Plasma PCSK9 Research Articles

An epigenetic editor targeting human PCSK9 efficiently and durably lowers Low Density Lipoprotein Cholesterol (LDL-C) in non-human primates

Abstract Background Reducing the risk of atherosclerotic cardiovascular disease is dependent on both the magnitude and cumulative duration of LDL-C lowering. However, in clinical practice achieving treatment goals is often hampered by low adherence to standard of care. Although PCSK9 inhibitors represent an effective option for LDL-C lowering, they require chronic life-long treatment. Epigenetic editing is a new therapeutic approach designed to durably silence genes by leveraging nature’s endogenous cellular mechanism for gene regulation via CpG methylation without the inherent genotoxic risks of genome editing approaches that nick or cut the DNA. We are developing a PCSK9 epigenetic editor (PCSK9-EE) to durably silence PCSK9, with the promise of lifelong reduction in LDL-C. Methods Here, we describe the development of an epigenetic editor targeting human PCSK9. This epigenetic editor consists of a DNA targeting component fused to a transcriptional repressor domain and a DNA methyltransferase domain. PCSK9-EE was first screened and evaluated for specificity and efficacy in primary human hepatocytes (PHH). In vivo efficacy and durability were then evaluated in a transgenic mouse carrying the human PCSK9 genomic locus (hPCSK9-Tg mouse) and non-human primates (NHP). Results Our initial screen of PCSK9-EEs identified several hits that efficiently suppressed secreted PCSK9 levels in immortalized liver cells. We confirmed that our top PCSK9-EE candidates robustly decreased PCSK9 secretion in PHH and were found to be highly specific at targeting PCSK9 as assessed by RNA-seq, methylation array, and whole-genome methylation sequencing. To examine in vivo activity, we treated hPCSK9-Tg mice with a single administration of a lipid nanoparticle delivering mRNA encoding our PCSK9-EE and observed >97% reduction in liver PCSK9 mRNA, and >98% reduction in plasma PCSK9 for the duration of the study (one year). We then delivered PCSK9-EE in NHP and observed a robust reduction in plasma PCSK9 with a concomitant reduction in LDL-C. These effects were found to be durable for at least 6 months following a single administration of PCSK9-EE in NHP. To establish a mechanistic relationship between PCSK9-EE’s molecular action and durable PCSK9 silencing, we performed serial liver biopsies in each NHP approximately 2 months apart. Robust CpG methylation at the PCSK9 genomic locus was observed in the first liver biopsy and was comparable to that observed in the second liver biopsy suggesting that PCSK9-EE induced stable CpG methylation in NHP livers. Conclusions We believe PCSK9-EE may offer an effective, safe, and durable approach to suppress hepatic PCSK9 expression and achieve long-lasting reduction in LDL-C. Furthermore, epigenetic editing may hold promise for one-and-done approaches for the treatment of atherosclerotic cardiovascular disease without cutting, nicking, or altering the DNA sequence.

Reduction of triglyceride-rich lipoproteins with retatrutide in type 2 diabetes may be explained by concurrent reduction in ANGPTL3/8 levels

Abstract Background/Introduction In the recent phase 2 study (NCT04867785) in patients with type 2 diabetes (T2D), retatrutide, an agonist of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP-1), and glucagon receptors, reduced triglycerides (TG) and non-high-density lipoprotein cholesterol (non-HDL-C) up to 35.0% and 20.7%, respectively. The angiopoietin-like 3/8 complex (ANGPTL3/8) is the most potent circulating inhibitor of lipoprotein lipase (LPL), and its serum levels have been shown to be directly correlated with TG and low-density lipoprotein cholesterol (LDL-C). Purpose In the aforementioned phase 2 study, we examined the effects of retatrutide on circulating ANGPTL3/8 complex concentrations to help understand the mechanism by which retatrutide reduced TG and non-HDL-C. Methods Patients with T2D were randomly assigned to receive once-weekly injections of placebo, 1.5 mg dulaglutide, or retatrutide doses of 0.5 mg, 4 mg, 8 mg, or 12 mg. Fasting serum samples were collected at baseline, week 2, week 4, week 8, week 12, week 16, and week 24, and ANGPTL3/8 levels were measured at all time points using a sensitive and selective immunoassay. Plasma PCSK9 levels were also measured at baseline and 24 weeks. Data were analyzed using a mixed model for repeated measures with log-transformation. Results Baseline ANGPTL3/8 levels (mean 26.3 ng/mL, IQR 17.4-33.0 ng/mL) were not different among all study groups. Mean percent changes of ANGPTL3/8 from baseline at week 24 were -1.3%, -12.0%, -48.7% and -51.0% in 0.5 mg, 4 mg, 8 mg and 12 mg retatrutide groups, respectively (Figure). Percent changes of ANGPTL3/8 in 8 mg and 12 mg retatrutide groups were significantly different from placebo group (p < 0.0001). Mean percent changes of PCSK9 levels from baseline to 24 weeks were not different from placebo for any treatment arm (+6.2%, -3.9%, -3.8% and -9.6% for retatrutide 0.5 mg, 4 mg, 8 mg and 12 mg groups, respectively, and -1.5% for dulaglutide and -6.5% for placebo). Conclusions In patients with T2D, retatrutide dose-dependently decreased serum concentrations of ANGPTL3/8, the most potent circulating LPL inhibitor. These data suggest that retatrutide may reduce TG-rich lipoproteins by decreasing ANGPTL3/8 levels.

Abstract 132: Immunosenescence-related Biomarkers And Potential Therapeutic Targets For Coronary Artery Disease Identified Via Plasma Proteome And Immune Cell Traits

There is a pressing need to identify novel biomarkers and therapeutic targets to address the epidemiological burden of coronary artery disease (CAD). Mendelian Randomization analysis (MR) leverages Genome-Wide Association Study (GWAS) data to infer the causal mechanism underlying the phenotype of interest. The proteome is the central mediator of the etiology and pathophysiology of multifactorial diseases such as CAD. Here, we screened for potential biomarkers and therapeutic targets for CAD through MR by using genetically predicted levels of plasma proteome followed by immune cell traits and transcriptomic analysis. The main workflow of our study is outlined in Figure 1. Briefly, MR analysis was performed with protein quantitative trait loci (pQTL) and meta-analysis of four independent GWASs. Sensitivity analysis was performed to ensure the robustness of identified proteins. Pathway Enrichment analysis on whole-blood transcriptome was performed with GSEA. Secondary MR explored the causal effect of 731 immune cell traits on CAD risk. In total, 11 overlapping candidate proteins (P<0.05, OR>1.0) were identified from the plasma proteome meta-analysis, including LPA, PLA2G7, and PCSK9, substantiating the validity of our MR analysis. Functional annotation of candidate proteins identified LCAT and CA11 as promising therapeutic targets and biomarkers. LCAT suppression is protective against atherosclerosis in mice models, while the functional significance of CA11 remains to be elucidated. Transcriptomic analysis shows enrichment in IL-2 signaling pathways reflective of the exhaustion of CD28 on cytotoxic T cells identified via secondary MR using immune-cell traits. Together, these results are indicative of immunosenescence as a risk factor for CAD. Overall, our results revealed that an altered immune landscape increased the risk for CAD and unraveled an encouraging avenue for early diagnosis and treatment of CAD.

Abstract 5169: Circulating levels of ANGPTL3, PCSK9, apoCIII and lipoprotein (a) in high-grade serous ovarian cancer

Abstract Background: Ovarian cancers require abundant amounts of lipids for their growth. Recent years have highlighted the role of ANGPTL3, PCSK9, ApoCIII and Lp(a) as key players in lipid metabolism. But circulating levels of these factors in ovarian cancers have not been thoroughly investigated to date. Objective: We assessed the profiles of plasma ANGPTL3, PCSK9, ApoCIII and Lp(a) in ovarian high-grade serous carcinoma (HGSC). Methods: Plasma samples collected from women diagnosed with an HGSC (n=31) or a benign ovarian lesion (BOL, n=40) were analyzed for plasma lipid profile (ApoB, total cholesterol, HDL, triglycerides, Lp(a) levels) on a clinical modular platform (Roche). ApoCIII, ANGPTL3 and PCSK9 levels were measured with commercially-available ELISA kits. Differences between HGSC and BOL were assessed by two-group comparisons. Pairwise correlation strength between variables was evaluated with Spearman’s rank order tests. Logistic regression modelling was used to further examine the associations between selected variables and HGSC diagnoses. Results: ANGPTL3 levels increased in HGSC (84.1 ng/ml, SD=29.2ng/ml, n=31) compared to BOL (66.9ng/ml, SD=30.7 ng/ml, n=40; HGSC vs BOL p=0.02). Receiver operating characteristic (ROC) curve suggested that plasma ANGPTL3 levels were moderately associated with HGSC (AUC=67.7%, p=0.005 1) and did not increase predictive performance of ovarian tumor biomarkers CA125 and HE4 in multivariable model. Associations between ANGPTL3 levels and cholesterol (HDL, non-HDL, LDL and total) could be observed in the control group but not in the HGSC group. The same dissociation was observed for the inverse correlation between HDL and triglycerides in HGSC. Furthermore, moderate associations were observed between PCSK9 & CA19-9 in the entire cohort (rho=0.34, n=53, p=0.013) and Lp(a) & CA125 in HGSC cases (rho=0.51, p= 0.017). Conclusion: In this cohort of 71 women, we report increased levels of ANGPTL3 in HGSC with an effect size (Cohen’s D) of 0.57. Confirmation of this result in larger cohort studies (minimal n>50 in each group) would warrant further investigation into the roles of circulating ANGPTL3. Its inhibition may allow to tackle deregulated lipid metabolic pathways contributing to ovarian cancer progression. Citation Format: Emilie Wong Chong, France-Hélène Joncas, Pierre Douville, Dimcho Bachvarov, Frédéric Calon, Nabil Seidah, Caroline Diorio, Anne Gangloff. Circulating levels of ANGPTL3, PCSK9, apoCIII and lipoprotein (a) in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5169.

Population pharmacokinetics of a novel PCSK9 antisense oligonucleotide.

The aim of this study was to characterize the population pharmacokinetics of AZD8233, an antisense oligonucleotide (ASO) that targets the PCSK9 transcript to reduce hepatocyte PCSK9 protein production and plasma levels. AZD8233 utilizes generation 2.5S-constrained ethyl motif (cET) chemistry and is conjugated to a triantennary N-acetylgalactosamine (GalNAc3) ligand for targeted hepatocyte uptake. A non-linear mixed-effect modelling approach utilizing NONMEM software was applied to AZD8233 concentration-time data from 3416 samples in 219 participants from four phase 1-2 studies, one in healthy volunteers (NCT03593785) and three in patients with dyslipidaemia (NCT04155645, NCT04641299 and NCT04823611). The final model described the AZD8233 plasma concentration-time profile from four phase 1-2 studies in healthy volunteers or participants with dyslipidaemia, covering a dose range of 4 to 120 mg. The pharmacokinetics of AZD8233 were adequately described by a two-compartment model with first-order absorption. The supra-proportional increase in maximum plasma concentration (Cmax) across the observed dose range was described by non-linear Michaelis-Menten elimination (maximum elimination rate, 9.9mg/h [12% relative standard error]; concentration yielding half-maximal elimination rate, 4.8mg/L [18% relative standard error]). Body weight, sex, estimated glomerular filtration rate and disease status (healthy participant vs. patient with dyslipidaemia) were identified as factors affecting exposure to AZD8233. Covariate analysis showed body weight to be the main factor affecting exposure to AZD8233, which largely explained the higher Cmax observed in the Asian population relative to non-Asians.

Extracellular vesicle-mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin-kexin type 9 (Pcsk9) in primary mouse hepatocytes.

The loss-of-function of the proprotein convertase subtilisin-kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low-density lipoprotein cholesterol (LDL-C) levels. Both CRISPR/Cas9 and CRISPR-based editor-mediated Pcsk9 inactivation have successfully lowered plasma LDL-C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle-mediated CRISPR delivery inactivating Pcsk9 affected low-density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV-mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin-interacting heterodimer FK506-binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12-rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNPdelivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low-density lipoprotein receptor and LDL-C. These readouts can be used in future experiments to assess the efficacy of vehicle-mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV-mediated CRISPR/Cas9 RNP delivery targeting Pcsk9.

Evaluating the effect of the antiPCSK9 vaccine on systemic inflammation and oxidative stress in CFA-challenged albino mice

Abstract Background Besides effective lipid lowering therapy aimed to reduce LDL-C, one should always look at other risk factors that may essentially increase the risk of residual cardiovascular disease risk, including diabetes, thrombotic factors and inflammation. Purpose To find whether the antiPCSK9 vaccine can affect the serum C-reactive protein (CRP) and oxidative stress during acute systemic inflammation. Methods Male albino mice were randomly divided into three groups, including non-treated mice (the sham group), mice treated with Freund's complete adjuvant (CFA) (the CFA group), and vaccinated mice treated with CFA (the vaccine group). The vaccine group was subcutaneously immunized with the antiPCSK9 formulation, four times in bi-weekly intervals. To induce inflammation, all mice were subjected to the CFA challenge after the vaccination plan. To determine the efficacy of the antiPCSK9 vaccine in albino mice, plasma antiPCSK9 antibody titer, plasma PCSK9 concentration, and PCSK9/LDLR interaction were assayed. The serum level of high-sensitive CRP (hs-CRP) and oxidative stress status were evaluated by a mouse CRP ELISA kit and the pro-oxidant antioxidant balance (PAB) assay, respectively. Results Upon three boosters, the vaccine induced a high-titer IgG antiPCSK9 antibody in albino mice, which was accompanied with a significant reduction in the plasma level of the free PCSK9 (-24.7% and -28.5% compared with the sham and CFA group, respectively), and the inhibition of PCSK9/LDLR interaction (-27.8% and -29.4% compared with the sham and CFA group, respectively). In addition, the serum level of hs-CRP was significantly increased in the vaccine and CFA groups by 225% and 274%, respectively, when compared with the sham group; however, it was non-significantly decreased (-18%) in the vaccine group in comparison with the CFA group (Figure 1A). Moreover, the PAB values indicated that oxidative stress was significantly increased in the CFA group (by 72.7%) and the vaccine group (by 76%) when compared to the sham group; however, there was no significant difference in the PAB values between the vaccine and CFA groups (Figure 1B). Conclusion Our results indicate that, despite inducing the production of the functional antiPCSK9 antibodies, the antiPCSK9 vaccine failed to significantly reduce the serum hs-CRP and oxidative stress induced in the CFA-challenged albino mice. Figure 1. The effect of the antiPCSK9 vaccine on (A) the serum level of hs-CRP in albino mice and (B) the serum PAB in albino mice. The sham group involved non-treated mice, the CFA group involved the CFA-treated mice, and the vaccine group involved mice who after vaccination were treated with the CFA. Pooling of samples was performed to obtain sufficient sample volume for assay, when needed. The PAB values were expressed in an arbitrary HK (Hamidi-Koliakos). Data are expressed as mean ±SD (10 mice per group). Statistical differences at a p-value <0.05 were considered as significant.Figure 1.

Exploring PCSK9 Genetic Impact on Lipoprotein(a) via Dual Approaches: Association and Mendelian Randomization.

Previous investigations have suggested an association between the PCSK9 common polymorphism E670G and Lipoprotein(a) (Lp(a)) levels, as well as a link between plasma PCSK9 levels and Lp(a) concentrations. However, the causal relationship between plasma PCSK9 and Lp(a) levels remains uncertain. In this study, we explored the association between PCSK9 E670G polymorphism and Lp(a) levels in 614 healthy Taiwanese individuals. Employing a two-sample Mendelian randomization (MR) analysis using openly accessible PCSK9 and Lp(a) summary statistics from the genome-wide association studies (GWAS) and UK Biobank, we aimed to determine if a causal link exists between plasma PCSK9 levels and Lp(a) concentrations. Our findings reveal that the E670G G allele is independently associated with a decreased likelihood of developing elevated Lp(a) levels. This association persists even after adjusting for common cardiovascular risk factors and irrespective of lipid profile variations. The MR analysis, utilizing six PCSK9 GWAS-associated variants as instrumental variables to predict plasma PCSK9 levels, provides compelling evidence of a causal relationship between plasma PCSK9 levels and Lp(a) concentration. In conclusion, our study not only replicates the association between the PCSK9 E670G polymorphism and Lp(a) levels but also confirms a causative relationship between PCSK9 levels and Lp(a) concentrations through MR analysis.

Evolocumab prevents atrial fibrillation in rheumatoid arthritis rats through restraint of PCSK9 induced atrial remodeling

IntroductionProprotein convertase subtilisin/kexin type 9 (PCSK9) is implicated in the pathogenesis and progression of autoimmune disease. Patients with rheumatoid arthritis (RA) are at high risk of developing atrial fibrillation (AF), while whether PCSK9 is involved in the onset of AF among RA patients remains unclear. ObjectivesTo explore the role of PCSK9 in the occurrence of AF in RA patients and decipher the underlying mechanism. MethodsWe established a rat model of collagen-induced arthritis (CIA) by immunization with type II collagen in Freund’s incomplete adjuvant. Atrial electrophysiological test was used to evaluate AF susceptibility. We performed a clinical study to examine the correlation between PCSK9 level and AF, which recruited healthy control, RA patients and RA patients complicated with AF. Evolocumab (a monoclonal antibody of PCSK9) is administered via intraperitoneal injection in CIA rats to assess the role of PCSK9 in RA-related AF. LPS-RS (LPS inhibitor), clodronate liposomes (depletion of macrophages), and cell co-culture model were used to dissect the mechanism underlying PCSK9 promotes AF. ResultsAF inducibility and duration were higher in CIA rats, accompanied by elevated plasma and atrial PCSK9. Interestingly, compared with healthy control subjects, patients with RA showed an increase in PCSK9, and the PCSK9 is much higher in RA patients complicated with AF. The level of PCSK9 was independently associated with AF risk in RA patients. In the in vivo experiment, evolocumab reduced AF susceptibility, and ameliorated atrial structural remodeling of CIA rats. Mechanistically, augmented LPS in CIA rats led to an increase in PCSK9, which exacerbated fibrosis of cardiac fibroblasts and apoptosis of cardiac myocytes by enhancement of M1 macrophages polarization and inflammation, thereby contributing to AF. ConclusionThis study suggests that elevated PCSK9 causes atrial structural remodeling by enhancement of M1 macrophages polarization in atria, and evolocumab can effectively protects CIA rats from AF.

Correlation between Plasma PCSK9 and Blood Lipids in Patients with Newly Diagnosed Primary Nephrotic Syndrome

Abstract Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a major post-transcriptional regulator of low-density lipoprotein receptor degradation. Recently, PCSK9 was shown to be overexpressed by liver cells in rats with proteinuria. However, the levels of PCSK9 in newly diagnosed primary nephrotic syndrome (PNS) patients and correlations involving PCSK9 and blood lipids are not clearly understood. Aim of the Work The aim of this study is to assess the association between plasma pcsk 9 and blood lipids in patients with newly diagnosed primary nephrotic syndrome Patients and Methods This was a case control study in Ain Shams University hospital for 4 months on 50 patients, 35 control. Results in our study elevated plasma PCSK9 levels had a positive linear correlation with elevated serum TC (p value <0.001) and LDL-C (p value <0.001), triglycerides (p value 0.003), HDL (p value 0.012), VLDL(p value 0.001) A comparison of plasma PCSK9 levels of MCD, MN And primary focal segmental patients showed that they were not significantly different (p value 0.954). In our study, we found that the plasma levels of pcsk9 were significantly higher than in healthy controls 314.580 ± 73.835 ng/ml vs 253.429 ±36.666, and this difference was statistically significant P-value <0.001. In our study plasma PCSK9 levels in patients with PNS had no linear correlation with the amount of proteinuria per 24 h, p value 0.070 or with albumin p value 0.549 and there was no statistical difference between the the two groups of proteinuria (3.5-8 (g)/24 h vs > 8 (g)/24 h) p value 0.378. Conclusion Plasma PCSK9 levels in newly diagnosed primary nephrotic patients were markedly increased and positively correlated with total cholesterol, VLDL, LDL, HDL. suggesting that PCSK9 may emerge as a novel therapeutic target in nephrotic syndrome associated hypercholesterolemia

The Immunogenic Potential of PCSK9 Peptide Vaccine in Mice.

To evaluate the immunogenic potential of the carrier-free peptide-based anti-PCSK9 (proprotein convertase subtilisin/kexin 9) vaccine in albino mice. The immunogenic pcsk9 peptide and 0.4% alum adjuvant were mixed thoroughly at a 1:1 ratio and used as a vaccine formulation. To assess the humoral immune response, animals' blood was sampled two weeks after the last immunization. The ELISA method was employed to measure serum anti-PCSK9 antibody titers, PCSK9 concentrations, and PCSK9/LDLR interaction. ELISA analysis showed significant induction of IgG antibody titers by PCSK9 peptide vaccine in vaccinated mice sera compared to the control mice (in male and female mice were 12000±586 and 11566±642, respectively, p<0.001). Mechanistic analyses showed a significant reduction in serum PCSK9 concentrations by vaccine-induced antibodies in vaccine groups compared to the control groups (in male mice by 29±5 ng/mL (22.4%), p<0.001 and female mice by 26±5 ng / mL (21.0%), p<0.001). Serum concentrations of PCSK9 in control and vaccine groups were 131±8.6 ng / mL and 102±8.1 ng/ml in male mice and 124±6 ng/ml and 98±10 ng/ml in female mice, respectively. Moreover, vaccine-induced antibodies inhibited the PCSK9-LDLR interaction in male and female groups by 34% and 26%, respectively. No significant difference was detected between the male and female groups in all tests (p>0.05). According to our results, the PCSK9 peptide vaccine provoked the humoral immune system in albino mice to produce functional antibodies that inhibit plasma PCSK9. These effects were seen in both genders without any significant difference.

PCSK9 Involvement in Autism Etiology: Sequence Variations, Protein Concentration, and Promoter Methylation

Besides its main role in the control of blood cholesterol, PCSK9 has a role in the regulation of neuronal development and apoptosis. We suggest, for the first time, the possible involvement of PCSK9 in autism. In this case-control study, Sanger sequencing was used to analyze sequence variations in the PCSK9 gene exons and their flanking intronic sequences. ELISA assay was used to determine the plasma concentration of PCSK9. The methylation percentage of the PCSK9 gene promoter was assessed by methylation-specific PCR (MSP). Forty-three variants were found; out of them, seven showed differential frequency between patients and controls. rs.45448095, rs.45613943, rs.630431, rs.529500286, and rs.45439391 are risk factors for autism, while rs.11800231 and rs.483462 are protective variants. The concentration of plasma PCSK9 protein was significantly elevated and the methylation percentage of PCSK9 gene promoter was significantly lower in cases than in controls (P <0.001 and = 0.002, respectively). ROC curve analysis identified an area under the curve (AUC) of 0.915 for plasma protein concentration and 0.693 for percent gene promoter methylation. In addition, two new variants were identified (g.23809C>T in intron 11 and g.24071T>G in 3' UTR). This is the first study to investigate the correlation between PCSK9 protein and autism and suggests the potential involvement of PCSK9 as one of the susceptibility genes for autism. Further studies with a larger number of subjects are recommended.

Small molecule inhibitors of PCSK9. SAR investigations of head and amine groups

Our previous work on the optimization of a new class of small molecule PCSK9 mRNA translation inhibitors focused on empirical optimization of the amide tail region of the lead PF-06446846 (1). This work resulted in compound 3 that showed an improved safety profile. We hypothesized that this improvement was related to diminished binding of 3 to non-translating ribosomes and an apparent improvement in transcript selectivity. Herein, we describe our efforts to further optimize this series of inhibitors through modulation of the heterocyclic head group and the amine fragment. Some of the effort was guided by an emerging cryo electron microscopy structure of the binding mode of 1 in the ribosome. These efforts led to the identification of 15 that was deemed suitable for evaluation in a humanized PCSK9 mouse model and a rat toxicology study. Compound 15 demonstrated a dose dependent reduction of plasma PCSK9 levels. The rat toxicological profile was not improved over that of 1, which precluded 15 from further consideration as a clinical candidate.

A cVLP-Based Vaccine Displaying Full-Length PCSK9 Elicits a Higher Reduction in Plasma PCSK9 Than Similar Peptide-Based cVLP Vaccines.

Administration of PCSK9-specific monoclonal antibodies, as well as peptide-based PCSK9 vaccines, can lower plasma LDL cholesterol by blocking PCSK9. However, these treatments also cause an increase in plasma PCSK9 levels, presumably due to the formation of immune complexes. Here, we utilize a versatile capsid virus-like particle (cVLP)-based vaccine platform to deliver both full-length (FL) PCSK9 and PCSK9-derived peptide antigens, to investigate whether induction of a broader polyclonal anti-PCSK9 antibody response would mediate more efficient clearance of plasma PCSK9. This head-to-head immunization study reveals a significantly increased capacity of the FL PCSK9 cVLP vaccine to opsonize and clear plasma PCSK9. These findings may have implications for the design of PCSK9 and other vaccines that should effectively mediate opsonization and immune clearance of target antigens.

Targeting PCSK9 With Antibodies and Gene Silencing to Reduce LDL Cholesterol.

The discovery of PCSK9 and its role in regulating the low-density lipoprotein (LDL) receptor, and the effect of loss-of-function mutations of its gene, identified it as a therapeutic target in 2006. Fully humanized monoclonal antibodies to PCSK9 (alirocumab and evolocumab) proved effective for lowering LDL cholesterol and subsequently for reducing atherosclerotic events in large outcome trials. Suppressing PCSK9 synthesis via gene silencing using inclisiran, a small interfering RNA, is another approach that effectively reduces LDL cholesterol, and a cardiovascular outcome trial is in progress. These treatments are given subcutaneously on a background of maximally tolerated statin treatment and are long-lasting: dosing is once or twice a month, self-administered, for alirocumab and evolocumab, and every 6 months for inclisiran, in the clinic, with an extra dose at 3 months in the initial year of therapy. These 3 agents produce mean LDL reductions of about 55% with no important adverse effects detectable to date. They are indicated in patients with atherosclerotic vascular disease or familial hypercholesterolemia who cannot achieve LDL cholesterol targets with maximally tolerated statin treatment. Such therapy can produce very low plasma LDL cholesterol and PCSK9, but there is no evidence this is harmful. Introduction into clinical practice has been impeded by economic considerations. The barrier to their use has not been scientific or medical, but rather the impact on healthcare resources. Prices have been reduced, but whether they are now cost-effective varies from country to country.

ODP557 PCSK9 Level Correlated with Disease Progression in Patient with Non-small Cell Lung Cancer

Abstract Background Hyperlipidemia may be present in cancer patients despite their debilitated general condition. It has also been reported that several types of cancer cells produce PCSK9, and that in advanced non-small cell lung cancer, chemotherapy with immune checkpoint inhibitors is effective in cases with low PCSK9 levels. We administered PCSK9 inhibitor to patients with advanced non-small cell lung cancer with hyperlipidemia according to the indications for hyperlipidemia. Chemotherapy with immune checkpoint inhibitors was performed, the patient's condition and the level of PSCK9 were observed. CASE: A woman in her 60s underwent surgery for lung cancer in 2017. The pathological diagnosis of squamous cell carcinoma of the left lower bronchus and the postoperative diagnosis of pT2bN2M0 stage IIIA were confirmed. Adjuvant chemotherapy with CDDP and VNR was started after surgery for her, but in 2018, recurrence of pulmonary lymph nodes was confirmed by intrabronchial induced transbronchial needle aspiration. The immunostaining result for PD-L1 was 50%. The first-line chemotherapy, the immune checkpoint inhibitor pembrolizumab, was started but was ineffective. She had high serum LDL levels of 174 mg/dL and high serum PCSK9 levels of 471 ng /mL (Norm: 47.4-225.2 ng/mL) despite exhaustion during her lung cancer treatment process. Her plasma PCSK9 levels were correlated with lung cancer status, suggesting PCSK9 production in lung cancer cells. Ezetimibe and statins were also discontinued because they were reported to increase serum PCSK9. The PCSC9K inhibitor evolocumab was administered at 140 mg every 2 weeks. Three months after the start of evolocumab, her serum PCSK9 level decreased slightly to 441 ng /mL. The immune checkpoint inhibitor nivolumab was then started, but the chemotherapy was ineffective, and her general condition worsened, correlating with her serum PCSK9 levels rising to 1635 ng/mL, and she died in 2021. Discussion and Clinical Lessons This is the first report on the clinical course of non-small cell lung cancer and the course of serum PCSK9 and the administration of PCSK9 inhibitors. Although PSCK9 is thought to be involved in cancer progression, the use of PCSK9 inhibitors at the same doses and doses as in the treatment of conventional hyperlipidemia failed to control cancer progression. The anti-PCSK9 vaccine has been experimentally shown to be effective against tumor progression. It is expected that PCSK9 inhibition by various methods will bring good results for cancer patients. Further research is needed to elucidate the exact mechanism by which PCSK9 regulates cancer cell proliferation and apoptosis. PCSK9 inhibitors, initially developed to treat hypercholesterolemia, may have significant involvement in the treatment of various cancers and their metastases. Therefore, collaboration between endocrinologists and oncologists is important in the laboratory and clinical practice. Presentation: No date and time listed

Independent association of PCSK9 with platelet reactivity in subjects without statin or antiplatelet agents.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) levels could predict cardiovascular event in patients with well-controlled LDL-C levels, suggesting an LDL-independent mechanism of PCSK9 on the cardiovascular system. Accumulating evidence suggests PCSK9 might be associated with increased platelet reactivity. This study aimed to assess the relationship between PCSK9 levels and platelet reactivity in subjects not taking statins or antiplatelet agents. A cross-sectional study was conducted to investigate the independent contribution of PCSK9 to platelet activity by controlling for the potential confounding factors. The study population included 89 subjects from a health examination centre who underwent routine annual health check-ups or had an examination before a selective operation. Subjects taking statins or antiplatelet agents were excluded. Adenosine diphosphate (ADP)-induced platelet aggregation was determined by PL-11 platelet analyzer using impedance aggregometry and plasma PCSK9 levels were determined using an ELISA. Serum Lipid profile was assessed by measuring the concentration of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG), with low-density lipoprotein cholesterol (LDL-C) being directly measured using enzymatic techniques. The association between PCSK9 and platelet reactivity was investigated. The study subjects were composed of 53 males and 36 females with an average age of 55 (±11) years old. The univariate correlation analysis showed significant correlation between ADP-induced maximal aggregation rate (MAR) and PCSK9 (r = 0.55, p < 0.001) as well as TC (r = 0.23, p = 0.028), LDL-C (r = 0.27, p < 0.001), and PLT (r = 0.31, p = 0.005). Being male (41.2% vs. 46.6, p = 0.04) and smoking (37.4 vs. 46.2%, p = 0.016) were associated with lower ADP-induced MAR than being female and non-smoking. However, there is no correlation between PCSK9 and AA-induced platelet maximal aggregation rate (r = 0.17, p = 0.12). Multiple regression analysis suggested that PCSK9 contributed independently to ADP-induced maximal aggregation rate (β = 0.08, p = 0.004) after controlling for the effect of TC, LDL-C, PLT, being male, and smoking. PCSK9 is positively associated with platelet reactivity, which may partly account for the beneficial effect of PCSK9 inhibition in reducing the risk of major adverse cardiovascular events after acute coronary syndrome (ACS).

Circulating levels of PCSK9, ANGPTL3 and Lp(a) in stage III breast cancers

Background / synopsisCholesterol and lipids play an important role in sustaining tumor growth and metastasis in a large variety of cancers. ANGPTL3 and PCSK9 modify circulating cholesterol levels, thus availability of lipids to peripheral cells. Little is known on the role, if any, of circulating lipid-related factors such as PCSK9, ANGPTL3 and lipoprotein (a) in cancers.Objective/purposeTo compare circulating levels of PCSK9, ANGPTL3, and Lp(a) in women with stage III breast cancer versus women with premalignant or benign breast lesions.MethodsTwenty-three plasma samples from women diagnosed with a stage III breast cancer (ductal, lobular or mixed) were matched for age with twenty-three plasma samples from women bearing premalignant (stage 0, n = 9) or benign (n = 14) breast lesions. The lipid profile (Apo B, total cholesterol, HDL cholesterol and triglycerides levels) and Lp(a) were measured on a Roche Modular analytical platform, whereas LDL levels were calculated with the Friedewald formula. ANGPTL3 and PCSK9 plasma levels were quantitated by ELISA. All statistical analyses were performed using SAS software version 9.4.ResultsPCSK9 levels were significantly higher in women with stage III breast cancer compared to age-matched counterparts presenting a benign lesion (95.9 ± 27.1 ng/mL vs. 78.5 ± 19.3 ng/mL, p < 0.05, n = 14). Moreover, PCSK9 levels positively correlated with breast disease severity (benign, stage 0, stage III) (Rho = 0.34, p < 0.05, n = 46). In contrast, ANGPTL3 and Lp(a) plasma levels did not display any association with breast disease status and lipids did not correlate with disease severity.ConclusionIn this small cohort of 46 women, PCSK9 levels tended to increase with the severity of the breast disease. Given that PCSK9 plays an important role in maintaining cholesterolemia, and a potential role in tumor evasion, present results warrant further investigation into a possible association between PCSK9 levels and breast cancer severity in larger cohorts of women.

Hepatic inactivation of murine Surf4 results in marked reduction in plasma cholesterol.

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018). Here, we generated hepatic SURF4-deficient mice (Surf4fl/fl Alb-Cre+) to investigate the physiologic role of SURF4 in vivo. Surf4fl/fl Alb-Cre+ mice exhibited normal viability, gross development, and fertility. Plasma PCSK9 levels were reduced by ~60% in Surf4fl/fl Alb-Cre+ mice, with a corresponding ~50% increase in steady state LDLR protein abundance in the liver, consistent with SURF4 functioning as a cargo receptor for PCSK9. Surprisingly, these mice exhibited a marked reduction in plasma cholesterol and triglyceride levels out of proportion to the partial increase in hepatic LDLR abundance. Detailed characterization of lipoprotein metabolism in these mice instead revealed a severe defect in hepatic lipoprotein secretion, consistent with prior reports of SURF4 also promoting the secretion of apolipoprotein B (APOB). Despite a small increase in liver mass and lipid content, histologic evaluation revealed no evidence of steatohepatitis or fibrosis in Surf4fl/fl Alb-Cre+ mice. Acute depletion of hepatic SURF4 by CRISPR/Cas9 or liver-targeted siRNA in adult mice confirms these findings. Together, these data support the physiologic significance of SURF4 in the hepatic secretion of PCSK9 and APOB-containing lipoproteins and its potential as a therapeutic target in atherosclerotic cardiovascular diseases.

The immunogenic potential of PCSK9 peptide vaccine in mice

Abstract Background Proprotein convertase subtilisin/Kexin 9 (PCSK9) is a serum protein expressed by liver cells that regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by modulating LDL receptors (LDLR). Recent studies of our group indicated the efficient immunogenicity of the nanoliposomal PCSK9 peptide vaccine in different animal models. Purpose Herein, we sought to evaluate the immunogenic potential of the non-liposomal form of the vaccine due to its simplicity of processing and lower production cost. Methods PCSK9 immunogenic peptide was prepared by linking a short PCSK9 peptide to a tetanus peptide as B and T cell epitopes, respectively. The immunogenic peptide and 0.4% alum adjuvant, in a 1:1 ratio, were mixed thoroughly and homogeneously prior to administration as vaccine formulation. In order to assess the humoral immune response, mice were inoculated subcutaneously four times at bi-week intervals. Immunized animals' blood was sampled two weeks after the last injection. The titers of the anti-PCSK9 antibodies in the serum of vaccinated mice were evaluated using the ELISA method. ELISA was also used to investigate the effects of anti-PCSK9 vaccination on PCSK9 serum concentrations and its interaction with LDL receptor (LDLR). Results ELISA analysis showed significant induction of IgG antibody titers by PCSK9 peptide vaccine in vaccinated mice compared to the control group (586±12000 and 642±11566 in male and female vaccinated mice, respectively, p&amp;lt;0.001). No significant difference was found between the male and female vaccine groups (p&amp;gt;0.05). Mechanistic analyses showed that vaccine-induced antibodies decreased serum PCSK9 concentration in vaccine groups compared to the control group (in male mice by 26±5 ng/mL, p&amp;lt;0.01 and female mice by 29±5 ng/mL, p&amp;lt;0.01). Serum concentrations of PCSK9 in control and vaccine groups were found to be 131±8.6 ng/mL and 102±8.1 ng/ml in male mice, and 124±6 ng/ml and 98±10 ng/ml in female mice, respectively. Moreover, vaccine-induced antibodies inhibited the PCSK9/LDLR interaction in the presence of serum from vaccinated mice compared to control mice serum (in male and female groups by 34% and 26%, respectively). The PCSK9 peptide vaccine significantly reduced LDL-C in immunized mice in both male (16.93±2.5 mg/dL, p=0.001) and female groups (22±2.7 mg/dL, p=0.001) compared with the control group. Conclusions According to our results, the PCSK9 peptide vaccine stimulates the humoral immune response in albino mice to produce active antibodies that inhibit plasma PCSK9 resulting in plasma LDL-C reduction. Funding Acknowledgement Type of funding sources: None.