VERVE-101, a CRISPR base-editing therapy designed to permanently inactivate hepatic PCSK9 and reduce LDL-cholesterol

KEYWORDS:

• Base editing
• CRISPR
• LDL
• LDL-cholesterol
• PCSK9
• PCSK9 inhibitor
• VERVE-101
• VERVE-102

1. Introduction

Just over two decades ago, a new member of the secretory subtilase family, neural apoptosis-regulated convertase 1 (NARC-1), now known as proprotein convertase subtilisin/kexin type 9 (PCSK9), was discovered by Seidah et al. [1]. The PCSK9 gene, located on chromosome 1p32, encodes a 692 amino acid proteinase K-like subtilase, which is mainly expressed in hepatocytes and enterocytes. Three primary domains in PCSK9, namely the prodomain, catalytic, and C-terminal domain, are preceded by an N-terminal signal peptide that targets the protein for secretion, before being cleaved, releasing the functional protein from the endoplasmic reticulum. Interestingly, PCSK9 only acts once as a protease, during the autocatalytic processing of the N-terminal domain; its role in LDL-receptor regulation is as a chaperone protein in targeting the LDL-receptor to lysosomes for subsequent degradation [2].

The discovery of rare ‘gain-of-function’ missense variants causing hypercholesterolemia in two French families established PCSK9 as the third causative gene for familial hypercholesterolemia (FH), a condition associated with premature atherosclerotic cardiovascular disease (ASCVD), primarily coronary artery disease (CAD), and death [3]. Subsequent studies demonstrated that more common ‘loss-of-function’ PCSK9 variants lower LDL-cholesterol and protect against ASCVD. Cohen et al. sequenced PCSK9 in individuals with low plasma LDL-cholesterol and identified two nonsense variants, Y142X and C679X [4]. These variants were found in 2.6% of African Americans and associated with a 28% reduction in LDL-cholesterol and an 88% reduction in the risk of coronary heart disease [5]. Similarly, in a study of 653 individuals in antenatal clinics in Zimbabwe, 24 participants (3.6%) with the C679X variant were identified and found to have a 27% decrease in LDL-cholesterol [6]. Rare individuals with compound heterozygous or homozygous ‘loss of function’ PCSK9 variants were found to have extremely low LDL-cholesterol (15 mg/dL or 0.4 mmol/L) but were otherwise well, with normal reproductive capacity [6,7]. These discoveries paved the way for the development of the first-generation PCSK9 inhibitors, namely alirocumab and evolocumab, in the form of human monoclonal antibodies, as a means of increasing LDL-receptor availability, to lower plasma LDL-cholesterol.

The current AHA/ACC guidelines on the management of blood cholesterol describe a role for PCSK9 inhibitors in very high-risk ASCVD patients [8]. In this cohort with LDL-cholesterol ≥1.8 mmol/L or non-HDL-cholesterol ≥2.6 mmol/L on maximally tolerated LDL-cholesterol lowering therapy, PCSK9 inhibitors can be considered. The first randomized controlled outcomes trial, FOURIER, enrolled over 27,000 patients with ASCVD with LDL-cholesterol ≥1.8 mmol/L on optimized statin therapy. At just 48 weeks of follow-up, major adverse cardiac events (MACE) were significantly lower in the group treated with subcutaneous injections of evolocumab (hazard ratio 0.85; 0.79–0.92, p < 0.001) [9]. The degree of risk reduction for MACE increased over time, from 12% in the first year to 19% thereafter. The ODYSSEY OUTCOMES trial showed comparably low rates of MACE among patients treated with alirocumab, and LDL-cholesterol similarly reduced by ~60% from baseline [10]. Small interfering RNA (siRNA) is another strategy to target PCSK9; by binding to PCSK9 mRNA in hepatocytes, its expression is silenced. In the ORION-10 and ORION-11 trials, the siRNA inclisiran resulted in approximately 50% reductions in LDL-cholesterol from baseline [11]. The above reductions in LDL-cholesterol were sustained up to 1.5 years for alirocumab and inclisiran. Further developments in PCSK9 inhibition include research into small molecules and vaccines [12]. The lower cost and ease of administration of small molecules are, however, juxtaposed against the challenges in their design. Peptide-based vaccines prevent PCSK9 from binding to the LDL-receptor or via structural breakdown of PCSK9.

In the search for PCSK9-targeted therapies that offer both efficacy and sustainability, gene transfer and gene editing approaches have garnered considerable interest [13]. Gene transfer involves using vectors, commonly in the viral form, to transport the gene of interest as an adjunct to existing in vivo genes. Gene editing, however, conveys an editing mechanism that acts on genes directly to either reinstate or disrupt select sequences. With gene transfer therapies using the adeno-associated virus (AAV) vector, the risk of incorporation into endogenous DNA sequences is low, and importantly, AAV has no potential for replication. Gene editing therapies can be administered via lipid nanoparticle technology. Whilst the potentially long-standing effects of both approaches are advantageous, the longer-term health and safety aspects of these therapies remain paramount, as these genetic therapies cannot be withdrawn or reversed. Several gene editing techniques are currently available, however, the CRISPR/Cas9 system has gained significant attention in view of its practicality and efficacy. This method uses guide RNAs for sequence specificity and bacterial Cas proteins to facilitate breaks in double-stranded DNA. Although modeled to be specific, possible issues include interaction with alternate sites and integration of Cas DNA. The potential for insertions, deletions, and oncogenesis have placed notable boundaries with regard to clinical application of CRISPR/Cas9 systems [13].

VERVE-101 (Verve Therapeutics Inc) is an investigational drug, using CRISPR base-editing technology, which is designed to alter a single DNA base in PCSK9, to permanently turn off hepatic PCSK9 production, and hence durably lower LDL-cholesterol [14]. A ‘first-in-human’ Phase Ib, Heart-1 (VT-1001; NCT05398029), open-label, single-ascending dose study was undertaken to evaluate the safety and tolerability and pharmacodynamic profile of VERVE-101 administered to heterozygous FH (heFH) patients (aged 18–75 years) with established ASCVD and not controlled on maximal lipid-lowering treatment (LLT). Patients received pre-medication with dexamethasone and antihistamines. VERVE-101 was given as a single intravenous infusion of a CRISPR adenine base editor composed of an mRNA and a guide RNA strand targeting PCSK9 contained in a lipid nanoparticle (LNP). The genetic sequence editing involved a base pair change from A-T to G-C within a splice donor site, which effectively silenced PCSK9 and hence its expression in hepatocytes. The study is currently recruiting, with a study duration of 1 year, an estimated enrollment of 44 participants, and a completion date of December 2024. As mandated by the US FDA, participants in the Heart-1 trial will be followed for another 14 years.

Interim data from the Heart-1 clinical trial, conducted in the United Kingdom (UK) and New Zealand, were reported on 12 November 2023 at the AHA Scientific Sessions in Philadelphia, PA [14]. That dataset included 10 heFH patients (8 male, 2 female, mean age 54 years) with an average LDL-cholesterol of 193 mg/dL (5.0 mmol/L), enrolled as of a data cutoff date of 16 October 2023; six of whom were treated with VERVE-101 infusion at sub-therapeutic doses (0.1 mg/kg (n = 3) and 0.3 mg/kg (n = 3)), whereas the other four patients received potentially therapeutic doses (0.45 mg/kg (n = 3) and 0.6 mg/kg (n = 1)). One of the ten patients, who had received VERVE-101 at 0.45 mg/kg, failed to reach the minimum 28 days of follow-up and hence was excluded from the analysis.

VERVE-101 decreased circulating PCSK9 levels in two patients who received the 0.45 mg/kg dose by 59% and 84%, respectively, whereas a 47% reduction was observed in the single patient at the 0.6 mg/kg dose. Time-averaged LDL-cholesterol was lowered by 39% and 48% with the 0.45 mg/kg VERVE-101 dose, and a sustained 55% reduction, which extended out to 6 months, was observed with the 0.6 mg/kg dose.

Transient mild-to-moderate infusion-related reactions and transient, asymptomatic liver enzyme (ALT) elevations (mean peak > 5× the upper reference limit within the first week after dosing) with normal bilirubin levels, at the higher doses of VERVE-101. Two out of the ten patients experienced a total of three cardiovascular serious adverse events, two of which were deemed to be unrelated to the study. One patient, with history of ischemic cardiomyopathy and previous cardiac arrest, who received the 0.3 mg/kg dose, had a fatal cardiac arrest ~5 weeks post-VERVE-101 infusion, which was deemed unrelated to the study treatment. The other patient, with an unreported history of unstable symptomatic chest pain, had a myocardial infarction 1 day after receiving VERVE-101 at a dose of 0.45 mg/kg, which was thought to be potentially related to the study treatment, in addition to non-sustained ventricular tachycardia, which occurred more than 4 weeks post-infusion, and which was considered unrelated.

Thirteen patients, six of whom had been dosed with VERVE-101 at the 0.45 mg/kg, were enrolled in Heart-1 as of a data cut-off date of 18 March 2024 [15]. One of the six patients, who received VERVE-101 at 0.45 mg/kg, failed to reach the minimum 28 days of follow-up and hence was excluded from the interim analysis, which showed an average LDL-cholesterol reduction of 46% (range of 21 to 73%), with sustained reductions out to 270 days, for those with the longest follow-up. However, on 2 April 2024, Verve announced, that in consultation with its independent data and safety monitoring board, it had halted enrollment into the Heart-1 clinical trial, after the sixth patient who received VERVE-101 in the 0.45 mg/kg cohort experienced a serious drug-related adverse event, namely transient ALT elevation as well as thrombocytopenia, which occurred within the first 4 days after dosing, and which fully resolved after a few days [15]. Verve reported that it is investigating the laboratory abnormalities and, depending on the findings, will work with regulatory authorities to try to pave a way forward for VERVE-101.

As PCSK9 is thought to exert a pro-thrombotic effect in patients with cardiometabolic disease, one would expect that the opposite would hold true for PCSK9 inhibition [16]. So why in the Heart-1 clinical trial did some heFH patients develop cardiovascular adverse events, thrombocytopenia, and elevated ALT levels relatively soon after receiving an infusion of VERVE-101? In 2020, the sudden onset of severe thrombocytopenia was observed with the clinical use of evolocumab in a patient with CAD, which provided the first indication for careful monitoring of changes in platelet counts during PCSK9 inhibitor therapy [17].

PCSK9 is known to target the free fatty acid (FFA) transporter and scavenger receptor, CD36, and VLDL receptor (VLDLR) for degradation [2]. Thus, PCSK9 inhibition may increase levels of CD36 and/or VLDLR, resulting in an increase in FFA uptake, especially in skeletal muscle, the heart and endothelial cells, which may, in part, explain the adverse effects on the function of these tissues, and possibly also affect platelet count [18,19]. In a murine model, PCSK9-mediated CD36 degradation has been shown to limit FFA uptake and hepatic fat accumulation and to prevent liver steatosis [20]. Thus, PCSK9 inhibition could, theoretically, result in increased CD36-mediated hepatic fat uptake and an increased risk of metabolic-associated fatty liver disease.

2. Expert opinion

ASCVD is a leading cause of morbidity and mortality globally. Lowering of LDL-cholesterol is one of the most important strategies to reduce ASCVD risk. FH is a relatively common, often underdiagnosed and undertreated, dominantly inherited disorder characterized by lifelong elevation of LDL-cholesterol, associated with increased risk of premature ASCVD and death. Statins plus ezetimibe are the conventional treatment for FH; however, LDL-cholesterol targets are not achieved in a large proportion of patients. PCSK9 inhibitors significantly reduce LDL-cholesterol in individuals with FH who are receiving the maximal tolerated LLT.

CRISPR base editing, a potentially safer alternative to genome editing, allows permanent inactivation of hepatic PCSK9 production resulting in long-lasting LDL-cholesterol lowering. However, the interim analysis of the ‘proof of concept’ Heart-1 study raises some safety concerns, with transient mild-to-moderate infusion-related reactions and transient, asymptomatic ALT elevations, at the higher VERVE-101 doses, despite pre-medication with dexamethasone and antihistamines, and more importantly, two of the trial participants had cardiovascular events. However, after enrolling only 13 patients, Verve has recently halted enrollment into the Heart-1 trial, after another patient experienced a serious drug-related adverse event, with elevated ALT and thrombocytopenia, requiring hospitalization, and has now shifted their priority to the development of VERVE-102.

VERVE-102 uses the same base editor and guide mRNA as VERVE-101 but a different LNP delivery system. This delivery system includes a different ionizable lipid, which has been well tolerated in other clinical trials, and incorporates N-acetylgalactosamine (GalNAc)-LNP to target the liver [15]. VERVE-102 will be assessed in the Heart-2 trial (VT-10201; NCT06164730). This is an open-label, Phase Ib, single-ascending dose study that will evaluate the safety and pharmacodynamic profile of VERVE-102 administered to patients with heFH or premature CAD who require additional LDL-cholesterol lowering. The study is scheduled to start recruiting in the UK and Canada in May 2024, with a study duration of 1 year, an estimated enrollment of 36 participants, and a completion date of August 2026.

In conclusion, although CRISPR base-editing therapy is a promising technology, it is not without risks, including off-target effects and uncertain long-term health and safety consequences, as demonstrated with the VERVE-101 in the Heart-1 clinical trial. This contrasts with current PCSK9 inhibitors in use, which are generally well tolerated with low rates of adverse events, including those related to injection sites. With transient biochemical and hematological derangements thought to be related to the LNP delivery system, the outcomes of the Heart-2 trial with a distinct ionizable lipid and a GalNAc liver-targeting ligand are awaited with interest.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.