Journal: Nature Medicine

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

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: 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 .

Article Snippet: Cells were resuspended in Hepatocyte Plating Medium (Lonza) and plated at a density of 80,000 cells per well (Collagen I–coated 96-well plate; STEMCELL Technologies).

Techniques: Transfection, Methylation, Methylation Sequencing, Activity Assay, Isolation, Control

Journal: Nature Medicine

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

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: 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.

Article Snippet: Cells were resuspended in Hepatocyte Plating Medium (Lonza) and plated at a density of 80,000 cells per well (Collagen I–coated 96-well plate; STEMCELL Technologies).

Techniques: In Vivo, Transgenic Assay, Formulation, Clinical Proteomics, CpG Methylation Assay, Methylation, Expressing, Saline, Control, Two Tailed Test

Journal: Nature Medicine

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

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: 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.

Article Snippet: Cells were resuspended in Hepatocyte Plating Medium (Lonza) and plated at a density of 80,000 cells per well (Collagen I–coated 96-well plate; STEMCELL Technologies).

Techniques: In Vivo, Formulation, Control, Saline, CpG Methylation Assay, Methylation, Expressing

Journal: Nature Medicine

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

doi: 10.1038/s41591-025-03508-x

Figure Lengend Snippet: 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 .

Article Snippet: Cells were resuspended in Hepatocyte Plating Medium (Lonza) and plated at a density of 80,000 cells per well (Collagen I–coated 96-well plate; STEMCELL Technologies).

Techniques: Formulation, CpG Methylation Assay, Methylation, Expressing

Journal: Current Research in Toxicology

Article Title: Microphysiological system to address the opioid crisis: A novel multi-organ model of acute opioid overdose and recovery

doi: 10.1016/j.crtox.2024.100209

Figure Lengend Snippet: Immunocytochemical characterization of opioid receptor expression of the multi-organ system components under flow condition. A) opioid receptor expression of cardiomyocytes, B) opioid receptor expression of hepatocytes, C) opioid receptor expression of skeletal muscle, D) opioid receptor expression of preBötC neurons; scale bars = 100um.

Article Snippet: Supernatant was aspirated, cells were resuspended in 4 mL of hepatocyte plating medium (Novabiosis, NHPM), and a cell count was performed.

Techniques: Expressing

Journal: Current Research in Toxicology

Article Title: Microphysiological system to address the opioid crisis: A novel multi-organ model of acute opioid overdose and recovery

doi: 10.1016/j.crtox.2024.100209

Figure Lengend Snippet: Phase images of organ components inside the multi-organ system. A) hepatocytes after assembly into the multi-organ system, B) cardiomyocyte cantilevers after assembly into multi-organ system, C) skeletal muscle cantilevers after assembly into the multi-organ system, D) patterned cardiomyocyte MEA after assembly into the multi-organ system, E) patterned preBötC MEA after assembly into the multi-organ system; scale bars = 50um.

Article Snippet: Supernatant was aspirated, cells were resuspended in 4 mL of hepatocyte plating medium (Novabiosis, NHPM), and a cell count was performed.

Techniques:

Journal: Current Research in Toxicology

Article Title: Microphysiological system to address the opioid crisis: A novel multi-organ model of acute opioid overdose and recovery

doi: 10.1016/j.crtox.2024.100209

Figure Lengend Snippet: Skeletal muscle contractile force and hepatocyte enzymatic activity. A) skeletal muscle amplitude on cantilevers, B) no significant change was observed in hepatocyte enzymatic activity; data collected from more than 3 independent biological replicates per condition, data is shown as mean with error bars indicating SEM.

Article Snippet: Supernatant was aspirated, cells were resuspended in 4 mL of hepatocyte plating medium (Novabiosis, NHPM), and a cell count was performed.

Techniques: Activity Assay

Journal: Current Research in Toxicology

Article Title: Microphysiological system to address the opioid crisis: A novel multi-organ model of acute opioid overdose and recovery

doi: 10.1016/j.crtox.2024.100209

Figure Lengend Snippet: Viability assays. A) Cardiac, SKM, and preBötC viability determined via Alamar Blue, B) hepatocyte viability determined via MTT (control: gray, methadone: orange, naloxone: green, methadone + naloxone: blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: Supernatant was aspirated, cells were resuspended in 4 mL of hepatocyte plating medium (Novabiosis, NHPM), and a cell count was performed.

Techniques: Control