Haojie Yu

Prime editing enables the precise modification of genomes, thereby holding great potential for the treatment of genetic diseases. Despite substantial advancements in prime editing technology and the initiation of the first clinical trial for treating chronic granulomatous disease, further enhancement of editing efficiency across edit types is still urgently needed. Here, we developed a compact prime editor, PE2ΔR, by deleting the RNase H domain of the MMLV reverse transcriptase (MMLV-RT). We then conducted a saturated mutagenesis screen targeting two DNA interacting regions within the PE2ΔR-RT Fingers domain. By integrating three highly effective mutations (I61R, V101R, S67W) into PEmax lacking RNase H domain (termed PEmaxΔRM3), we achieved up to a 90% increase in editing efficiency across editing types compared to PEmax. Structural modelling using AlphaFold 3 suggests that these mutations enhance primer–template stabilization and guide the RNA/DNA hybrid into a catalytically favourable trajectory, providing a mechanistic explanation for the enhanced activity. Taken together, our study demonstrates proof-of-concept for the application of unbiased mutagenesis screen to identify novel mutations that enhance prime editor performance. Furthermore, we discovered that RT variants (I61R, V101R, S67W) synergize with PEmax and epegRNA to improve prime editing efficiency across edit types, with the strongest improvement observed in introducing small deletions.

Cardiovascular disease (CVD) remains the leading cause of global mortality, prompting urgent efforts to explore novel therapeutic approaches. The remarkable success of mRNA technology brings new possibilities for CVD treatment, with numerous preclinical studies establishing proof-of-concept, and several have advanced into clinical trials. Leveraging cumulative experience in mRNA therapeutics, improved understanding of cardiovascular pathophysiology and developments in nanotechnology, genome editing and RNA synthetic biology, mRNA medicine presents promising opportunities for addressing CVDs. Here, we provide an overview of the advancements of mRNA-based biotechnologies for CVDs, including mRNA modifications, mRNA delivery platforms, mRNA-encoded genomic and epigenomic editing, and mRNA-based chimeric antigen receptors for immune cell engineering. We also summarize preclinical and clinical applications of mRNA medicine in various CVDs, including hypercholesterolemia, atherosclerosis, ischemic cardiac injury, cardiac fibrosis and cardiac amyloidosis. Finally, we discuss the challenges and highlight the future directions for bench-to-bedside translation of mRNA technology in the field of CVDs.

The limited therapeutic options for metabolic dysfunction-associated steatotic liver disease (MASLD) underscore the need for deeper mechanistic insight and new treatment strategies. Here, we identify the orphan G protein-coupled receptor GPR146 as a regulator of hepatic steatosis through adipose–liver crosstalk. Human genetic analyses link the GPR146 locus to circulating markers of liver injury and inflammation. In mice, both constitutive and acute GPR146 depletion protect against diet-induced obesity and hepatic steatosis. Notably, adipose-specific, but not liver-specific, GPR146 deletion reduces hepatic lipid accumulation by limiting free fatty acid (FFA) influx. Mechanistically, GPR146 promotes adipogenesis in preadipocytes via Gαq-PKC-AKT signaling, increasing lipid storage capacity, and enhances lipolysis in mature adipocytes through ERK activation, elevating circulating FFA. Together, these coordinated actions increase FFA delivery to the liver, promoting triglyceride accumulation. Our findings establish GPR146 as a pleiotropic regulator of adipose tissue biology and a potential therapeutic target for MASLD.

The application of messenger RNA (mRNA) beyond infectious diseases is challenged by inefficient protein production. Whereas the engineering of secondary mRNA structures has been shown to increase mRNA half-life, it remains unclear whether tertiary mRNA structures influence therapeutic efficacy. Here we develop a metal-ion-assisted RNA folding (MARF) strategy and show that, when delivered with lipid nanoparticles (LNPs), specific metals promote mRNA folding architectures that result in the amplification of protein expression by up to 7.3-fold compared with control mRNA. This effect is due to altered mechanical interactions between the mRNA LNPs and the surrounding biosystem, resulting in enhanced intracellular processing and prolonged retention of delivered mRNA in targeted cells. Administered intravenously, MARF LNPs achieved effective and durable genome editing of the clinically relevant Pcsk9 gene through treatment with a single dose. Overall, this work provides a new MARF technology for more effective mRNA therapy and highlights the potential of mechanical cues in designing nanoparticles for improved mRNA delivery.

Several vascular diseases including coronary artery disease, hypertension, stroke, and abdominal aortic aneurysm, have significant genetic underpinnings. Genome-wide association studies have unveiled many genetic loci associated with one or more of these diseases. However, the causative genes at most of these loci are yet to be determined, which hampers the translation of the genetic findings into a better understanding of the disease mechanisms and the identification of new therapeutic targets. Here, in an integrative functional genomics analysis of these loci, we identify a panel of likely causal genes, some of which are pleiotropic for more than one of these vascular diseases. Pooled CRISPR knockout screen analyses of these likely causal genes indicate that many of them influence vascular smooth muscle cell behaviour, and validation experiments of selected genes confirm that FES , BCAR1, CARF and SMARCA4 exert such effects. Further functional experiments focusing on FES , a pleiotropic gene for both coronary artery disease and hypertension, show that it modulates the expression of genes involved in vascular remodeling and that Fes knockout in mice promotes atherosclerosis as well as raises blood pressure. These findings provide an insight into the genetic basis of vascular diseases and inform targets for therapeutic development.

Lipolysis of triglyceride‐rich lipoproteins by peripheral lipoprotein lipase (LPL) plays an essential role in maintaining systemic cholesterol/lipid homeostasis. Human genetic studies have unequivocally demonstrated that activation of LPL pathway reduces risks for both coronary artery disease (CAD) and type 2 diabetes (T2D). Although sterol regulatory element‐binding protein 2 (SREBP2) is well established as the master transcription factor that regulates the hepatic biosynthesis of both cholesterol and fatty acids, whether and how its activity in liver interacts with peripheral LPL pathway remains unknown. Here, it is demonstrated that acute liver‐specific depletion of SREBP2 results in divergent effects on the regulation of peripheral LPL activity in mice, depending on the presence or absence of low‐density lipoprotein receptors (LDLR). SREBP2 deficiency drastically elevates peripheral LPL activity through downregulation of plasma angiopoietin‐related protein 3 (ANGPTL3) levels in LDLR‐deficient mice. Moreover, in addition to SREBP2's transcriptional regulation of ANGPTL3, it is found that SREBP2 promotes proteasome‐based degradation of ANGPTL3 in the presence of LDLR. Remarkably, acute depletion of hepatic SREBP2 protects against hypercholesterolemia and atherosclerosis, in which atherosclerotic lesions are reduced by 45% compared to control littermates. Taken together, these findings outline a liver‐peripheral crosstalk mediated by SREBP2‐ANGPTL3‐LPL axis and suggest that SREBP2 inhibition can be an effective strategy to tackle homozygous familial hypercholesterolemia (HoFH).

Biallelic mutations of the chromatin regulator SMARCAL1 cause Schimke Immunoosseous Dysplasia (SIOD), characterized by severe growth defects and premature mortality. Atherosclerosis and hyperlipidemia are common among SIOD patients, yet their onset and progression are poorly understood. Using an integrative approach involving proteomics, mouse models, and population genetics, we investigated SMARCAL1’s role. We found that SmarcAL1 interacts with angiopoietin-like 3 (Angptl3), a key regulator of lipoprotein metabolism. In vitro and in vivo analyses demonstrate SmarcAL1’s vital role in maintaining cellular lipid homeostasis. The observed translocation of SmarcAL1 to cytoplasmic peroxisomes suggests a potential regulatory role in lipid metabolism through gene expression. SmarcAL1 gene inactivation reduces the expression of key genes in cellular lipid catabolism. Population genetics investigations highlight significant associations between SMARCAL1 genetic variations and body mass index, along with lipid-related traits. This study underscores SMARCAL1’s pivotal role in cellular lipid metabolism, likely contributing to the observed lipid phenotypes in SIOD patients.

Summary: Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we have substantially improved the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function of OTX2 leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.

Background: Genome-wide association studies have discovered a link between genetic variants on human chromosome 15q26.1 and increased coronary artery disease (CAD) susceptibility; however, the underlying pathobiological mechanism is unclear. This genetic locus contains the FES (FES proto-oncogene, tyrosine kinase) gene encoding a cytoplasmic protein-tyrosine kinase involved in the regulation of cell behavior. We investigated the effect of the 15q26.1 variants on FES expression and whether FES plays a role in atherosclerosis. Methods and Results: Analyses of isogenic monocytic cell lines generated by CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing showed that monocytes with an engineered 15q26.1 CAD risk genotype had reduced FES expression. Small-interfering-RNA-mediated knockdown of FES promoted migration of monocytes and vascular smooth muscle cells. A phosphoproteomics analysis showed that FES knockdown altered phosphorylation of a number of proteins known to regulate cell migration. Single-cell RNA-sequencing revealed that in human atherosclerotic plaques, cells that expressed FES were predominately monocytes/macrophages, although several other cell types including smooth muscle cells also expressed FES . There was an association between the 15q26.1 CAD risk genotype and greater numbers of monocytes/macrophage in human atherosclerotic plaques. An animal model study demonstrated that Fes knockout increased atherosclerotic plaque size and within-plaque content of monocytes/macrophages and smooth muscle cells, in apolipoprotein E-deficient mice fed a high fat diet. Conclusions: We provide substantial evidence that the CAD risk variants at the 15q26.1 locus reduce FES expression in monocytes and that FES depletion results in larger atherosclerotic plaques with more monocytes/macrophages and smooth muscle cells. This study is the first demonstration that FES plays a protective role against atherosclerosis and suggests that enhancing FES activity could be a potentially novel therapeutic approach for CAD intervention.