Insulin resistance is a hallmark of diabetes and an unmet clinical need. Insulin inhibits hepatic glucose production and promotes lipogenesis by suppressing FOXO1-dependent activation of G6pase and ...inhibition of glucokinase, respectively. The tight coupling of these events poses a dual conundrum: mechanistically, as the FOXO1 corepressor of glucokinase is unknown, and clinically, as inhibition of glucose production is predicted to increase lipogenesis. Here, we report that SIN3A is the insulin-sensitive FOXO1 corepressor of glucokinase. Genetic ablation of SIN3A abolishes nutrient regulation of glucokinase without affecting other FOXO1 target genes and lowers glycemia without concurrent steatosis. To extend this work, we executed a small-molecule screen and discovered selective inhibitors of FOXO-dependent glucose production devoid of lipogenic activity in hepatocytes. In addition to identifying a novel mode of insulin action, these data raise the possibility of developing selective modulators of unliganded transcription factors to dial out adverse effects of insulin sensitizers.
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•Discovery of SIN3a as the FOXO corepressor of hepatic glucokinase•SIN3a regulates hepatic insulin sensitivity•Corepressor clearance as a novel mechanism of gene induction by insulin•Selective targeting of the activator and repressor functions of FOXO1
The transcriptional output of FOXO1 can be selectively modulated in a way that might reduce adverse effects of insulin sensitizers.
Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), ...which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles.
To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified.
Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.
Abstract
Streptococcus pyogenes
Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal ...rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized
PCSK9
mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from
Parasutterella secunda
(PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the
PCSK9
site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.
α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In ...humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene edited PiZ mice. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.
•α1-antitrypsin (AAT) is a circulating protein secreted from the liver and important in preventing tissue damage in lungs.•We used CRISPR/Cas9 to disrupt the gene of a mutant version of the protein to reverse liver pathology in a mouse model of human AAT deficiency (AATD)•Our gene editing approach reverted the AATD pathology and genomic analysis confirmed significant disruption to the gene.
In an attempt to treat a pathologic liver disease called α1-antitrypsin deficiency (AATD) in a mouse model of the human disease, we used CRISPR/Cas9 technology to remove a mutant form of hSERPINA1, which causes AATD in the mouse. Our gene editing approach reverted the disease associated with the mutated gene and we saw a reversal in liver fibrosis and mutant protein aggregation. Our findings in a mouse model indicate that therapeutic gene removal, by editing out a mutated form of the gene, in liver cells is possible.
We present a methodology for gene functional prediction based on extraction of physiologically relevant growth variables from all viable haploid yeast knockout mutants. This quantitative phenomics ...approach, here applied to saline cultivation, identified marginal but functionally important phenotypes and allowed the precise determination of time to adapt to an environmental challenge, rate of growth, and efficiency of growth. We identified ≈500 salt-sensitive gene deletions, the majority of which were previously uncharacterized and displayed salt sensitivity for only one of the three physiological features. We also report a high correlation to protein-protein interaction data; in particular, several salt-sensitive subcellular networks indicating functional modules were revealed. In contrast, no correlation was found between gene dispensability and gene expression. It is proposed that high-resolution phenomics will be instrumental in systemwide descriptions of intragenomic functional networks.
To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a ...model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes.
Apolipoprotein L1 (APOL1) high-risk genotypes are associated with increased risk of chronic kidney disease (CKD) in people of West African ancestry. Given the importance of endothelial cells (ECs) in ...CKD, we hypothesized that APOL1 high-risk genotypes may contribute to disease via EC-intrinsic activation and dysfunction.
Single cell RNA sequencing (scRNA-seq) analysis of the Kidney Precision Medicine Project dataset revealed APOL1 expression in ECs from various renal vascular compartments. Utilizing two public transcriptomic datasets of kidney tissue from African Americans with CKD and a dataset of APOL1-expressing transgenic mice, we identified an EC activation signature; specifically, increased intercellular adhesion molecule 1 (ICAM-1) expression and enrichment in leukocyte migration pathways. In vitro, APOL1 expression in ECs derived from genetically modified human induced pluripotent stem cells and glomerular ECs triggered changes in ICAM-1 and platelet endothelial cell adhesion molecule 1 (PECAM-1) leading to an increase in monocyte attachment. Overall, our data suggest the involvement of APOL1 as an inducer of EC activation in multiple renal vascular beds with potential effects beyond the glomerular vasculature.
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•APOL1 is expressed in multiple renal vascular beds•APOL1 is an inducer of endothelial cell (EC) activation in humans•APOL1 RV expression in ECs induces ICAM1 and reduces PECAM1•APOL1 RV expression in ECs promotes monocyte attachment to ECs
Nephrology
In nonalcoholic fatty liver disease (NAFLD) the patatin‐like phospholipase domain‐containing 3 (PNPLA3) rs738409 variant is a contributor. In mice, the Pnpla3 148M variant accumulates on lipid ...droplets and probably leads to sequestration of a lipase cofactor leading to impaired mobilization of triglycerides. To advance our understanding of the localization and abundance of PNPLA3 protein in humans, we used liver biopsies from patients with NAFLD to investigate the link to NAFLD and the PNPLA3 148M genotype. We experimentally qualified an antibody against human PNPLA3. Hepatic PNPLA3 protein fractional area and localization were determined by immunohistochemistry in biopsies from a well‐characterized NAFLD cohort of 67 patients. Potential differences in hepatic PNPLA3 protein levels among patients related to degree of steatosis, lobular inflammation, ballooning, and fibrosis, and PNPLA3 I148M gene variants were assessed. Immunohistochemistry staining in biopsies from patients with NAFLD showed that hepatic PNPLA3 protein was predominantly localized to the membranes of small and large lipid droplets in hepatocytes. PNPLA3 protein levels correlated strongly with steatosis grade (p = 0.000027) and were also significantly higher in patients with lobular inflammation (p = 0.009), ballooning (p = 0.022), and significant fibrosis (stage 2–4, p = 0.014). In addition, PNPLA3 levels were higher in PNPLA3 rs738409 148M (CG, GG) risk allele carriers compared to 148I (CC) nonrisk allele carriers (p = 0.0029). Conclusion: PNPLA3 protein levels were associated with increased hepatic lipid content and disease severity in patients with NAFLD and were higher in PNPLA3 rs738409 (148M) risk allele carriers. Our hypothesis that increased hepatic levels of PNPLA3 may be part of the pathophysiological mechanism of NAFLD is supported.
A specific variant in the PNPLA3 gene is a known genetic risk factor for NAFLD. We validated an antibody recognizing the PNPLA3 protein and used it to show that the PNPLA3 protein is primarily located on hepatic lipid droplets in human liver samples from patients with NAFLD. We also showed that PNPLA3 protein levels are associated with NAFLD and that carriers of the specific PNPLA3 gene variant had higher hepatic levels of the protein compared to subjects without the mutation. The hypothesis that increased hepatic PNPLA3 protein levels in subjects carrying the mutation contributes to their increased risk of NAFLD is supported.
In this chapter, we describe a series of genome-wide, cell-based assays that provide a solid basis for understanding drug-gene interactions, gene function, and for defining the mechanism of action of ...small molecules. Each of these assays takes advantage of the ability to grow complex pools competitively and to use high-density microarrays that report the results of such screens. The assays described here take advantage of alterations in gene dosage of Saccharomyces cerevisiae, and include HIP (haploinsufficiency profiling), HOP (homozygous profiling), and MSP (multicopy suppression profiling) as genetic tools to understand gene function and drug mechanism. The common experimental theme is that, in each assay, strains are pooled and screened in parallel to investigate the relative contribution of each gene product to sensitivity or resistance to a drug or environmental perturbation across the genome in a single assay. Further, the compendium of results from these screens can inform large-scale network analysis of genetic function, gene-gene interactions, and mechanism of drug action.
Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo1,2-apyridines and imidazo1,2-apyrimidines compose a class of compounds that ...target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo1,2-apyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo1,2-apyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo1,2-apyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical-genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure-activity relationships.
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