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At least 73 records · Page 4Linked to original sources

PubMed2025

Engineered virus-like particle-assembled Vegfa-targeting Cas9 ribonucleoprotein treatment alleviates neovascularization in wet age-related macular degeneration.

BACKGROUND: Age-related macular degeneration, particularly the wet form, is a leading cause of vision loss, characterized by vascular endothelial growth factor A (VEGFA) overproduction. Engineered virus-like particles (eVLPs) combine the efficiency of viral systems with the transient nature of non-viral platforms to offer a potential solution for delivering VEGFA-targeting genome editing enzymes in a safe and efficient manner. Here, we investigate the therapeutic efficacy of eVLPs for transient delivery of Vegfa-targeting Cas9 ribonucleoprotein in a laser-induced choroidal neovascularization mouse model of wet age-related macular degeneration. RESULTS: We find that Cas9-eVLPs enables efficient intracellular delivery in vitro, achieving up to 99% insertion and deletion frequency at Vegfa target locus and significant VEGFA protein downregulation in NIH/3T3 cells. A single subretinal injection of Cas9-eVLPs into the mouse retinal pigment epithelium effectively disrupts Vegfa expression, achieving an average indel efficiency of 16.7%. Compared to control groups, the laser-induced choroidal neovascularization mouse model exhibits significantly reduced choroidal neovascularization formation following Cas9-eVLPs intervention, and decreased VEGFA protein levels are detected in the retinal pigment epithelium. Furthermore, the retinal anatomical and functional toxicity are not affected after treatment. CONCLUSIONS: eVLPs exhibit the potential as a safe and efficient delivery platform for Cas9 ribonucleoproteins, achieving precise Vegfa downregulation and significant reduction in choroidal neovascularization in a mouse model of wet age-related macular degeneration. With transient delivery of gene editing enzymes, high editing efficiency, and minimal risk of genomic integration, eVLPs present a promising alternative to conventional delivery systems for advancing genome editing therapies in retinal diseases.

CRISPR-Associated Protein 9↗

PubMed2026

Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.

CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.

Humans↗

PubMed2026

Redox-activated cholesterol-dependent cytolysin enables cytosolic release of liposomal cargo.

Precise intracellular delivery of biologic therapeutics remains a major challenge due to endosomal entrapment and inefficient delivery systems. Here, we develop a bioinspired platform that uses Streptolysin O (SLO), a member of the cholesterol-dependent cytolysin (CDC) family, for cytosolic cargo delivery. This delivery system incorporates an affibody for selective targeting and endocytosis and a redox-cleavable PEG-conjugated dithiol-ethyl carbonate linker (PEG-DEC) that reversibly inactivates SLO extracellularly. After endosomal uptake, the reductive intracellular environment removes the PEG layer, reactivating SLO to induce localized endosomal disruption and cargo release. This mechanism minimizes off-target toxicity while promoting efficient cytosolic delivery of diverse cargo, including doxorubicin (DOX), the fluorescent protein GFP and mApple, and the enzyme NanoLuciferase (NanoLuc) and lactate oxidase (LOX). PEGylated SLO exhibited significantly improved cytosolic release efficiency compared with conventional liposomal formulations, confirming the advantage as a controllable intracellular delivery module.

Liposomes↗

PubMed2026

Emerging Nucleic Acid-Based Therapies for Hypercholesterolemia with Focus on a New Modality, Liver-Directed miR-30c Analog C2.

Despite major advances in lipid-lowering therapies, a significant unmet need remains, particularly for patients with homozygous familial hypercholesterolemia (HoFH), severe heterozygous familial hypercholesterolemia (HeFH), and those who fail to achieve guideline-recommended LDL-C targets. Nucleic acid-based therapeutics have emerged as a transformative approach for treating hypercholesterolemia. Antisense oligonucleotides and small interfering RNAs (siRNAs) have demonstrated durable hepatic gene silencing and have led to approved therapies, while gene replacement and in vivo genome-editing strategies offer the potential for long-lasting, and possibly one-time, interventions. In parallel, microRNAs (miRNAs) have attracted increasing interest because of their ability to coordinately regulate multiple genes involved in lipoprotein metabolism, cholesterol transport, and lipid homeostasis. Human genetic studies further support the importance of miRNA-mediated regulation, exemplified by a rare ~2.5 kb deletion in the distal LDLR 3'UTR ("del2.5") that disrupts miRNA-binding sites and is associated with lifelong low LDL-C levels. This review summarizes recent advances, mechanisms of action, clinical progress, and remaining challenges across antisense oligonucleotides, siRNAs, gene therapy, genome editing, and emerging miRNA-based therapeutics for hypercholesterolemia. As an example of the latter approach, the liver-directed miR-30c analog C2 has demonstrated preclinical activity by coordinately reducing hepatic lipoprotein secretion and lipogenesis while enhancing cholesterol elimination, resulting in reduced LDL-C and atherosclerosis. However, it must be noted that these findings remain preclinical, and further optimization of delivery, pharmacokinetics, safety, and long-term efficacy will be required before clinical evaluation. Continued advances in RNA chemistry, targeted delivery, and genome engineering are expected to further expand the therapeutic landscape for dyslipidemia and cardiovascular disease.

Humans↗

PubMed2026

Base editing for precision therapeutics.

Base editing (BE), the precise installation of single-nucleotide changes in DNA or RNA without inducing double-strand breaks, holds substantial therapeutic promise for correcting single-nucleotide variants, which constitute more than half of the known pathogenic genetic variants. Recent advances have improved base editor specificity, efficiency, and delivery, enabling clinically oriented procedures. Clinically, BE has shown early success or strong translational promise in sickle cell disease, β-thalassemia, leukemia (via CAR T and epitope engineering), hypercholesterolemia (PCSK9 and ANGPTL3), alpha-1-antitrypsin deficiency, and glycogen storage disease type Ia. Key remaining challenges include bystander editing within the activity window, residual off-target DNA and RNA editing, delivery constraints (payload size, tissue targeting, and redosing limits), immunogenicity, and the need for durable long-term safety evidence across relevant cell types and disease contexts. Continued technological refinements, careful preclinical validation, and rigorous clinical assessment will be essential to fully realize BE's transformative potential in precision medicine.

Humans↗

PubMed2026

Closed-loop insulin delivery for glycaemic control in hospitalised and perioperative adults: A systematic review and meta-analysis of randomised controlled trials.

We evaluated whether closed-loop insulin delivery improves glycaemic control in hospitalised and perioperative adults. PubMed/MEDLINE, Embase, CENTRAL, and ClinicalTrials.gov were searched from inception to 29 June 2026 for randomised controlled trials comparing closed-loop or automated insulin delivery with usual care or conventional insulin therapy. Random-effects meta-analyses were conducted; risk of bias was assessed using RoB 2 and certainty of evidence using GRADE. Seven trials involving 375 analysed participants were included. Closed-loop insulin delivery increased time in target glucose range by 23.91 percentage points (95% CI 19.40 to 28.43; I2 = 0%) and reduced mean glucose by 1.79 mmol/L (95% CI 1.06 to 2.53 lower; I2 = 36.3%); certainty was moderate for both outcomes. Two trials involving 69 participants reported compatible participant-level data for clinically significant hyperglycaemia, and both estimates favoured closed-loop insulin delivery, although the evidence was exploratory and imprecise. No severe hypoglycaemic events occurred in either group, precluding reliable estimation of comparative safety. Closed-loop insulin delivery may improve glycaemic process measures, but larger pragmatic trials are needed to establish clinical benefits, safety, and implementation feasibility.

Humans↗

PubMed2026

MYC-bound enhancer RNAs in cis regulate gene transcription and tumorigenesis.

Emerging evidence suggests that MYC binds RNAs, but its functional consequences remain unclear. Here, we integrate multiomics data and reveal that MYC broadly binds enhancer RNAs (eRNAs), which exhibit high cancer- and tissue-specific expression in cancer cell lines and patient tumors. Moreover, we developed a computational pipeline to identify potential cis-regulatory MYC-eRNA target genes, with most predicted eRNA-target pairs supported by RNA polymerase II-mediated chromatin interaction data. Among these, we functionally characterized MERG1 as an oncogenic eRNA that promotes breast cancer tumorigenesis. Mechanistically, MERG1 interacts with MYC to enhance its occupancy at the GREB1 promoter, driving chromatin remodeling and epigenetic activation. This process specifically amplifies GREB1 expression and promotes tumor progression. Last, nanoparticle-mediated delivery of antisense oligonucleotides targeting MERG1 suppresses MYC-mediated breast cancer growth. These results advance our understanding of the enhancer-driven regulation of gene expression and tumorigenesis and provide insights into the regulatory landscape of MYC in cancer.

Humans↗

PubMed2008

Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.

Delivery of genes to the brain and spinal cord across the blood-brain barrier (BBB) has not yet been achieved. Here we show that adeno-associated virus (AAV) 9 injected intravenously bypasses the BBB and efficiently targets cells of the central nervous system (CNS). Injection of AAV9-GFP into neonatal mice through the facial vein results in extensive transduction of dorsal root ganglia and motor neurons throughout the spinal cord and widespread transduction of neurons throughout the brain, including the neocortex, hippocampus and cerebellum. In adult mice, tail vein injection of AAV9-GFP leads to robust transduction of astrocytes throughout the entire CNS, with limited neuronal transduction. This approach may enable the development of gene therapies for a range of neurodegenerative diseases, such as spinal muscular atrophy, through targeting of motor neurons, and amyotrophic lateral sclerosis, through targeting of astrocytes. It may also be useful for rapid postnatal genetic manipulations in basic neuroscience studies.

Animals↗

PubMed2026

CRISPR-Cas and Infectious Diseases: A Decade of Translational Advances in Molecular Biotechnology.

CRISPR-Cas systems have emerged as a versatile tool for diagnosing, treating, and preventing infectious diseases. This review highlights translational advancements in CRISPR-Cas-based applications, concentrating on the past decades in diagnostics, therapeutic genome editing, and vaccine development. The article highlights key platforms like DETECTR and SHERLOCK, which enable rapid, sensitive pathogen detection, and explores CRISPR-Cas9 systems in therapeutic strategies for directly targeting viral genomes and combating antimicrobial resistance. It also examines the role of CRISPR-Cas9 in engineering live-attenuated and personalized neoantigen vaccines. Principal findings demonstrate a clear progression from experimental proof-of-concept to preclinical applications primarily in CRISPR-based diagnostics and the engineering of live-attenuated vaccine candidates, whereas translation in CRISPR-based therapeutics and personalized neoantigen vaccines for infectious diseases remains at earlier, more exploratory stages. CRISPR-based diagnostics have progressed further toward clinical evaluation than therapeutics due to delivery and safety constraints, while personalized neoantigen vaccines are included mainly as an emerging, comparative concept for infectious diseases rather than a mature application. This review uniquely integrates CRISPR-based diagnostics, therapeutics, and vaccine development within a single infectious disease framework, critically assesses their current maturity, and systematically highlights technical, regulatory, and ethical barriers alongside realistic future priorities. The review concludes that while CRISPR-Cas holds transformative potential for infectious disease management, significant challenges in delivery efficiency, off-target effects, and ethical regulation must be addressed to ensure safe and equitable clinical translation.

Humans↗

PubMed2026

Ligand-Mediated Reprogramming Redirects Liver-Tropic Ionizable Lipid Nanoparticles for Lung-Selective mRNA Delivery.

Systemic delivery of messenger RNA (mRNA) to target tissues and cells using lipid nanoparticles (LNPs) holds transformative potential for gene therapy. However, most clinically validated LNP exhibit strong liver tropism, and redirecting their organ specificity without redesigning entirely new chemistries remains challenging. Here we present a ligand-mediated lipid reprogramming approach that repurposes chemically defined, liver-tropic, ionizable lipids (lipidoids) for mRNA delivery beyond the liver. From a library of 90 degradable lipidoids, we identified 2-t6b as a potent liver-targeting platform. By site-specific displaying of small molecule ligands onto 2-t6b headgroup, we engineered a series of reconfigured lipidoids that achieve lung-specific targeting while retaining the parent delivery scaffold. Ligand7-2-t6b-lipid-functionalized LNP achieved over 200-fold higher mRNA translation in the lungs compared to the parent liver-tropic LNP. Proteomics and molecular docking analysis revealed enhanced binding of the modified lipid to vitronectin, a serum glycoprotein that improves integrin binding and thus promotes cellular uptake and translation efficiency. Ligand-mediated 2-t6b/ligand7 LNPs achieved outperformed efficacy and therapeutic potential in lung-specific genome editing relative to SORT-constructed 2-t6b LNP system. Our modular reprogramming strategy provides a generalizable framework to upgrade existing liver-biased LNPs into lung-selective mRNA carriers, advancing next-generation tissue-specific mRNA therapies for gene editing, protein replacement therapy, and regenerative medicine.

RNA, Messenger↗

PubMed2026

Crown ethers as artificial decoys: A supramolecular strategy to block SARS-CoV-2 entry via host-guest interactions.

Coronavirus disease (COVID-19) remains a major global health challenge, highlighting the need for antiviral strategies that act at the earliest stages of infection. Given that viral entry and spike-receptor interaction are critical steps in the coronavirus life cycle, targeting these processes represents a powerful strategy to block infection at its earliest stage. Inspired by the glycan-recognition and extracellular viral-trapping functions of pulmonary surfactant collectins (SP-A and SP-D), this work integrates supramolecular chemistry, pulmonary surfactant biology, and antiviral research to establish a biomimetic supramolecular molecular-decoy framework based on crown ethers, cyclodextrins, and related macrocyclic architectures. Through host-guest molecular recognition, these macrocyclic scaffolds can be engineered to mimic sialylated host receptors and multivalent glycan motifs, enabling competitive binding to viral spike proteins, virion capture, and sequestration away from epithelial surfaces. By redirecting viruses toward artificial host-mimetic structures, supramolecular decoys could intercept SARS-CoV-2 and other enveloped respiratory viruses before host-cell attachment, membrane fusion, or genome release. Acting upstream of intracellular replication, this strategy may prevent initiation of the viral replication cycle and subsequent hijacking of the host protein synthesis machinery, while potentially minimizing interference with host metabolic pathways and reducing the likelihood of resistance development. Furthermore, it can be translated into inhalation nanoformulations for pulmonary delivery and localized formulations targeting the upper respiratory tract. Overall, by integrating the biological principles of pulmonary surfactant immunity with supramolecular host-guest chemistry, this work provides a conceptual foundation for biomimetic molecular-decoy antivirals and highlights a promising direction for next-generation broad-spectrum antiviral design against emerging respiratory viruses.

Antiviral Agents↗

PubMed2023

Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models.

Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.

Humans↗

PubMed2026

Targeting of the oncogenic fusion EWSR1-FLI1 in Ewing sarcoma by CRISPR/dCas9 silencers.

Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle to clinical applications. Here, we utilize precision molecular targeting and delivery strategies based on CRISPR-nuclease-dead Cas9 (dCas9) systems adapted for epigenetic repression (dCas9-Krüppel-associated box [KRAB]) to silence oncogenic drivers with high selectivity. As proof of principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing sarcoma (EWS)-an aggressive childhood malignancy. We describe the development of a programmable, non-viral polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in EWS-related patient-derived xenografts (PDXs) of EWS. We show that silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. Collectively, we characterize an effective non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which could be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.

CRISPR-dCas9↗

PubMed2026

Bifunctional covalent organic framework for rapid isolation of extracellular vesicles and proteomics-based biomarker discovery.

Extracellular vesicles (EVs), serving as crucial carriers of biomarkers for tumor diagnosis and prognostic evaluation, as well as drug delivery vehicles and therapeutic targets, making it a research hotspot. The isolation methods represent a key aspect of EV-associated research. In this work, an alkynyl-functionalized covalent organic framework (COF) was synthesized under acidic conditions at room temperature and further modified by photo-initiated thiol-yne click reaction, yielding a bifunctionalized COF material decorated with distearoyl phosphatidylethanolamine (DSPE) and Ti4+. This bifunctional COF material (COF-DSPE-Ti) can leverage the bifunctional synergistic effect between DSPE and Ti4+ sites, thereby facilitating the efficient isolation of EVs. This synergistic effect enables the efficient isolation of EVs within 3 min. Proteomic analysis reveals that this isolation method significantly outperforms ultracentrifugation, and an effective EV isolation and analysis can be completed using only 10 μL of plasma sample. For clinical liquid biopsy, the integration of the COF-DSPE-Ti method with proteomics lead to the identification of 64 upregulated proteins in plasma samples from colorectal cancer (CRC) patients, among which S100A9 emerged as a potential EV biomarker. In addition, KLK2, KLK3, and FOLH1, which have been established as diagnostic markers for prostate cancer (PCa), are successfully identified in EVs isolated from the urine of PCa patients. These findings demonstrate the reliability of this approach for screening EV-associated biomarkers and provide a novel strategy for the early diagnosis and prognostic assessment of CRC and PCa.

Proteomics↗

PubMed2026

[Research Advances on Mechanisms and Interventions of DNA Methylation-Regulated Aging-Related Imbalance in Bone Metabolism].

Aging can induce age-related bone diseases such as osteoporosis. DNA methylation, a core epigenetic regulatory mechanism, participate in the pathological process of aging-induced bone metabolism imbalance by modulating gene expression at the epigenetic level. Using S-adenosylmethionine as a methyl donor, it exhibits characteristics of hypomethylation in genomic repetitive regions and abnormal methylation in CpG islands of promoters of key bone metabolism genes with advancing age. The "epigenetic clock" constructed based on these features can accurately predict an individual's biological age. In bone metabolism, DNA methylation disrupts the osteoblast-osteoclast balance by targeting key factors. Such abnormalities are driven by aging-related inflammation and oxidative stress, while bone loss feedback exacerbates epigenetic disorders, forming a vicious cycle. Targeted intervention strategies have demonstrated significant potential in addressing bone metabolism-related issues. Low-dose DNA methyltransferase inhibitors can improve bone metabolism; nutrients such as folate and cobalamin maintain methylation homeostasis by optimizing one-carbon metabolism pathways; while CRISPR/dCas technology enables precise regulation in the cellular and animal levels, thereby affecting bone metabolism. However, existing strategies still face challenges such as off-target effects and low delivery efficiency. Future research needs to deepen mechanistic studies, optimize intervention methods, and promote their translation into clinical prevention and treatment of osteoporosis.

DNA Methylation↗

PubMed2009

A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection.

To engineer gene vectors that target striated muscles after systemic delivery, we constructed a random library of adeno-associated virus (AAV) by shuffling the capsid genes of AAV serotypes 1 to 9, and screened for muscle-targeting capsids by direct in vivo panning after tail vein injection in mice. After 2 rounds of in vivo selection, a capsid gene named M41 was retrieved mainly based on its high frequency in the muscle and low frequency in the liver. Structural analyses revealed that the AAVM41 capsid is a recombinant of AAV1, 6, 7, and 8 with a mosaic capsid surface and a conserved capsid interior. AAVM41 was then subjected to a side-by-side comparison to AAV9, the most robust AAV for systemic heart and muscle gene delivery; to AAV6, a parental AAV with strong muscle tropism. After i.v. delivery of reporter genes, AAVM41 was found more efficient than AAV6 in the heart and muscle, and was similar to AAV9 in the heart but weaker in the muscle. In fact, the myocardium showed the highest gene expression among all tissues tested in mice and hamsters after systemic AAVM41 delivery. However, gene transfer in non-muscle tissues, mainly the liver, was dramatically reduced. AAVM41 was further tested in a genetic cardiomyopathy hamster model and achieved efficient long-term delta-sarcoglycan gene expression and rescue of cardiac functions. Thus, direct in vivo panning of capsid libraries is a simple tool for the de-targeting and retargeting of viral vector tissue tropisms facilitated by acquisition of desirable sequences and properties.

Animals↗

PubMed2026

A compact GAD67 promoter enables inhibitory neuron-targeted AAV gene therapy for seizure suppression.

Epilepsy arises from disruption of excitation-inhibition (E/I) balance, typically due to excessive excitatory activity. Despite available therapies, a substantial proportion of patients remain treatment resistant. Enhancing inhibitory neuron activity via gene therapy can restore E/I balance and may therefore provide a therapeutic strategy for treatment-resistant epilepsy. Here, we developed a compact 410-bp glutamic acid decarboxylase 67 promoter (cmGAD67) that enables strong, selective transgene expression in inhibitory neurons while preserving adeno-associated virus (AAV) packaging capacity. Systemic delivery of AAV vectors carrying cmGAD67 preferentially targeted parvalbumin interneurons and enabled efficient circuit modulation. To evaluate therapeutic potential, we expressed glutamic acid decarboxylase 65 (GAD65) under the control of cmGAD67 (AAV-GAD65). AAV-GAD65 suppressed abnormal delta oscillations, reduced seizure-like activity, normalized anxiety-like behavior, and improved survival in seizure models. Biochemical analyses confirmed increased GABA levels in the cortex and hippocampus, linking functional improvements to enhanced inhibitory neurotransmitter synthesis. Together, these findings establish the cmGAD67 promoter as a versatile platform for inhibitory neuron-targeted AAV gene delivery and identify AAV-GAD65 as a promising strategy for seizure control and disorders associated with E/I imbalance.

Glutamate Decarboxylase↗

PubMed2026

Efficient prime editing in vivo and in vitro using lipid nanoparticles.

Prime editing is a versatile clinical genome editing method that enables precise substitutions, small insertions and deletions at specified locations in the genomes of living systems including human cells. Although non-viral lipid nanoparticle (LNP) delivery of RNA in vivo has become a preferred method for gene editing in animals and patients, its application to complex, three-component prime editing systems has yielded low editing efficiencies. Here we developed a systematic prime editing LNP (PE-LNP) optimization platform that addresses key bottlenecks in cargo design that limit editing efficiency. This generalizable workflow yielded PE-LNPs that can achieve 49% average in vivo prime editing in the bulk mouse liver with a single dose of 2 mg kg-1. We applied our workflow to the correction of PAH R408W, a cause of phenylketonuria, in a mouse model and achieved prime editing efficiencies and serum phenylalanine levels anticipated to be curative. We also show that PE-LNPs minimize off-target editing compared with DNA delivery methods, induce only transient elevation of liver enzymes and can be dosed repeatedly to improve editing efficiencies. These PE-LNP systems provide an attractive alternative to viral delivery by offering transient expression that minimizes off-target editing, no observed long-term toxicity and high levels of non-viral in vivo liver prime editing.

Animals↗