variants of PKU.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive connective tissue disorder characterized by ectopic calcification of elastic fibers throughout the skin, retina, and arteries. It is caused by pathogenic variants in ABCC6, which encodes a transmembrane transporter that primarily localizes to hepatocytes. Loss of ABCC6 function in hepatocytes leads to systemic deficiency of inorganic pyrophosphate (PPi), a potent inhibitor of calcification; such depletion of PPi from the circulation is responsible for multisystemic ectopic calcification seen in PXE. Therefore, liver-targeted variant correction by genome editing and subsequent restoration of systemic PPi may offer a one-and-done therapeutic approach for PXE. The ABCC6 c.3490C>T (p.R1164X) variant is one of the most common variants found in PXE patients. Here, we show that liver-directed correction of the R1164X variant by adenine base editing restores plasma PPi and prevents ectopic skin calcification in mice fed a standard diet or an "acceleration diet" that exacerbates ectopic calcification. These results provide fundamental insight into the molecular etiology of PXE and provide a proof-of-principle that genetic correction of ABCC6 defects through adenine base editing may represent a novel, permanent therapy for the treatment of PXE.

The base editor (BE) and prime editing guide RNA (pegRNA)-based prime editor (PE) technologies relying on the CRISPR/Cas system are very efficient gene editors that have been developed in recent years. The BEs include cytosine base editors (CBEs) that mediate the conversion of C to T, adenine base editors (ABEs) that mediate the conversion of A to G, glycosylase base editors (GBEs) that mediate the conversion of C to G, and the dual-base editors (DBEs) that mediate the simultaneous conversion of C to T and A to G. The BEs and PEs have been successfully applied for genome editing of various animals, plants, and microorganisms due to their advantages of high efficiency and independence of DNA double-strand breaks or donor DNA. The development process and characteristics of various BEs and PEs and their effectiveness of application are systematically introduced to provide a reference for selecting appropriate genome editing technologies. Moreover, the urgent issues that need to be addressed for more efficient and precise editing are summarized and prospected.

[This corrects the article DOI: 10.1016/j.omtn.2025.102697.].

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the DMD gene that disrupt the production of functional dystrophin proteins. Intellectual disability and neurobehavioral complications including autism spectrum disorder, attention-deficit disorders, and anxiety cumulatively occur in 33%-43% of the patients due to deficiency of multiple dystrophin isoforms produced in brain. Previous work also identified behavioral abnormalities in the mdx23 mouse model of DMD. In this work we mapped the expression of the different dystrophin isoforms in different areas of the mouse brain. Next, we determined the behavioral phenotypes that best differentiate mdx23 (lacking the Dp427 isoform) and wild-type mice. Finally, we investigated the response to intracisternal magna (ICM) injection of exon-skipping phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides, which induces skipping of exon 23 and restores the reading frame on these phenotypes. PMO administration led to low, detectable, restoration of dystrophin protein and DMD exon skipping in different brain regions. Treated mdx23 male mice exhibited a small but significant rescue of their enhanced fear response. We conclude that ICM delivery of PMO leads to low levels of dystrophin restoration, but these levels are sufficient to elicit a modest behavioral phenotype in mdx23 mice.

MicroRNAs (miRNAs) are small, non-coding RNAs that influence various cellular activities through post-transcriptional gene silencing. Recent research has shown that miRNA modifications, including N6-methyladenosine (m6A), 5-methylcytidine (m5C), 2'-O-methylation (Nm), N7-methylguanosine (m7G), pseudouridylation (Ψ), phosphorylation, RNA editing (adenosine to inosine [A to I]), acetylation, and oxidation, play crucial roles in fine-tuning miRNA expression and function. This review examines the impact of nucleotide modifications on miRNA biogenesis, particularly their role in regulating RNA interactions with the Drosha-DiGeorge syndrome critical region 8 (DGCR8) and Dicer complexes, thereby influencing primary miRNA (pri-miRNA) processing, pre-miRNA export, and miRNA maturation. It also examines whether these modifications assist miRNA recognition by RNA-binding proteins (RBPs) in controlling miRNA processing and stability, as well as their impact on miRNA strand selection, target recognition, and the recruitment of regulatory proteins to the miRNA-induced silencing complex (miRISC), which facilitates the silencing of miRNA-targeted messenger RNAs (mRNAs). Additionally, the review discusses the role of miRNA modifications in various human diseases and considers how advanced sequencing technologies and chemical biology approaches enable detailed mapping of these modifications. Furthermore, it provides new insights into the challenges of understanding the dynamic nature of miRNA modifications and their context-dependent effects. It also highlights future directions, including innovative detection methods and epigenetic crosstalk with potential therapeutic applications in human diseases.

We developed siRMSD, a predictive parameter for off-target effects induced by chemical modifications, to optimize siRNA therapeutics. In RNA interference, small interfering RNA (siRNA) suppresses gene function by degrading mRNA with perfect sequence complementarity, providing therapeutic potential through the targeted inhibition of disease-related genes. However, off-target effects on unintended mRNAs pose a significant challenge to clinical application. While chemical modifications improve nuclease stability and reduce off-target effects, the underlying mechanisms remain unclear. Here, we show that structural distortions caused by chemical modifications determine off-target effects. Modifications, including 2'-O-methoxyethyl, 2'-O-methyl, and 2'-formamido, at positions 2-5 disrupted the A-form RNA duplex on argonaute 2, preventing stable binding to target mRNA. In contrast, modifications at positions 6-8 had minimal impact on off-target effect resulting from changes in thermodynamic stability.