Objective

To investigate the safety, side-effect profile, and efficacy of single ascending doses of CTX310, a lipid-nanoparticle–encapsulated formulation of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats–Cas9 endonuclease) components for in vivo gene editing of the target gene, ANGPTL3.

Study design

Phase 1A, multicentre, open-label trial of single ascending doses of CTX310.

Study population

Adults aged 18 to 75 years with a diagnosis of uncontrolled hypercholesterolemia, moderate-to-severe hypertriglyceridemia, or mixed dyslipidemia. Subjects were refractory to maximally tolerated doses of lipid-lowering therapies, as defined by at least one of the following criteria: a fasting serum triglyceride (TG) >150 mg/dL (1.7 mmol/L), low-density lipoprotein cholesterol (LDL-C) >100 mg/dL (2.6 mmol/L) or >70 mg/dL (1.8 mmol/L) in subjects with atherosclerotic cardiovascular disease, apolipoprotein B >100 mg/dL, or non–high-density lipoprotein cholesterol (non-HDL-C) >160 mg/dL (4.1 mmol/L). Major exclusion criteria were women of childbearing potential, individuals with familial chylomicronemia syndrome with <5% lipoprotein lipase activity, or individuals with an estimated glomerular filtration rate <60 ml/minute/1.73 m² of body-surface area.

Main study variables

·       Primary safety endpoints: Incidence of adverse events as assessed by investigators, including dose-limiting toxic effects, which were graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, of the National Cancer Institute. Events were graded 1 to 5, with a higher grade indicative of greater severity.  Dose-limiting toxic effects were defined as any of the following: an increase in liver aminotransferase levels to CTCAE grade 3 or higher, with increases persisting for more than 14 days after infusion; an increase in bilirubin levels or international normalized ratio to CTCAE grade 3 or higher; any abnormal grade 3 laboratory-assessed result persisting for 7 days or longer; or any abnormal laboratory-assessed result of severity equal to CTCAE grade 4.

·       Secondary safety endpoints: Frequency and severity of adverse events, including adverse events of special interest (infusion-related reactions, abnormal bleeding, thrombotic events, or hemorrhagic events; increases in aminotransferase levels; allergic or localized reactions; or new malignant condition).

·       Secondary efficacy endpoints: Percent change from baseline over time in LDL-C, TGs, apolipoprotein B, HDL-C, and non-HDL-C.

Methods

Subjects were initially enrolled into one of four ascending CTX310-dose cohorts (0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, or 0.8 mg/kg).  Doses were calculated by the total amount of RNA administered and based on estimated lean body weight. CTX310 was administered as a single intravenous infusion. Escalation of the doses was approved by the safety review committee after a minimum of three subjects were treated at each dose level and completed at least 30 days of follow-up. Subsequently, a cohort was added to receive 0.7 mg/kg, and the cohort that received 0.8 mg/kg was expanded after the first three subjects completed CTX310 infusion, to better define the dose–response relationship.

All subjects were observed for at least 24 hours after infusion, with daily safety assessments conducted for 3 days after treatment, weekly follow-up for the first 30 days after treatment, and subsequent visits scheduled at days 60, 90, 180, 270, and 360. At the time of publication, all subjects had completed at least 60 days of follow-up.

Results

Of 27 individuals screened, 15 were eligible and received CTX310 (3 each at a dose of 0.1 mg/kg, 0.3 mg/kg or 0.6 mg/kg, 2 at 0.7 mg/kg, and 4 at 0.8 mg/kg). The median age of the study cohort was 53 years (range, 31 to 68 years); 6 (40%) had atherosclerotic cardiovascular disease, and 6 (40%) had a clinical diagnosis of familial hypercholesterolemia, including 5 with confirmed pathogenic genetic mutations. Mean (± standard deviation) LDL-C before treatment was 155±79 mg/dL and the median TG level was 192 mg/dL (interquartile range, 109 to 252 mg/dL).

Single dose infusion of CTX310 was associated with few adverse events (Table 1).

ANGPLT3 levels were reduced by a mean of 32.7% at the 0.6 mg/kg dose ranging to 79.7% at the 0.7 mg/kg dose and 73.2% at the 0.8 mg/kg dose. LDL-C and TG levels were reduced at doses of 0.6 mg/kg or higher, by up to 50% and up to 60%, respectively. Reductions in these lipid parameters appeared within the first two weeks of treatment and were sustained through to at least 60 days, the latest follow-up data included in this analysis.

Table 1. Key safety results

* One subject received CTX310 0.3 mg/kg and was hospitalized for a spinal disk herniation 7 months after treatment, and one subject died 179 days after administration of CTX310 0.1 mg/kg

** Two subjects who received CTX310 0.6 mg/kg and one who received CTX310 0.8 mg/kg

Author conclusions

Editing of ANGPTL3 was associated with few adverse events and resulted in reductions from baseline in ANGPTL3 levels.