Durable and efficient gene silencing in vivo by hit-and-run epigenome editing.

Permanent epigenetic silencing using programmable editors equipped with transcriptional repressors holds great promise for the treatment of human diseases1-3. However, to unlock its full therapeutic potential, an experimental confirmation of durable epigenetic silencing after the delivery of transient delivery of editors in vivo is needed. To this end, here we targeted Pcsk9, a gene expressed in hepatocytes that is involved in cholesterol homeostasis. In vitro screening of different editor designs indicated that zinc-finger proteins were the best-performing DNA-binding platform for efficient silencing of mouse Pcsk9. A single administration of lipid nanoparticles loaded with the editors' mRNAs almost halved the circulating levels of PCSK9 for nearly one year in mice. Notably, Pcsk9 silencing and accompanying epigenetic repressive marks also persisted after forced liver regeneration, further corroborating the heritability of the newly installed epigenetic state. Improvements in construct design resulted in the development of an all-in-one configuration that we term evolved engineered transcriptional repressor (EvoETR). This design, which is characterized by a high specificity profile, further reduced the circulating levels of PCSK9 in mice with an efficiency comparable with that obtained through conventional gene editing, but without causing DNA breaks. Our study lays the foundation for the development of in vivo therapeutics that are based on epigenetic silencing.

In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.

Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.

CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.

Exacerbated atherosclerosis in progeria is prevented by progerin elimination in vascular smooth muscle cells but not endothelial cells.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here, we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice.

Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq.

Discovery of off-target CRISPR-Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR-Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.

The mutational constraint spectrum quantified from variation in 141,456 humans.

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes1. Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.

Inhibition of PCSK9 potentiates immune checkpoint therapy for cancer.

Despite its success in achieving the long-term survival of 10-30% of treated individuals, immune therapy is still ineffective for most patients with cancer1,2. Many efforts are therefore underway to identify new approaches that enhance such immune 'checkpoint' therapy3-5 (so called because its aim is to block proteins that inhibit checkpoint signalling pathways in T cells, thereby freeing those immune cells to target cancer cells). Here we show that inhibiting PCSK9-a key protein in the regulation of cholesterol metabolism6-8-can boost the response of tumours to immune checkpoint therapy, through a mechanism that is independent of PCSK9's cholesterol-regulating functions. Deleting the PCSK9 gene in mouse cancer cells substantially attenuates or prevents their growth in mice in a manner that depends on cytotoxic T cells. It also enhances the efficacy of immune therapy that is targeted at the checkpoint protein PD1. Furthermore, clinically approved PCSK9-neutralizing antibodies synergize with anti-PD1 therapy in suppressing tumour growth in mouse models of cancer. Inhibiting PCSK9-either through genetic deletion or using PCSK9 antibodies-increases the expression of major histocompatibility protein class I (MHC I) proteins on the tumour cell surface, promoting robust intratumoral infiltration of cytotoxic T cells. Mechanistically, we find that PCSK9 can disrupt the recycling of MHC I to the cell surface by associating with it physically and promoting its relocation and degradation in the lysosome. Together, these results suggest that inhibiting PCSK9 is a promising way to enhance immune checkpoint therapy for cancer.

Accelerated discovery and miniaturization of novel single-stranded cytidine deaminases.

Cytidine base editors (CBEs) hold significant potential in genetic disease treatment and in breeding superior traits into animals. However, their large protein sizes limit their delivery by adeno-associated virus (AAV), given its packing capacity of <4.7 kb. To overcome this, we employed a web-based fast generic discovery (WFG) strategy, identifying several small ssDNA deaminases (Sdds) and constructing multiple Sdd-CBE 1.0 versions. SflSdd-CBE 1.0 demonstrated high C-to-T editing efficiency, comparable to AncBE4max, while SviSdd-CBE 1.0 exhibited moderate C-to-T editing efficiency with a narrow editing window (C3 to C5). Utilizing AlphaFold2, we devised a one-step miniaturization strategy, reducing the size of Sdds while preserving their efficiency. Notably, we administered AAV8 expressing PCSK9 targeted sgRNA and SflSdd-CBEs (nSaCas9) 2.0 into mice, leading to gene-editing events (with editing efficiency up to 15%) and reduced serum cholesterol levels, underscoring the potential of Sdds in gene therapy. These findings offer new single-stranded editing tools for the treatment of rare genetic diseases.

Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications.

The precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here, we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a). We engineer the nuclease (termed AiEvo2) for increased specificity, protospacer adjacent motif recognition, and efficacy on a variety of human clinical targets. AiEvo2 is highly precise and able to efficiently discriminate between normal and disease-causing alleles in Huntington's patient-derived cells by taking advantage of a single nucleotide polymorphism on the disease-associated allele. AiEvo2 efficiently edits several liver-associated target genes including PCSK9 and TTR when delivered to primary hepatocytes as mRNA encapsulated in a lipid nanoparticle. The enzyme also engineers an effective CD19 chimeric antigen receptor-T-cell therapy from primary human T cells using multiplexed simultaneous editing and chimeric antigen receptor insertion. To further ensure precise editing, we engineered an anti-CRISPR protein to selectively inhibit off-target gene editing while retaining therapeutic on-target editing. The engineered AiEvo2 nuclease coupled with a novel engineered anti-CRISPR protein represents a new way to control the fidelity of editing and improve the safety and efficacy of gene editing therapies.

Association of lipid-lowering drugs with risk of sarcopenia: a drug target mendelian randomization study and meta-analysis.

BACKGROUND: Lipid-lowering drugs are widely used among the elderly, with some studies suggesting links to muscle-related symptoms. However, the causality remains uncertain. METHODS: Using the Mendelian randomization (MR) approach, we assessed the causal effects of genetically proxied reduced low-density lipoprotein cholesterol (LDL-C) through inhibitions of hydroxy-methyl-glutaryl-CoA reductase (HMGCR), proprotein convertase subtilisin/kexin type 9 (PCSK9), and Niemann-Pick C1-like 1 (NPC1L1) on sarcopenia-related traits, including low hand grip strength, appendicular lean mass, and usual walking pace. A meta-analysis was conducted to combine the causal estimates from different consortiums. RESULTS: Using LDL-C pooled data predominantly from UK Biobank, genetically proxied inhibition of HMGCR was associated with higher appendicular lean mass (beta = 0.087, P = 7.56 × 10- 5) and slower walking pace (OR = 0.918, P = 6.06 × 10- 9). In contrast, inhibition of PCSK9 may reduce appendicular lean mass (beta = -0.050, P = 1.40 × 10- 3), while inhibition of NPC1L1 showed no causal impact on sarcopenia-related traits. These results were validated using LDL-C data from Global Lipids Genetics Consortium, indicating that HMGCR inhibition may increase appendicular lean mass (beta = 0.066, P = 2.17 × 10- 3) and decelerate walking pace (OR = 0.932, P = 1.43 × 10- 6), whereas PCSK9 inhibition could decrease appendicular lean mass (beta = -0.048, P = 1.69 × 10- 6). Meta-analysis further supported the robustness of these causal associations. CONCLUSIONS: Genetically proxied HMGCR inhibition may increase muscle mass but compromise muscle function, PCSK9 inhibition could result in reduced muscle mass, while NPC1L1 inhibition is not associated with sarcopenia-related traits and this class of drugs may serve as viable alternatives to sarcopenia individuals or those at an elevated risk.

PCSK9 dysregulates cholesterol homeostasis and triglyceride metabolism in olanzapine-induced hepatic steatosis via both receptor-dependent and receptor-independent pathways.

Schizophrenia, affecting approximately 1% of the global population, is often treated with olanzapine. Despite its efficacy, olanzapine's prolonged use has been associated with an increased risk of cardiovascular diseases and nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanism remains unclear. Proprotein convertase subtilisin kexin type 9 (PCSK9) plays a crucial role in lipid metabolism and is involved in NAFLD pathogenesis via an unknown mechanism. This study aims to investigate the role of PCSK9 in olanzapine-induced NAFLD. C57BL/6J mice and HepG2 and AML12 cell lines were treated with varying concentrations of olanzapine to examine the effects of olanzapine on PCSK9 and lipid metabolism. PCSK9 levels were manipulated using recombinant proteins, plasmids, and small interfering RNAs in vitro, and the effects on hepatic lipid accumulation and gene expression related to lipid metabolism were assessed. Olanzapine treatment significantly increased PCSK9 levels in both animal and cell line models, correlating with elevated lipid accumulation. PCSK9 manipulation demonstrated its central role in mediating hepatic steatosis through both receptor-dependent pathways (impacting NPC1L1) and receptor-independent pathways (affecting lipid synthesis, uptake, and cholesterol biosynthesis). Interestingly, upregulation of SREBP-1c, rather than SREBP-2, was identified as a key driver of PCSK9 increase in olanzapine-induced NAFLD. Our findings establish PCSK9 as a pivotal factor in olanzapine-induced NAFLD, influencing both receptor-related and metabolic pathways. This highlights PCSK9 inhibitors as potential therapeutic agents for managing NAFLD in schizophrenia patients treated with olanzapine.

Characterising metabolomic signatures of lipid-modifying therapies through drug target mendelian randomisation.

Large-scale molecular profiling and genotyping provide a unique opportunity to systematically compare the genetically predicted effects of therapeutic targets on the human metabolome. We firstly constructed genetic risk scores for 8 drug targets on the basis that they primarily modify low-density lipoprotein (LDL) cholesterol (HMGCR, PCKS9, and NPC1L1), high-density lipoprotein (HDL) cholesterol (CETP), or triglycerides (APOC3, ANGPTL3, ANGPTL4, and LPL). Conducting mendelian randomisation (MR) provided strong evidence of an effect of drug-based genetic scores on coronary artery disease (CAD) risk with the exception of ANGPTL3. We then systematically estimated the effects of each score on 249 metabolic traits derived using blood samples from an unprecedented sample size of up to 115,082 UK Biobank participants. Genetically predicted effects were generally consistent among drug targets, which were intended to modify the same lipoprotein lipid trait. For example, the linear fit for the MR estimates on all 249 metabolic traits for genetically predicted inhibition of LDL cholesterol lowering targets HMGCR and PCSK9 was r2 = 0.91. In contrast, comparisons between drug classes that were designed to modify discrete lipoprotein traits typically had very different effects on metabolic signatures (for instance, HMGCR versus each of the 4 triglyceride targets all had r2 < 0.02). Furthermore, we highlight this discrepancy for specific metabolic traits, for example, finding that LDL cholesterol lowering therapies typically had a weak effect on glycoprotein acetyls, a marker of inflammation, whereas triglyceride modifying therapies assessed provided evidence of a strong effect on lowering levels of this inflammatory biomarker. Our findings indicate that genetically predicted perturbations of these drug targets on the blood metabolome can drastically differ, despite largely consistent effects on risk of CAD, with potential implications for biomarkers in clinical development and measuring treatment response.

Is It Ever Wise to Edit Wild-Type Alleles? Engineered CRISPR Alleles Versus Millions of Years of Human Evolution.

The tremendous burden of lipid metabolism diseases, coupled with recent developments in human somatic gene editing, has motivated researchers to propose population-wide somatic gene editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) within the livers of otherwise healthy humans. The best-characterized molecular function of PCSK9 is its ability to regulate plasma LDL (low-density lipoprotein) levels through promoting LDL receptor degradation. Individuals with loss-of-function PCSK9 variants have lower levels of plasma LDL and reduced cardiovascular disease. Gain-of-function variants of PCSK9 are strongly associated with familial hypercholesterolemia. A new therapeutic strategy delivers CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR-associated protein 9) specifically to liver cells to edit the wild-type alleles of PCSK9 with the goal of producing a loss-of-function allele. This direct somatic gene editing approach is being pursued despite the availability of US Food and Drug Administration-approved PCSK9 inhibitors that lower plasma LDL levels. Here, we discuss other characterized functions of PCSK9 including its role in infection and host immunity. We explore important factors that may have contributed to the evolutionary selection of PCSK9 in several vertebrates, including humans. Until such time that more fully understand the multiple biological roles of PCSK9, the ethics of permanently editing the gene locus in healthy, wild-type populations remains highly questionable.

Evolocumab Treatment in Pediatric Patients With Homozygous Familial Hypercholesterolemia: Pooled Data From Three Open-Label Studies.

BACKGROUND: Pediatric patients with homozygous familial hypercholesterolemia (HoFH) have an increased risk of atherosclerotic cardiovascular disease and difficulty meeting low-density lipoprotein cholesterol (LDL-C) goals. In this post hoc analysis, we evaluated pooled safety and efficacy data from 3 studies in pediatric patients with HoFH treated with the PCSK9 (proprotein convertase subtilisin/kexin type 9) monoclonal antibody inhibitor evolocumab. METHODS: Patients with HoFH aged 10 to 17 years received treatment with open-label evolocumab 420 mg subcutaneously monthly or biweekly in the TAUSSIG, RAMAN, or HAUSER-OLE clinical studies. All patients received background statins with or without ezetimibe. Study duration ranged from 12 to 260 weeks. The primary end point was treatment-emergent adverse events per 100 patient-years. Efficacy end points were changes from baseline to week 12 in lipids and PCSK9. RESULTS: Of the 39 patients in the pooled analysis, 69.2% were males, median age was 13.0 years, and 79.5% (31/39) had genotyped HoFH with LDLR pathogenic variants. Overall, median exposure to evolocumab was 18.2 (Q1, Q3: 3.0, 18.5) months. Treatment-emergent adverse events with an exposure-adjusted patient incidence rate of ≥5% were upper respiratory tract infection (6.6%), influenza (5.2%), and acne (5.0%) per 100 patient-years. Exposure-adjusted patient incidence of serious treatment-emergent adverse events was 13.3% per 100 patient-years. Excluding 4 patients receiving lipoprotein apheresis, week 12 median percentage change from baseline in LDL-C was -2.9% (Q1, Q3: -21.7, 1.5); however, 42.9% (15/35) of patients achieved ≥15% reduction in LDL-C from baseline. Residual LDLR (LDL receptor) activity was not associated with a reduction in LDL-C. CONCLUSIONS: In this pooled data analysis from 3 studies in pediatric patients with HoFH, evolocumab was well tolerated, with no new safety signals reported. These safety findings are consistent with findings from previous studies of evolocumab. Patients showed marked variability in LDL-C reduction. Results from this pooled analysis support guidelines suggesting a trial of PCSK9 inhibitor therapy regardless of estimated residual LDLR function. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01624142, NCT03403374, and NCT02624869.

How safe are proprotein convertase subtilisinekexin type 9 inhibitors in diabetes?

PURPOSE OF REVIEW: To examine the safety of proprotein convertase subtilisinekexin type 9 (PCSK9) inhibitors in patients with diabetes, specifically focusing on their impact on glucose metabolism. RECENT FINDINGS: Patients with diabetes often require intensified lipid-lowering therapy. PCSK9 inhibitors can reduce low-density lipoprotein cholesterol (LDL-C) concentrations by approximately 60%, and significantly reduce cardiovascular risk when added to statin therapy. Some studies have suggested an association between low LDL-C levels and an increased risk of new-onset diabetes, and genetics has almost consistently shown an increased glucose concentration and risk of diabetes. Most clinical trials have not demonstrated a deterioration in glycaemic control in patients with diabetes after the use of PCSK9 inhibitors, and they do not lead to other significant treatment-emergent adverse events. SUMMARY: Although the majority of patients with diabetes are undergoing background statin therapy, which may mask potential adverse effects of PCSK9 inhibitors on glycaemic control, current data suggest that the benefits outweigh the risks for diabetic patients using PCSK9 inhibitors. Considering the different nature of genetic studies and of clinical trials, close monitoring of glucose parameters is necessary, especially in individuals with prediabetes.

Proprotein convertase subtilisin/kexin type 9-inhibition across different patient populations.

PURPOSE OF REVIEW: Monoclonal antibodies (mAb) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) have been established in cardiovascular risk prevention. The purpose of this review is to summarize the effects of PCSK9 inhibitors across different patient populations. RECENT FINDINGS: Long-term data on the use of evolocumab and alirocumab shows persisting low- density lipoprotein cholesterol (LDL-C) lowering and good tolerability. PCSK9 inhibitors are effective and safe in both sexes, in pediatric patients as well as in the elderly. Initiation of PCSK9 mAb during acute myocardial infarction is safe and leads to beneficial morphological plaque changes. The PCSK9 inhibitors evolocumab, alirocumab and inclisiran lower LDL-C in patients with heterozygous familial hypercholesterolemia (FH), while the response of patients with homozygous FH is heterogeneous. New areas of application beyond lipid lowering are currently investigated. SUMMARY: PCSK9 inhibitors are safe, well tolerated, and effective in primary and secondary prevention in a wide range of patient populations.

APOE and familial hypercholesterolemia.

PURPOSE OF REVIEW: Autosomal dominant hypercholesterolemia is a common cause of cardiovascular disease. In addition to the classic genes that cause hypercholesterolemia, LDLR, APOB and PCSK9 , a new locus has emerged as a candidate to be the cause of this hyperlipidemia, the p.(Leu167del) mutation in the APOE gene. RECENT FINDINGS: Various studies have demonstrated the involvement of the p.(Leu167del) mutation in the APOE gene in hypercholesterolemia: Studies of family segregation, lipoprotein composition by ultracentrifugation and proteomic techniques, and functional studies of VLDL-carrying p.(Leu167del) internalization with cell cultures have demonstrated the role of this mutation in the cause of hypercholesterolemia. The phenotype of individuals carrying the p.(Leu167del) in APOE is indistinguishable from familial hypercholesterolemia individuals with mutations in the classic genes. However, a better response to lipid-lowering treatment has been demonstrated in these APOE mutation carrier individuals. SUMMARY: Therefore, APOE gene should be considered a candidate locus along with LDLR, APOB , and PCSK9 to be investigated in the genetic diagnosis of familial hypercholesterolemia.

PCSK9-directed therapies: an update.

PURPOSE OF REVIEW: Two large cardiovascular outcomes trials of monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9) demonstrated that therapeutic inhibition of extracellular PCSK9 markedly reduces LDL cholesterol concentration and cardiovascular risk. Several novel strategies to inhibit PCSK9 function are in development. Different mechanisms of action may determine specific properties with potential relevance for patient care. RECENT FINDINGS: For the monoclonal antibodies evolocumab und alirocumab as first-generation PCSK9 inhibitors, follow-up data of up to 8 years of exposure complement the information on efficacy and safety available from outcome trials. For the small-interfering RNA inclisiran as second-generation PCSK9 inhibitor, several phase III trials have been published and a cardiovascular outcome trial has completed recruitment and is ongoing. Third-generation PCSK9 inhibitors encompass, among others, orally available drugs such as MK-0616 and the fusion protein lerodalcibep. Additional strategies to inhibit PCSK9 include vaccination and gene editing. SUMMARY: Long-term inhibition of PCSK9 with monoclonal antibodies is safe and conveys sustained cardiovascular benefit. Novel strategies to inhibit PCSK9 function such as orally available drugs, RNA targeting, and one-time treatment with gene editing may further enhance the therapeutic armamentarium and enable novel preventive strategies.

Proprotein convertase subtilisin/kexin type 9 as a drug target for abdominal aortic aneurysm.

PURPOSE OF REVIEW: There are no current drug therapies to limit abdominal aortic aneurysm (AAA) growth. This review summarizes evidence suggesting that inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) may be a drug target to limit AAA growth. RECENT FINDINGS: Mendelian randomization studies suggest that raised LDL and non-HDL-cholesterol are causal in AAA formation. PCSK9 was reported to be upregulated in human AAA samples compared to aortic samples from organ donors. PCSK9 gain of function viral vectors promoted aortic expansion in C57BL/6 mice infused with angiotensin II. The effect of altering PCSK9 expression in the aortic perfusion elastase model was reported to be inconsistent. Mutations in the gene encoding PCSK9, which increase serum cholesterol, were associated with increased risk of human AAA. Patients with AAA also have a high risk of cardiovascular death, myocardial infarction and stroke. Recent research suggests that PCSK9 inhibition would substantially reduce the risk of these events. SUMMARY: Past research suggests that drugs that inhibit PCSK9 have potential as a novel therapy for AAA to both limit aneurysm growth and reduce risk of cardiovascular events. A large multinational randomized controlled trial is needed to test if PCSK9 inhibition limits AAA growth and cardiovascular events.

Geranylgeranyl isoprenoids and hepatic Rap1a regulate basal and statin-induced expression of PCSK9.

LDL-C lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is now a validated therapeutic strategy that lowers serum LDL-C and reduces coronary events. Ironically, the most widely used medicine to lower cholesterol, statins, has been shown to increase circulating PCSK9 levels, which limits their efficacy. Here, we show that geranylgeranyl isoprenoids and hepatic Rap1a regulate both basal and statin-induced expression of PCSK9 and contribute to LDL-C homeostasis. Rap1a prenylation and activity is inhibited upon statin treatment, and statin-mediated PCSK9 induction is dependent on geranylgeranyl synthesis and hepatic Rap1a. Accordingly, treatment of mice with a small-molecule activator of Rap1a lowered PCSK9 protein and plasma cholesterol and inhibited statin-mediated PCSK9 induction in hepatocytes. The mechanism involves inhibition of the downstream RhoA-ROCK pathway and regulation of PCSK9 at the post-transcriptional level. These data further identify Rap1a as a novel regulator of PCSK9 protein and show that blocking Rap1a prenylation through lowering geranylgeranyl levels contributes to statin-mediated induction of PCSK9.