Targeting Proprotein Convertase Subtilisin/kexin Type 9 Research Articles

Increase of PCSK9 expression in diabetes promotes VEGFR2 ubiquitination to inhibit endothelial function and skin wound healing.

Diabetic foot ulcers (DFUs) are a serious vascular disease. Currently, no effective methods are available for treating DFUs. Pro-protein convertase subtilisin/kexin type 9 (PCSK9) regulates lipid levels to promote atherosclerosis. However, the role of PCSK9 in DFUs remains unclear. In this study, we found that the expression of PCSK9 in endothelial cells (ECs) increased significantly under high glucose (HG) stimulation and in diabetic plasma and vessels. Specifically, PCSK9 promotes the E3 ubiquitin-protein ligase NEDD4 binding to vascular endothelial growth factor receptor 2 (VEGFR2), which led to the ubiquitination of VEGFR2, resulting in its degradation and downregulation in ECs. Furthermore, PCSK9 suppresses the expression and activation of AKT, endothelial nitric oxide synthase (eNOS), and ERK1/2, leading to decreased nitric oxide (NO) production and increased superoxide anion (O2._) generation, which impairs vascular endothelial function and angiogenesis. Importantly, using evolocumab to limit the increase in PCSK9 expression blocked the HG-induced inhibition of NO production and the increase in O2._ production, as well as inhibited the phosphorylation and expression of AKT, eNOS, and ERK1/2. Moreover, evolocumab improved vascular endothelial function and angiogenesis, and promoted wound healing in diabetes. Our findings suggest that targeting PCSK9 is a novel therapeutic approach for treating DFUs.

The multifaceted role of PCSK9 in cancer pathogenesis, tumor immunity, and immunotherapy.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a well-known regulator of cholesterol metabolism and cardiovascular diseases, has recently garnered attention for its emerging involvement in cancer biology. The multifunctional nature of PCSK9 extends beyond lipid regulation and encompasses a wide range of cellular processes that can influence cancer progression. Studies have revealed that PCSK9 can modulate signaling pathways, such as PI3K/Akt, MAPK, and Wnt/β-catenin, thereby influencing cellular proliferation, survival, and angiogenesis. Additionally, the interplay between PCSK9 and cholesterol homeostasis may impact membrane dynamics and cellular migration, further influencing tumor aggressiveness. The central role of the immune system in monitoring and controlling cancer is increasingly recognized. Recent research has demonstrated the ability of PCSK9 to modulate immune responses through interactions with immune cells and components of the tumor microenvironment. This includes effects on dendritic cell maturation, T cell activation, and cytokine production, suggesting a role in shaping antitumor immune responses. Moreover, the potential influence of PCSK9 on immune checkpoints such as PD1/PD-L1 lends an additional layer of complexity to its immunomodulatory functions. The growing interest in cancer immunotherapy has prompted exploration into the potential of targeting PCSK9 for therapeutic benefits. Preclinical studies have demonstrated synergistic effects between PCSK9 inhibitors and established immunotherapies, offering a novel avenue for combination treatments. The strategic manipulation of PCSK9 to enhance tumor immunity and improve therapeutic outcomes presents an exciting area for further investigations. Understanding the mechanisms by which PCSK9 influences cancer biology and immunity holds promise for the development of novel immunotherapeutic approaches. This review aims to provide a comprehensive analysis of the intricate connections between PCSK9, cancer pathogenesis, tumor immunity, and the potential implications for immunotherapeutic interventions.

Biparatopic anti-PCSK9 antibody enhances the LDL-uptake in HepG2 cells

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target to reduce lipids. In 2020, we reported a chimeric camelid-human heavy chain antibody VHH-B11-Fc targeting PCSK9. Recently, it was verified that VHH-B11 binds one linear epitope in the PCSK9 hinge region. To enhance its druggability, we have developed a novel biparatopic B11-H2-Fc Ab herein. Thereinto, surface plasmon resonance (SPR) confirmed the epitope differences in binding-PCSK9 among VHH-B11, VHH-H2 and the approved Repatha. Additionally, SPR revealed the B11-H2-Fc exhibits an avidity of approximately 0.036 nM for PCSK9, representing a considerable increase compared to VHH-B11-Fc (~ 0.69 nM). Moreover, we found the Repatha and B11-H2-Fc exhibited > 95% PCSK9 inhibition efficiency compared to approximately 48% for the VHH-Fc at 7.4 nM (P < 0.0005). Further, we verified its biological activity using the human hepatoma cells G2 model, where the B11-H2-Fc exhibited almost 100% efficiency in PCSK9 inhibition at only 0.75 μM. The immunoblotting results of low-density lipoprotein cholesterol (LDL-c) uptake assay also demonstrated the excellent performance of B11-H2-Fc on recovering the LDL-c receptor (LDLR), as strong as the Repatha (P > 0.05). These findings provide the first evidence of the efficacy of a novel Ab targeting PCSK9 in the field of lipid-lowering drugs.

PCSK9 inhibition attenuates alcohol-associated neuronal oxidative stress and cellular injury

Alcohol Use Disorder (AUD) is a persistent condition linked to neuroinflammation, neuronal oxidative stress, and neurodegenerative processes. While the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has demonstrated effectiveness in reducing liver inflammation associated with alcohol, its impact on the brain remains largely unexplored. This study aimed to assess the effects of alirocumab, a monoclonal antibody targeting PCSK9 to lower systemic low-density lipoprotein cholesterol (LDL-C), on central nervous system (CNS) pathology in a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for six weeks in 32 male rats subjected to a 35 % ethanol liquid diet or a control liquid diet (n = 8 per group). The study evaluated PCSK9 expression, LDL receptor (LDLR) expression, oxidative stress, and neuroinflammatory markers in brain tissues. Chronic ethanol exposure increased PCSK9 expression in the brain, while alirocumab treatment significantly upregulated neuronal LDLR and reduced oxidative stress in neurons and brain vasculature (3-NT, p22phox). Alirocumab also mitigated ethanol-induced microglia recruitment in the cortex and hippocampus (Iba1). Additionally, alirocumab decreased the expression of pro-inflammatory cytokines and chemokines (TNF, CCL2, CXCL3) in whole brain tissue and attenuated the upregulation of adhesion molecules in brain vasculature (ICAM1, VCAM1, eSelectin). This study presents novel evidence that alirocumab diminishes oxidative stress and modifies neuroimmune interactions in the brain elicited by chronic ethanol exposure. Further investigation is needed to elucidate the mechanisms by which PCSK9 signaling influences the brain in the context of chronic ethanol exposure.

Targeting PCSK9 to upregulate MHC-II on the surface of tumor cells in tumor immunotherapy.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), the last member of the proprotein convertase family, functions as a classic regulator of low-density lipoprotein (LDL) by interacting with low-density lipoprotein receptor (LDLR). Recent studies have shown that PCSK9 can affect the occurrence and development of tumors and can be used as a novel therapeutic target. However, a comprehensive pan-cancer analysis of PCSK9 has yet to be conducted. The potential oncogenic effects of PCSK9 in 33 types of tumors were explored based on the datasets of The Cancer Genome Atlas (TCGA) dataset. In addition, the immune regulatory role of PCSK9 inhibition was evaluated via in vitro cell coculture and the tumor-bearing mouse model. Finally, the antitumor efficacy of targeted PCSK9 combined with OVA-II vaccines was verified. Our results indicated that PCSK9 was highly expressed in most tumor types and was significantly correlated with late disease stage and poor prognosis. Additionally, PCSK9 may regulate the tumor immune matrix score, immune cell infiltration, immune checkpoint expression, and major histocompatibility complex expression. Notably, we first found that dendritic cell (DC) infiltration and major histocompatibility complex-II (MHC-II) expression could be upregulated by PCSK9 inhibition and improve CD8+ T cell activation in the tumor immune microenvironment, thereby achieving potent tumor control. Combining PCSK9 inhibitors could enhance the efficacies of OVA-II tumor vaccine monotherapy. Conclusively, our pan-cancer analysis provided a more comprehensive understanding of the oncogenic and immunoregulatory roles of PCSK9 and demonstrated that targeting PCSK9 could increase the efficacy of long peptide vaccines by upregulating DC infiltration and MHC-II expression on the surface of tumor cells. This study reveals the critical oncogenic and immunoregulatory roles of PCSK9 in various tumors and shows the promise of PCSK9 as a potent immunotherapy target.

Small-interfering RNA targeting proprotein convertase subtilisin/kexin type 9 might promote fatty liver disease and hepatocellular carcinoma through upregulation of CD36.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipoprotein (LDL) receptor and fatty acid translocase CD36, inducing lysosomal degradation of these two receptors in the liver cells. Both monoclonal antibody (mAb) and small-interfering RNA (siRNA) targeting PCSK9 have been designed for treatment of familial hypercholesterolemia recently, with elevating LDL receptors on the liver cell surface and increasing LDL uptake as the main beneficial mechanism. However, given that the binding domains of PCSK9 for LDL receptor and CD36 are different, and PCSK9 mAb only attacks the domain for LDL receptor, CD36 expression remains partially controlled under PCSK9 mAb treatment. In contrast, PCSK9 siRNA brings on complete loss of PCSK9, resulting in overexpression of CD36. Based on the fact that CD36 is a key factor in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and subsequent hepatocellular carcinoma (HCC), the risk of developing NAFLD and HCC on long-term use of PCSK9 siRNA is thus raised as a hypothesis. Additionally, because CD36 is also involved in the promotion of malignant diseases other than HCC, such as acute myeloid leukemia, gastric cancer, breast cancer, and colorectal cancer, the speculative danger of flourishing these malignancies by PCSK9 siRNA is discussed as well.

Targeting PCSK9 and Beyond for the Management of Low-Density Lipoprotein Cholesterol.

Reducing low-density lipoprotein cholesterol (LDL-C) levels is crucial to the prevention of atherosclerotic cardiovascular disease (ASCVD). However, many patients, especially those at very high ASCVD risk or with familial hypercholesterolemia (FH), do not achieve target LDL-C levels with statin monotherapy. The underutilization of novel lipid-lowering therapies (LLT) globally may be due to cost concerns or therapeutic inertia. Emerging approaches have the potential to lower LDL-C and reduce ASCVD risk further, in addition to offering alternatives for statin-intolerant patients. Shifting the treatment paradigm towards initial combination therapy and utilizing novel LLT strategies can complement existing treatments. This review discusses innovative approaches including combination therapies involving statins and agents like ezetimibe, bempedoic acid, cholesterol ester transfer protein (CETP) inhibitors as well as strategies targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) and angiopoietin-like protein 3 (ANGPTL3) inhibition. Advances in nucleic acid-based therapies and gene editing are innovative approaches that will improve patient compliance and adherence. These strategies demonstrate significant LDL-C reductions and improved cardiovascular outcomes, offering potential for optimal LDL-C control and reduced ASCVD risk. By addressing the limitations of statin monotherapy, these approaches provide new management options for elevated LDL-C levels.

Pleiotropy of PCSK9: Functions in Extrahepatic Tissues.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a central role in the metabolism of LDL receptors and mainly acts in the liver. However, there are accumulating data that PCSK9 involves in several functions in different organs beyond the liver. Herein we aimed to summarize the effects of PCSK9 in tissues other than the liver. PCSK9 has crucial roles in heart, brain and kidney in addition to the cholesterol metabolism. Targeting PCSK9 for the treatment of hypercholesterolemia is effective in the prevention from cardiovascular diseases and PCSK9 inhibitors are getting to be administered in more cases. Therefore understanding the effects of PCSK9 in other tissues gained importance in the use of PCSK9 inhibitors era. PCSK9 participates in cardiac, renal, and neurologic functions however, current literature reveals that use of PSCSK9 inhibitors have beneficial or neutral effects on these organs. Inhibition of PCSK9 is assigned to be associated with new onset diabetes in experimental studies whereas real world data with PCSK9 inhibitors established no relationship between PCSK9 inhibitors and new onset diabetes. PCSK9 might be used as a target for the treatment of nephrotic syndrome and heart failure in the future.

Effect of Proprotein Convertase Subtilisin/Kexin Type 9 Inhibition on Podocytes in Mouse Nephrotic Syndrome

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is known to play a crucial role in dyslipidemia, and an increase in serum PCSK9 levels has also been reported in patients with nephrotic syndrome (NS). However, the specific effects of PCSK9 in kidney disease and the therapeutic potential of targeting PCSK9 in NS remain elusive. We thus investigated the effects of evolocumab (EVO) in mice with adriamycin (ADR)-induced NS. Male BALB/c mice were divided into the following 4 groups: Control, N = 11; EVO (monoclonal antibody for PCSK9), N = 11; ADR, N = 11; and ADR+EVO, N = 11. We also performed in vitro experiments using immortalized murine podocyte cells to validate the direct effects of PCSK9 on podocytes. EVO decreased urinary albumin levels and ameliorated podocytopathy in mice with ADR nephropathy. Further, EVO suppressed the Nod-like receptor protein 3 (NLRP3) inflammasome pathway in podocytes. PCSK9 expression upregulated CD36, a scavenger receptor of oxidized low-density lipoprotein (Ox-LDL), which in turn stimulated the absorption of Ox-LDL in vitro. EVO downregulated CD36 expression in podocytes both in vitro and in vivo. Immunofluorescence staining analysis reveals that CD36 and PCSK9 colocalized in the glomerular tufts of mice with ADR nephropathy. In the patients with focal segmental glomerulosclerosis, the CD36+ area in glomerular tufts increased compared with those diagnosed with minor glomerular abnormalities. This study revealed that EVO ameliorated mouse ADR nephropathy through the regulation of CD36 and NLRP3 inflammasome signaling. EVO treatment represents a potential therapeutic strategy for human NS.

New Approaches for Targeting PCSK9: Small-Interfering Ribonucleic Acid and Genome Editing.

There is overwhelming clinical and genetic evidence supporting the concept that low-density-lipoprotein cholesterol should be as low as possible for as long as possible in patients at very high cardiovascular risk. Despite the wide availability of effective lipid-lowering therapies, the majority of patients still fail to reach guideline-based lipid goals. Advances in novel approaches targeting PCSK9 (proprotein convertase subtilisin/kexin type 9) through small-interfering RNA and genome editing hold the potential to bridge this gap, by offering long-acting alternatives, which may overcome adherence and other challenges in the current chronic care model. In this review, we discuss the history of targeting PCSK9 with the use of mRNA and small-interfering ribonucleic acid. We also shed light on targeting PCSK9 with genome editing, including discussion of the VERVE-101 clustered regularly interspaced short palindromic repeats-base editing medicine currently being evaluated in a clinical trial and others in development.

Targeting PCSK9 reduces cancer cell stemness and enhances antitumor immunity in head and neck cancer

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been demonstrated to play a critical role in regulating cholesterol homeostasis and Tcell antitumor immunity. However, the expression, function, and therapeutic value of PCSK9 in head and neck squamous cell carcinoma (HNSCC) remain largely unexplored. Here, we found that the expression of PCSK9 was upregulated in HNSCC tissues, and higher PCSK9 expression indicated poorer prognosis in HNSCC patients. We further found that pharmacological inhibition or siRNA downregulating PCSK9 expression suppressed the stemness-like phenotype of cancer cells in an LDLR-dependent manner. Moreover, PCSK9 inhibition enhanced the infiltration of CD8+ Tcells and reduced the myeloid-derived suppressor cells (MDSCs) in a 4MOSC1 syngeneic tumor-bearing mouse model, and it also enhanced the antitumor effect of anti-PD-1 immune checkpoint blockade (ICB) therapy. Together, these results indicated that PCSK9, a traditional hypercholesterolemia target, may be a novel biomarker and therapeutic target to enhance ICB therapy in HNSCC.

Efficacy and Safety of an Investigational Single-Course CRISPR Base-Editing Therapy Targeting PCSK9 in Nonhuman Primate and Mouse Models.

VERVE-101 is an investigational in vivo CRISPR base-editing medicine designed to alter a single DNA base in the PCSK9 gene, permanently turn off hepatic protein production, and thereby durably lower low-density lipoprotein cholesterol. We test the efficacy, durability, tolerability, and potential for germline editing of VERVE-101 in studies of nonhuman primates and a murine F1 progeny study. Cynomolgus monkeys were given a single intravenous infusion of a vehicle control (n=10) or VERVE-101 at a dose of 0.75 mg/kg (n=4) or 1.5 mg/kg (n=22) with subsequent follow-up up to 476 days. Two studies assessed the potential for germline editing, including sequencing sperm samples from sexually mature male nonhuman primates treated with VERVE-101 and genotyping offspring from female mice treated with the murine surrogate of VERVE-101 (VERVE-101mu). Liver biopsies 14 days after dosing noted mean PCSK9 editing of 46% and 70% in monkeys treated with VERVE-101 at 0.75 and 1.5 mg/kg, respectively. This translated into mean reductions in blood PCSK9 (proprotein convertase subtilisin/kexin type 9) of 67% and 83% and reductions of low-density lipoprotein cholesterol of 49% and 69% at the 0.75 and 1.5 mg/kg doses, respectively, assessed as time-weighted average change from baseline between day 28 and up to 476 days after dosing. Liver safety monitoring noted a transient rise in alanine aminotransferase and aspartate aminotransferase concentrations after infusion that fully resolved by day 14 with no accompanying change in total bilirubin. In a subset of monkeys necropsied 1 year after dosing, no findings related to VERVE-101 were identified on macroscopic and histopathologic assessment of the liver and other organs. In the study to assess potential germline editing of male nonhuman primates, sperm samples collected after VERVE-101 dosing showed no evidence of PCSK9 editing. Among 436 offspring of female mice treated with a saturating dose of VERVE-101mu, the PCSK9 edit was transmitted in 0 of 436 animals. VERVE-101 was well tolerated in nonhuman primates and led to 83% lower blood PCSK9 protein and 69% lower low-density lipoprotein cholesterol with durable effects up to 476 days after dosing. These results have supported the initiation of a first-in-human clinical trial in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease.

Inhibition of PCSK9 enhances the antitumor effect of PD-1 inhibitor in colorectal cancer by promoting the infiltration of CD8+ T cells and the exclusion of Treg cells.

Immunotherapy especially immune checkpoint inhibitors (ICIs) has brought favorable clinical results for numerous cancer patients. However, the efficacy of ICIs in colorectal cancer (CRC) is still unsatisfactory due to the poor median progression-free survival and overall survival. Here, based on the CRC models, we tried to elucidate novel relapse mechanisms during anti-PD-1 therapy. We found that PD-1 blockade elicited a mild antitumor effect in these tumor models with both increased CD8+ T cells and Treg cells. Gene mapping analysis indicated that proprotein convertase subtilisin/kexin type 9 (PCSK9), low-density lipoprotein receptor, transforming growth factor-β (TGF-β), and CD36 were unexpectedly upregulated during PD-1 blockade. To investigate the critical role of these proteins especially PCSK9 in tumor growth, anti-PCSK9 antibody in combination with anti-PD-1 antibody was employed to block PCSK9 and PD-1 simultaneously in CRC. Data showed that neutralizing PCSK9 during anti-PD-1 therapy elicited a synergetic antitumor effect with increased CD8+ T-cell infiltration and inflammatory cytokine releases. Moreover, the proportion of Treg cells was significantly reduced by co-inhibiting PCSK9 and PD-1. Overall, inhibiting PCSK9 can further enhance the antitumor effect of anti-PD-1 therapy in CRC, indicating that targeting PCSK9 could be a promising approach to potentiate ICI efficacy.

†Efficacy and Safety of Inclisiran in Patients with Established Cerebrovascular Disease: Pooled Post hoc Analysis of the Phase 3 ORION-9, ORION-10, and ORION-11 Randomized Controlled Trials

<h3>Lead Author's Financial Disclosures</h3> Wolfgang Koenig, MD has received consulting fees and lecture fees from AstraZeneca, Novartis and Amgen; consulting fees from Pfizer, the Medicines Company, DalCor Pharmaceuticals, Genentech, Esperion, OMEICOS, Novo Nordisk, LIB Therapeutics, Kowa, Corvidia Therapeutics and Daiichi Sankyo; lecture fees from Berlin- Chemie, Bristol-Myers Squibb and Sanofi; and grant support and provision of reagents from Singulex, Abbott, Roche Diagnostics and Dr. Beckmann Pharma. <h3>Study Funding</h3> Novartis Pharma AG, Basel, Switzerland. <h3>Background/Synopsis</h3> Patients with hyperlipidemia and established cerebrovascular disease (CeVD) are at increased risk of future strokes or other cardiovascular events (1). In ischemic stroke survivors, statins and inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce recurrent cardiovascular events, including stroke (2,3). With guidelines advocating increasingly lower low-density lipoprotein cholesterol (LDL-C) goals, adding lipid-lowering therapies to statins may be needed. Inclisiran, a first-in-class small interfering RNA targeting PCSK9 messenger RNA, when added to maximally tolerated statin therapy for patients with established CeVD, may provide further LDL-C lowering, with a convenient, infrequent dosing schedule (4). <h3>Objective/Purpose</h3> To assess the efficacy and safety of inclisiran in patients with established CeVD. <h3>Methods</h3> This was a post hoc analysis of patients with heterozygous familial hypercholesterolemia or atherosclerotic cardiovascular disease or its risk equivalents and established CeVD (ischemic stroke, carotid artery stenosis by angiography or ultrasound greater than 70%, and/or percutaneous or surgical carotid artery revascularization) from ORION-9 (NCT03397121), ORION-10 (NCT03399370), and ORION-11 (NCT03400800), randomized 1:1 to receive inclisiran sodium 300 mg (equivalent to 284mg inclisiran) or placebo at Day 1, Day 90, and 6 monthly thereafter to Day 540. Evaluations included LDL-C percentage change from baseline to Day 510 and corresponding time-averaged percentage change from baseline after Day 90 to Day 540; safety was assessed over 540 days. <h3>Results</h3> Of 202 patients with established CeVD at baseline, 90.0% (99/110) of patients receiving inclisiran and 84.8% (78/92) on placebo reported prior ischemic stroke; others reported prior carotid artery stenosis and/or carotid revascularization (Table 1). Mean (95% CI) placebo- corrected LDL-C percentage change from baseline at Day 510 with inclisiran was −55.2% (−64.5 to −45.9); corresponding time-averaged change from baseline between Days 90 and 540 was −55.2% (−62.4 to −47.9) (P less than 0.0001 for both; Table 2). Treatment-emergent adverse events (TEAEs) and serious TEAEs were more frequent with inclisiran vs placebo. Clinically relevant injection site TEAEs were more frequent with inclisiran (3.6% [4/110]) vs placebo (0% [0/92]) and none was severe. Few patients had clinically relevant laboratory measurements (Table 2). <h3>Conclusions</h3> In patients with established CeVD, twice-yearly dosing with inclisiran (after the initial and 3-month doses) provided a sustained additional LDL-C reduction of ∼55% and a modest increase in mild/moderate injection site TEAEs. The cardiovascular benefits of inclisiran in patients with established CeVD are being evaluated in ongoing trials.

†Efficacy and Safety of Inclisiran in Patients with Polyvascular Disease: Pooled Post hoc Analysis of the phase 3 ORION-9, ORION-10, and ORION-11 Randomized Controlled Trials

<h3>Lead Author's Financial Disclosures</h3> Wolfgang Koenig, MD has received consulting fees and lecture fees from AstraZeneca, Novartis and Amgen; consulting fees from Pfizer, the Medicines Company, DalCor Pharmaceuticals, Genentech, Esperion, OMEICOS, Novo Nordisk, LIB Therapeutics, Kowa, Corvidia Therapeutics and Daiichi Sankyo; lecture fees from Berlin- Chemie, Bristol-Myers Squibb and Sanofi; and grant support and provision of reagents from Singulex, Abbott, Roche Diagnostics and Dr. Beckmann Pharma. <h3>Study Funding</h3> Novartis Pharma AG, Basel, Switzerland. <h3>Background/Synopsis</h3> Approximately 25% of patients with atherosclerotic cardiovascular disease (ASCVD) have polyvascular disease (PVD) involving 2 or more coronary, cerebrovascular, and peripheral artery beds. PVD is an independent predictor of major adverse cardiovascular events (MACE) and death. Agents that inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein cholesterol (LDL-C) concentrations and MACE incidence in patients with PVD. Inclisiran is a small interfering RNA agent targeting PCSK9 messenger RNA that is well tolerated and provides effective and sustained LDL-C reductions. <h3>Objective/Purpose</h3> To describe the effect of inclisiran vs placebo in patients with and without PVD. <h3>Methods</h3> This was a post hoc analysis of patients with heterozygous familial hypercholesterolemia or ASCVD or its risk equivalents randomized 1:1 to 300mg inclisiran sodium (equivalent to 284mg inclisiran) or placebo at baseline, Day 90, and 6 monthly thereafter in ORION-9 (NCT03397121), ORION-10 (NCT03399370), and ORION-11 (NCT03400800). Evaluations included LDL-C percentage change from baseline to Day 510 and corresponding time-averaged change from Day 90 to Day 540, with both evaluated by PVD presence or absence, and safety over 540 days. <h3>Results</h3> Baseline characteristics for 470 patients (13.6%) with PVD and 2984 (86.4%) without PVD were generally balanced between treatment arms in both groups, with higher cardiovascular risk factors and lower LDL-C concentrations in patients with PVD (Table 1). Mean (95% CI) placebo-corrected LDL-C percentage changes from baseline to Day 510 with inclisiran were −48.9% (−55.6 to −42.2) in patients with PVD and −51.5% (−53.9 to −49.1) in those without (Table 2). Treatment-emergent adverse events (TEAEs) and treatment-emergent serious adverse events (TESAEs) were similar between treatment arms irrespective of PVD status, although reported TESAEs trended higher with PVD in both treatment arms (Table 2). Clinically relevant injection site TEAEs were more frequent with inclisiran vs placebo in both groups; all were mild or moderate. Few patients had clinically relevant laboratory measurements (Table 2). <h3>Conclusions</h3> Twice-yearly dosing with inclisiran (after the initial and 3-month doses) provided effective and sustained LDL-C lowering in patients with ASCVD irrespective of their PVD status, with a safety profile similar to that of placebo except for a modest increase in mainly mild TEAEs at the injection site. Notably, TESAEs were reported more frequently in patients with PVD, which was likely due to their more advanced disease. Since patients with PVD are at high risk of cardiovascular events, intensive LDL-C lowering may be beneficial to reduce this risk.

Targeting PCSK9 Ameliorates Graft Vascular Disease in Mice by Inhibiting NLRP3 Inflammasome Activation in Vascular Smooth Muscle Cells.

BackgroundGraft vascular disease (GVD), which limits the long-term survival of patients after solid-organ transplantation, is associated with both immune responses and nonimmune factors, including dyslipidemia. Recent studies have shown that inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), a U.S. Federal Drug Administration-approved treatment for hyperlipidemia, reduces cardiovascular events, regulates inflammatory responses, and enhances the efficacy of immune checkpoint therapy in cancer treatment through a cholesterol-independent mechanism. However, whether targeting PCSK9 is a potential therapeutic strategy for GVD remains unknown.MethodsSerum samples and grafts were harvested from male mice undergoing abdominal aortic transplantation. The pathological alterations in the aortic grafts were detected by hematoxylin and eosin staining, Verhoeff’s Van Gieson staining, and Masson staining. Inflammatory cell infiltration and proinflammatory cytokine expression in the aortic grafts were detected by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The regulatory effects of PCSK9 on vascular smooth muscle cell (VSMC) migration and proliferation were examined by transwell, EdU, and western blot assays. The effect of Evolocumab, a PCSK9 inhibitor, on GVD in humanized PCSK9 mice was also evaluated.ResultsPCSK9 was upregulated in the serum, grafts, and liver of mice in the allograft group subjected to abdominal aortic transplantation. Pcsk9 knockout significantly reduced vascular stenosis, the intimal hyperplasia area and collagen deposition. Pcsk9 depletion also inhibited macrophage recruitment and the mRNA expression of proinflammatory cytokines in aortic grafts. Furthermore, Pcsk9 knockout suppressed the migration and proliferation of VSMCs, which was related to the inhibition of NLRP3 inflammasome activation. Meanwhile, Evolocumab significantly ameliorated GVD in humanized PCSK9 mice.ConclusionPCSK9 is upregulated in a mouse model of GVD, and Pcsk9 knockout reduces vascular occlusion, suggesting that PCSK9 may be a promising target for the treatment of GVD.

Gene Therapy Targeting PCSK9.

The last decades of research in cardiovascular prevention have been characterized by successful bench-to-bedside developments for the treatment of low-density lipoprotein (LDL) hypercholesterolemia. Recent examples include the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with monoclonal antibodies, small interfering RNA and antisense RNA drugs. The cumulative effects of LDL cholesterol on atherosclerosis make early, potent, and long-term reductions in LDL cholesterol desirable—ideally without the need of regular intake or application of medication and importantly, without side effects. Current reports show durable LDL cholesterol reductions in primates following one single treatment with PCSK9 gene or base editors. Use of the CRISPR/Cas system enables precise genome editing down to single-nucleotide changes. Provided safety and documentation of a reduction in cardiovascular events, this novel technique has the potential to fundamentally change our current concepts of cardiovascular prevention. In this review, the application of the CRISPR/Cas system is explained and the current state of in vivo approaches of PCSK9 editing is presented.

Lipid Nanoparticles Delivery of CRISPR/Cas9 Targeting PCSK9 and ANGTPL3 as New Therapeutic Gene Editing Modalities for Potential Long-Lasting Treatment Of Dyslipidemia

Dyslipidemia is the main risk factor for atherosclerosis leading to cardiovascular disease, one of the important health problems in the Asia Pacific region. Several dyslipidemia treatment modalities such as statins and monoclonal antibodies were considered less effective both from the aspect of cost or toxic side effect. The aim of this study is to describe the potential of lipid nanoparticle-mediated efficient delivery of clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (CRISPR/Cas9) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) and angiopoietin-like protein 3 (ANGPTL3) as new therapeutic genome editing modalities for potential long-lasting treatment of dyslipidemia. The method used in this study is to explore the literature in the form of systematic reviews, meta-analysis, and randomized control trials (RCTs) through several search engines such as Sciencedirect, Pubmed, and Google Schoolar in the last 10 years. The outcome of this study is to review the effectiveness of PCSK9 and ANGPTL3 inhibition in lowering cholesterol levels making both genes as a major therapeutic target for the treatment of dyslipidemia. Currently, an efficient way to permanently inhibit both genes has been developed using CRISPR-Cas9 genome editing delivered by lipid nanoparticles (LNPs), the most effective non-viral delivery modalities that work specifically on the liver. A single administration of LNPs-CRISPR/Cas9 in mice produced undetectable PCSK9 serum levels more than 80% and a drop of total cholesterol by 35%-40%. Meanwhile, CRISPR/Cas9 targeting ANGPTL3 resulted in a greater decrease in triglycerides on 7 day post-treatment. As a conclusion, genome editing therapy based on CRISPR/Cas9 lipid nanoparticles targeting PCSK9 and ANGPTL3 is promising for the treatment of dyslipidemia.

Abstract 9747: Safety, Pharmacokinetics and Pharmacodynamics of Multiple Ascending Doses of AZD8233, Targeting PCSK9, in Patients with Dyslipidemia

Introduction: Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces cardiovascular risk by lowering low-density lipoprotein-cholesterol (LDL-C) levels. AZD8233 is an antisense oligonucleotide in development for the treatment of dyslipidemia. In a single ascending dose study (NCT03593785), AZD8233 was well tolerated and effectively reduced PCSK9 and LDL-C levels in patients with elevated LDL-C. Here, we report results from a multiple ascending dose study of AZD8233. Methods: This was a phase 1, randomized, single-blind, placebo-controlled study (NCT04155645) in patients with dyslipidemia. Participants were receiving statins for ≥ 3 months, had an LDL-C in the range 70-190 mg/dL, and were 18-65 years old. Three cohorts of 11 participants received four subcutaneous injections over an 8-week period (days 1, 8, 29, and 57) of either AZD8233 (15 mg, 30 mg, or 90 mg; n = 8) or placebo (n = 3), and were followed up for 16 weeks post dose. The primary objective was to assess safety and tolerability; secondary objectives included assessment of pharmacokinetics and pharmacodynamics of AZD8233; circulating LDL-C was estimated using the Friedewald formula. Results: Overall, 34 patients were randomized and data are available from 33 patients; 15 were male, the mean age was 58 years, and baseline PCSK9 and LDL-C levels were comparable across treatment groups. Treatment with AZD8233 did not result in any clinically relevant safety or tolerability findings. Peak plasma concentrations of AZD8233 were reached within 0.5 to 5 hours post dosing, followed by a biphasic decline. A decrease from baseline in PCSK9 (69.7%, 79.9%, and 95.3%) and LDL-C levels (63.8%, 65.7%, and 83.3%) was observed at 12 weeks for the AZD8233 15 mg, 30 mg, and 90 mg groups, respectively vs placebo ( Figure ). Conclusions: Multiple doses of AZD8233 were generally safe, well tolerated, and reduced PCSK9 and LDL-C levels in a dose-dependent manner in statin-treated patients with dyslipidemia.

Inclisiran: a small interfering RNA strategy targeting PCSK9 to treat hypercholesterolemia

ABSTRACT Introduction Inclisiran is a novel posttranscriptional gene silencing therapy that inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9) synthesis by RNA interference and has a potent, dose-dependent, durable effect in lowering LDL-C, and therefore is an effective drug to treat dyslipidemia, reducing the risk for acute cardiovascular (CV) events. It is safe and well-tolerated. Areas covered This paper aims to review the mechanism of action of inclisiran while evaluating its efficacy and safety in the treatment of dyslipidemia from data of the clinical trials in the ORION program. Expert opinion Data from the clinical trials in the ORION program demonstrated efficacy and safety of inclisiran in patients with dyslipidemia. Adverse events were similar in the inclisiran and placebo groups in the clinical trials, although injection-site reactions were more frequent with inclisiran than with placebo. Although the combination of efficacy and safety makes inclisiran a good option for the treatment of dyslipidemia compared to other PCSK9 targeting therapeutic strategies, however, further studies should exclude the possibility that inclisiran, through lower-affinity interactions, may influence other mRNAs in the physiological milieu.