A potent epigenetic editor targeting human PCSK9 for durable reduction of low-density lipoprotein cholesterol levels

Abstract

Epigenetic editing holds the promise of durable therapeutic effects by silencing disease-causing genes without changing the underlying DNA sequence. In this study, we designed an epigenetic editor to target human PCSK9 and thereby induce DNA methylation at this locus. A single administration of lipid nanoparticles encapsulating mRNA encoding this epigenetic editor was sufficient to drive near-complete silencing of human PCSK9 in transgenic mice. Silencing was durable for at least 1 year and was fully maintained after partial hepatectomy-induced liver regeneration. In addition, we showed reversibility of epigenetic editing in mice with previously silenced PCSK9 upon treatment with a targeted epigenetic activator designed to demethylate the PCSK9 locus. Notably, in cynomolgus monkeys, a single administration of the epigenetic editor potently and durably decreased circulating PCSK9 protein levels by approximately 90% with concomitant reduction in low-density lipoprotein cholesterol levels by approximately 70%. These findings demonstrate the therapeutic potential of durable and reversible epigenetic editing in vivo and support the development of epigenetic editor-based treatment for hypercholesterolemia.

Fig. 1. In vitro activity of EEs targeting human PCSK9 in immortalized cells and PHHs.

a, Schematic outline of the architecture of PCSK9 EEs b, Primary screen evaluating 240 candidate gRNAs targeting the human PCSK9 gene using a spCas9-based EE. Each point represents the average of two independent measurements of secreted PCSK9 protein levels 7 d after transfection; the location of each point along the x axis indicates the position of the gRNA relative to the distance (in nucleotides) to the PCSK9 gene TSS. PCSK9 protein levels in cells transfected with a non-targeting (NT) gRNA or effector only (no gRNA) are shown with a dotted line. CpG location (first row); methylation percentage (0–100%) of each CpG dinucleotide at the PCSK9 locus measured by whole-genome bisulfite sequencing (WGBS) or hybrid capture in neurons (second row); HeLa cells (third row); liver hepatocytes (fourth row); and DNaseI accessibility in human liver (fifth row) are shown below the graph and mapped onto the CpG island (CGI) and PCSK9 5′ gene region. c, The top 40 gRNA were selected from b and were evaluated for their ability to potently and durably silence PCSK9 in HeLa cells for up to 28 d. Individual data points and means are shown (n = 2 replicates per experimental condition). Results are expressed as percent of secreted PCSK9 protein in cells treated with transfection (txn) reagent only. d, The top five gRNAs were selected based on their activity and durability in HeLa cells (from c) as well as having full cross-reactivity with the cynomolgus macaque PCSK9 gene. PHHs isolated from PXB mice were treated with LNPs containing selected gRNAs and EE mRNA. Results are shown as mean ± s.d. (n = 4 replicates per gRNA). For b and c, WT Cas9 served as a control for durable silencing of PCSK9; for c and d, CRISPRi served as a control for non-durable silencing of PCSK9.

Fig. 2. Specificity of the top-ranked human PCSK9 EE in PHHs.

a. Activity of the LNP formulation with the top-ranked PCSK9 EE (PCSK9-EE) in PHHs isolated from chimeric mice with a humanized liver. Each point represents the average of three independent measurements of secreted PCSK9 protein levels at baseline and at 6 d and 15 d after treatment. Results are shown as mean ± s.d. PHHs treated with APOE only or LNP formulation containing the effector without gRNA (effector only) served as negative controls. b–f, Assessment of the specificity of PCSK9-EE was performed using PHHs obtained at 15 d after treatment. b, Specificity testing was assessed using RNA-seq on three independent replicates of each control condition (ApoE Only and Effector Only) and PCSK9-EE. On-target PCSK9 TPM from RNA-seq for each replicate are shown in the dot plot. c, Volcano plot of RNA-seq data comparing PCSK9-EE versus Effector Only control. Thresholds for differential expression: adjusted P value (DEseq2 Wald test, two-sided, Benjamini–Hochberg multiple comparisons adjustment) < 1×10⁻⁵, log₂FC > 1 or log₂FC < −1. PCSK9 is shown as a yellow circle; off-target (upregulated) DEG is shown as a navy blue circle; all other genes below the thresholds are shown as light blue circles. d, Specificity of methylation at CpG-enriched sites was measured using a Twist Human Methylome Hybrid Capture Methylation Sequencing assay. Volcano plot of CpG methylation comparing PCSK9-EE versus Effector Only control. Individual CpGs are colored according to whether they were called as a DMR at the PCSK9 locus (yellow), at an off-target genomic region (navy blue) or were not part of a DMR (light blue). DMR thresholds were set as P value (DSS Wald test, two-sided, unadjusted) < 1 × 10⁻¹⁰, beta value difference < −0.2 or beta value difference > 0.2. e, Manhattan plot of genome-wide methylation, as determined by a WGMS assay, comparing PCSK9-EE versus Effector Only control. Benjamini–Hochberg (false discovery rate (FDR)) adjusted P values for each CpG (DSS Wald test, two-sided) are plotted versus genomic coordinate for each CpG. Differentially methylated CpGs within the PCSK9 DMR are shown in yellow. The DMR threshold was set as P value (DSS Wald test, two-sided, unadjusted) < 1 × 10⁻¹⁰. f, Scatterplot showing methylation difference of DMRs from WGMS (y axis) versus log₂FC from RNA-seq (x axis) of all genes within 20 kb of each DMR for the PCSK9-EE versus Effector Only control comparison. PCSK9 gene is shown in yellow. Thresholds (gray dashed lines) are set as methylation (beta value) difference > 0.2 or < −0.2, RNA-seq log₂FC > 1 or < −1. DEG, differentially expressed gene.

Fig. 3. In vivo durability of PCSK9 silencing and effects on DNA methylation in liver.

a, Schematic outline of the in vivo study in transgenic mice (PCSK9-Tg) carrying the human PCSK9 genomic locus. The mice were treated with LNPs formulated with the top-ranked PCSK9 EE (PCSK9-EE), CRISPRi or WT Cas9 payload. Illustration was created with BioRender. b, Circulating PCSK9 protein levels in PCSK9-Tg over a 1-year period after a single administration of an LNP formulation with PCSK9-EE (n = 6 mice per group). c–e, Effect of a single administration of an LNP formulation with PCSK9-EE on PCSK9 mRNA (c), total plasma cholesterol (d) and CpG methylation levels in liver (e) in PCSK9-Tg mice 1 month after treatment (n = 4 mice per group). For e, liver methylation data from all PCSK9-Tg mice treated with PCSK9-EE for 1 year (n = 6, from b) are also included. CpG location (first row) and the methylation percentage (0–100%) of each CpG dinucleotide at the PCSK9 locus as measured by WGBS in cells expressing PCSK9 (liver hepatocyte WGBS; second row) or not expressing PCSK9 (neuron WGBS; third row) are shown. Vehicle-treated animals received a single administration of saline solution. CRISPRi served a control for robust but transient silencing of PCSK9, whereas WT Cas9 served as a control for durable silencing of PCSK9. For b–d, results are shown as mean ± s.d. For c and d, statistical analysis was performed by one-way ANOVA followed by two-tailed Dunnett’s test. For c, ***P = 0.000893; ****P = 000005 versus vehicle-treated mice. For d, ***P = 0.000253; ****P = 0.000024 versus vehicle-treated mice. For e, CpG methylation profiles for all analyzed samples are shown.

Fig. 4. In vivo durability of PCSK9 silencing after two-thirds PHx and effects on DNA methylation in liver.

a, Schematic outline of the in vivo PHx study. The timing of the PHx (or sham) procedure and the length of time allowed for full liver regeneration before liver sample collection are highlighted. Illustration was created with BioRender. b, Circulating PCSK9 protein levels after a single administration of an LNP formulation with the top-ranked PCSK9 EE (PCSK9-EE) in PCSK9-Tg mice before and after PHx (n = 6) or sham (n = 5) procedures. The PHx or sham procedure was performed on day 35. WT Cas9 served as a control for durable silencing before and after PHx (n = 6) or sham (n = 6) procedures. Control animals received saline (vehicle) and were also subjected to pre-PHx and post-PHx (n = 8) or sham (n = 3) procedures. c, Effect of a single administration of an LNP formulation with PCSK9-EE on CpG methylation levels in liver from PCSK9-Tg mice at 90 d after LNP treatment. Methylation data from the resected liver section after the PHx procedure at day 35 were also included in the analysis. CpG location (first row) and the methylation percentage (0–100%) of each CpG dinucleotide at the PCSK9 locus in cells expressing PCSK9 (liver hepatocyte; second row) or not expressing PCSK9 (neuron; third row) are shown. For b, results are shown as mean ± s.d. For c, CpG methylation profiles for all analyzed samples are shown.

Fig. 5. In vivo reversibility of the effects of the PCSK9 EE using a PCSK9 epigenetic activator.

a, Schematic outline of PCSK9 silencing in mice using a PCSK9 EE (PCSK9-EE), followed at more than 100 d by treatment with a PCSK9 activator (dCas-Tet). Illustration was created with BioRender. b, Circulating PCSK9 protein levels after a single administration of an LNP formulation with dCas-Tet in PCSK9-Tg previously treated with PCSK9-EE to silence PCSK9. Results are shown as mean ± s.d. (n = 5 mice). c, Effect of a single administration of LNP formulation with dCas-Tet in PCSK9-Tg mice previously treated with PCSK9-EE to silence PCSK9 on CpG methylation levels at 56 d after dCas-Tet treatment. CpG location (first row) and the methylation percentage (0–100%) of each CpG dinucleotide at the PCSK9 locus in cells expressing PCSK9 (liver hepatocyte; second row) or not expressing PCSK9 (neuron; third row). CpG methylation profiles for all analyzed samples are shown for c.

Fig. 6. Activity of a human PCSK9 EE in PCHs in vitro and cynomolgus monkeys in vivo.

a, In silico alignment of CpGs (the molecular target of the EE) between human and cynomolgus monkey around the PCSK9 TSS. Matched and unmatched CpGs are labeled in blue and yellow, respectively (84 out of 112 CpGs are matched). Note that the cynomolgus PCSK9 gene is located on the negative strand; hence, CpGs in the reverse complement (rev-comp) sequence are shown. b, Activity and potency of LNP formulation using the EE PCSK9-EE-V2 in cultured PHHs and PCHs were assessed by measuring secreted PCSK9 protein in the supernatant. IC₅₀ values are indicated. Results are shown as mean ± s.d. (n = 4 replicates per group). c, Dose–response of a single infusion of an LNP formulation with PCSK9-EE-V2 on circulating PCSK9 protein levels (left) and LDL-C (right) in cynomolgus macaques (n = 3 per group). Vehicle-treated animals received a single infusion of saline solution (n = 4). Results are shown as mean ± s.d. Plasma samples were obtained from two of the vehicle-treated animals at days 84 and 98. These data were averaged and plotted at day 91 to better visualize the group mean. d, Effect of a single administration of PCSK9-EE-V2 on CpG methylation levels in liver biopsy samples at 24 d after treatment. CpG location (first row) and the methylation percentage (0–100%) of each CpG dinucleotide at the cynomolgus PCSK9 locus for individual animals are shown. Note that liver biopsies were obtained for only two of the vehicle-treated animals shown in c. e, Pearson’s correlation (r) comparing average cynomolgus PCSK9 (cPCSK9) TSS methylation levels (day 24) versus cynomolgus PCSK9 protein levels (day 21) for individual animals shown in c and d. Two-sided P value is shown. Cyno, cynomolgus; IC₅₀, half-maximal inhibitory concentration.

Extended Data Fig. 1. Effect of statin treatment on in vitro activity of top-ranked human PCSK9 epigenetic editors in immortalized cells.

Top 5 gRNA selected based on their activity and durability in HeLa cells (from Fig. 1c), as well as having full cross-reactivity with the cynomolgus macaque PCSK9 gene were evaluated for their ability to maintain PCSK9 protein reduction in the presence of 1 mM simvastatin or DMSO (Vehicle). Simvastatin or DMSO was added in the last 24 hrs of incubation. Individual data points and means are shown, n = 2 replicates per experimental condition. Results are expressed as % of secreted PCSK9 protein in cells treated with transfection (txn) reagent only. WT Cas9 serves as a control for durable silencing of PCSK9.

Extended Data Fig. 2. In vivo activity of human PCSK9 epigenetic editors in mice.

a. CpG methylation levels in livers from untreated transgenic mice carrying the human PCSK9 genomic locus (PCSK9-Tg, in blue) and primary human hepatocytes (in red). b. Location of top 5 gRNAs is shown relative to the distance (in nucleotides) to the PCSK9 gene transcription start site (TSS). c. Effect of a single administration at near-saturating dose (0.75 mg/kg) of an LNP formulation evaluating an epigenetic editor with individual or combination of two gRNA candidates on circulating PCSK9 protein levels in PCSK9-Tg. Results are shown as mean ± s.d. (n = 6 mice/group). d. Effect of a single administration at a sub-saturating dose (0.2 mg/kg) of an LNP formulation evaluating epigenetic editor with best individual or combination of two gRNA candidates on circulating PCSK9 protein levels in PCSK9-Tg mice. Results are shown as mean ± s.d. (n = 4 mice in gRNA41 group and n = 5 mice in gRNA41 + 49 group).

Extended Data Fig. 3. Specificity follow-up of top human PCSK9 epigenetic editor in primary human hepatocytes.

a. WGMS CpG methylation for Effector Only (grey) and PCSK9-EE (blue) in the genomic region surrounding off-target DEG ENSG00000285976 (boxed gene) (left) and low level of baseline expression (TPM) of off-target DEG ENSG00000285976 from RNA-Seq is shown in the dot plot (n = 3) (right). b. Scatterplot of RNA-Seq Log2 fold-change of all genes within 20 kb of any DMR called in Twist Human Methylome Hybrid Capture Methylation Sequencing assay (PCSK9-EE vs Effector Only) vs methylation difference of the associated DMR for each gene (PCSK9-EE vs Effector Only). PCSK9 gene/DMR is shown in yellow. Thresholds (grey dashed lines) set as methylation (beta-value) difference > 0.2 or < -0.2, RNA-Seq Log2FC > 1 or < -1. c-i. For all DMRs meeting either the methylation or gene expression change thresholds in Fig. 2f and Extended Data Fig. 3b, WGMS CpG methylation for Effector Only (grey) and PCSK9-EE (blue) in the genomic region surrounding each off-target DMR (left) and expression (TPM) of all off-target genes with transcription start sites within 20 kb of each DMR from RNA-Seq is shown in the dot plot (n = 3) (right). RNA-seq padj values are from a DEseq2 Wald test, two-sided, with Benjamani-Hotchburg multiple comparison correction. c. DMR near WDR81/MIR22HG/SERPINF2, average methylation difference within DMR by Twist Human Methylome assay: 0.24. Average methylation difference within DMR by WGMS: 0.18. WDR81 RNA-Seq padj = 9.998728e-01, MIR22HG RNA-Seq padj = 9.998728e-01, SERPINF2 RNA-Seq padj = 5.354105e-02, d. DMR near RB1-DT/PPP1R26P1, average methylation difference within DMR by WGMS: 0.14. RB1-DT RNA-Seq padj = 9.998728e-01, PPP1R26P1 RNA-Seq padj = 9.998728e-01. e. DMR near MCF2, average methylation difference within DMR by Twist Human Methylome assay: 0.07, MCF2 RNA-Seq padj = 9.998728e-01, f. DMR near TCEA1P3, average methylation difference within DMR by Twist Human Methylome assay: -0.16, TCEA1P3 RNA-Seq padj = 9.998728e-01, g. DMR near SLC47A2, average methylation difference within DMR by WGMS: 0.13, SLC47A2 RNA-Seq padj = 9.998728e-01. h. DMR near L3MBTL1, average methylation difference within DMR by Twist Human Methylome assay: −0.22, L3MBTL1 RNA-Seq padj = 9.998728e-01, i. DMR near Y_RNA, average methylation difference within DMR by WGMS: 0.11, Y_RNA RNA-Seq padj = 9.998728e-01.

Extended Data Fig. 4. Liver safety monitoring of a human PCSK9 epigenetic editor in cynomolgus monkeys.

Serial measurements of blood alanine transaminase (a), aspartate transaminase (b), and total bilirubin (c) were performed in non-human primates following dosing with either a vehicle control or PCSK9-EE-V2. Results are shown as mean ± s.d. (n = 4 animals in vehicle group and n = 3 animals in each experimental group receiving PCSK9-EE-V2). Plasma samples were obtained from two of the vehicle-treated animals at day 84 and 98. These data have been averaged and plotted at day 91 to better visualize the group mean.

Extended Data Fig. 5. Activity of a human PCSK9 epigenetic editor in individual cynomolgus monkeys in vivo.

Time-course and dose-response effect of a single infusion of an LNP formulation with PCSK9-EE-V2 on circulating PCSK9 protein levels (b-d) and LDL-cholesterol (g-i) in individual cynomolgus macaque. Vehicle-treated animals receive a single infusion of saline solution (a and f). Average cPCSK9 (e) and LDL-cholesterol (j) change at day 91 post dose from baseline. For each experimental group, individual animals are represented by a different symbol (a-j). For e and j, results are shown as mean ± s.d. (n = 4 animals in vehicle group and n = 3 animals in each experimental group receiving PCSK9-EE-V2).