Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure 1. Identification of CCL2 as a potential chemokine in the DRGs after SNI. A, Color- coded heat maps of expression levels measured by chemokine PCR array. Fold changes in chemokine expression level relative to that in the control group were log2-transformed and color-coded based on the color scale shown at the top. The chemokines and chemotaxis- related molecules shown here belonged to the cluster of genes whose expression was already ele- vated1dafterSNI.Thesignalsfromthreeindependentarrayswereaveragedtoobtaintheexpression levelateachtimepoint.B,Aquantificationgraphofreal-timeRT-PCRresultsforCCL2geneexpression inDRGsamplesobtainedbefore(0d)or1,3,and7dafterSNI.N 3animalsforeachtimepoint. ***p0.001comparedwithcontrol(0d)valuesbyone-wayANOVAfollowedbyTukey’sposthoc analysis. C–F, Representative immunofluorescence images of L5 DRG tissue sections from an unin- juredcontrol(CTL)animal(C)orobtained1dafterSNI(D),dorsalcolumninjury(DCI;E),orrhizotomy (F).SectionswerestainedwithaCCL2antibody(red)andaNeuNantibody(green).Arrowsindicate cellspositiveforbothmarkers.Scalebars,50m.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Expressing, Control, Transformation Assay, Chemotaxis Assay, Immunofluorescence

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure 2. CCL2 plays an essential role in neuron–macrophage interactions in vitro. A, A quantification graph of real-time RT-PCR results for CCL2 expression in cultured DRG neurons treatedwithdb-cAMP(cAMP)orPBS.N 3independentculturesforeachcondition.***p 0.001comparedwithPBSbyunpairedttests.B,ELISAmeasurementoftheCCL2concentration in the cell culture media obtained from different culture conditions. N M, Neuron–mac- rophagecocultures;N,neuron-onlycultures;M,macrophage-onlycultures.N 3independent cultures for each condition. ***p 0.001 by unpaired t test. C, Representative images of III tubulin-positive cultured DRG neurons grown for 15 h with CM obtained from neuron–mac- rophage cocultures. To obtain the CM, neuron–macrophage cocultures were treated first for 24 h with PBS or cAMP together with control IgG or CCL2-neutralizing antibodies (CCL2) and then the IgG or CCL2-neutralizing antibodies were maintained in the cultures during the sub- sequent72hCMcollectionperiod(CM).Inotheroutgrowthassays,CCL2-neutralizingantibod- ies were added directly to CM obtained from neuron–macrophage cocultures treated with cAMPsothattheantibodieswerepresentonlyduringtheneuriteoutgrowthassay(NOA).D,E, Quantification graphs of neurite length in the experiments in which the antibodies were present during the neuron–macrophage cocultures (CM collection period; D) or only dur- ing the NOA (E). N 4 independent cultures using independent CMs for each condition. ***p 0.001 compared with PBS values by one-way ANOVA followed by Tukey’s post hoc analysis. Scale bars, 100 m.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Cell Culture, Control

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure 3. CCL2 in neurons and CCR2/CCR4 in macrophages are required for in vitro neuron–macrophage interactions to produce proregenerative activity. A, C, E, G, I, Representative images of neurite outgrowth in DRG neuron cultures treated with CM obtained from neuron–macrophage cocultures using WT, CCL2-deficient (CCL2 /), or CCR2-deficient (CCR2 /) neurons (N) or macrophages(M).GenotypeconditionsforthecoculturestoobtainCMwereindicatedattheleftoftherepresentativeimages.AllcoculturesweretreatedwitheitherPBSordb-cAMP(cAMP),and the CMs were collected for 72 h. I, C 021, CCR4 antagonist, was added at a concentration of 0.1 M during the coculture period. WT neurons were used for the neurite outgrowth assays for all conditions. DRG neurons and their neurites were visualized by immunofluorescence staining for III tubulin. Scale bars, 100 m. B, D, F, H, J, Quantification graphs of neurite outgrowth in the presence of CM from cultures of the different neuron–macrophage genotype combinations treated with PBS or cAMP. N 4 independent cultures using independent CMs for each condition. ***p 0.001 and **p 0.01 compared with PBS values by unpaired t test.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: In Vitro, Activity Assay, Concentration Assay, Immunofluorescence, Staining

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure4. ChangesinthenumberofmacrophagesinDRG,axongrowthcapacity,andexpressionofRAGsinCCL2-deficientmice. A,B,RepresentativeimagesofIba1-positivemacrophagesinDRGSsofWT(A)andCCL2/(B)miceatdifferenttimepointsafter SNI. C, Quantification of the number of macrophages in WT and CCL2/ mice 0 (CTL), 1, 3, and 7 d after SNI. N 3 animals for each condition. Scale bars, 50 m. D, E, Representative images of neurite outgrowth of DRG neurons taken from WT (D) and CCL2/(E)miceatdifferenttimepointsafterSNI.NeuronsfromtheL4,L5,andL6DRGswereculturedfor15hbeforebeingfixed fortheimmunofluorescentvisualizationofneuriteswithanti-IIItubulin.Scalebar,100m.F,Comparisonofthemeanneurite length between cultures from WT and CCL2/ mice 0 (CTL), 1, 3, and 7 d after SNI. N 4 animals for each condition. ***p 0.001 compared with WT values by unpaired t test. G, H, J, K, Representative immunofluorescence images of GAP-43 (G, H) or c-Jun(J,K)staininginL5DRGsectionsobtainedfromWT(G,J)andCCL2/(H,K)miceatdifferenttimepointsafterSNI.Scale bars, 50 m. I, L, Quantification graphs of the percentage of GAP-43-positive (F) or c-Jun-positive (I) cells in WT and CCL2 /

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Immunofluorescence

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure 5. Intraganglionic injection of CCL2 neutralizing antibodies abolishes conditioning effects. A, Representative images of Iba1 staining in L5 DRGs obtained 7 d after SNI of animals withintraganglionicinjectionofIgGorCCL2-neutralizingantibody(CCL2).Theinjectionwas performed immediately after SNI. Scale bars, 50 m. B, Comparison of the number of macro- phages.N 3animalspergroup.***p0.001.C,Representativeimagesofneuronscultured fromL5DRGswithintraganglionicinjectionofIgGorCCL2ofanimalssubjected7dpreviously to SNI. Scale bars, 100 m. D, Quantification of neurite outgrowth. N 3 animals per group. ***p 0.001.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Injection, Staining, Comparison

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure 6. CCL2 is sufficient for enhancement of axon regenerative capacity by mobilizing M2-likemacrophages.A,RepresentativeimagesofIba1staininginL5DRGsectionsobtained7d after intraganglionic injection of PBS or CCL2, CXCL1, and CCL3. Scale bars, 50 m. B, Repre- sentativeimagesofneuronsculturedfromL5DRGsdissectedfromanimalswithintraganglionic injection of PBS, or CCL2, CXCL1, and CCL3. Scale bars, 100 m. C, Quantitative comparison of thenumberofIba1-positivemacrophagesinDRGs.N 3animalspergroup.***p0.001.D, Quantitation of neurite outgrowth. The culture period was 15 h and DRG neurons and their neurites were visualized by immunofluorescence staining for III tubulin. N 3–4 animals per group. ***p 0.001. E, Real-time RT-PCR results for M1 and M2 markers in cultured macrophages treated with CCL2, CX3CL1, or CCL3 for 24 h. N 3 independent cultures per group. ***p 0.001 compared with the control (untreated) condition. F, Real-time RT-PCR results for of M1 and M2 marker gene expression in MACS-separated (using CD68 antibody) macrophages from the L4 and L5 DRGs at the indicated time points after SNI (CD68-positive fraction). N 4 animals per group. **p 0.01 and ***p 0.001 compared with control values, respectively, by one-way ANOVA followed by Tukey’s post hoc analysis.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Injection, Comparison, Quantitation Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Cell Culture, Control, Marker, Gene Expression

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure7. Intraganglionic gene delivery by AAV5. A–C, Confocal images of GFP-positive and NeuN-stained cells in the L5 DRG 7 d (A), 14 d (B), and 28 d (C) after intraganglionic injection of AAV5-GFP. Arrows indicate GFP/NeuN cells. Scale bars, 100 m. D–F, Confocal images of DRG sections double stained for CCL2 (green) and NeuN (red) 7 d (D), 14 d (E), and 28 d (F) after intraganglionic injection of AAV5-CCL2. Arrows indicate CCL2/NeuN cells. Scale bars, 100 m.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Staining, Injection

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure8. IntraganglionicAAV5-CCL2injectionincreasesthenumberofmacrophagesin theDRGsandenhancesneuriteoutgrowth.A,B,RepresentativeimagesofIba1stainingin L5DRGsectionsobtained7,14,and28dafterintraganglionicinjectionofAAV5-GFP(A)or AAV5-CCL2(B).Scalebars,50m.C,Aquantificationgraphcomparingthemeannumber of Iba1-positive macrophages in DRGs injected with AAV5-GFP or AAV5-CCL2. N 4 animals per group. *p 0.05 and ***p 0.001, respectively, by unpaired t test. D, Representative images of DRG sections stained with neurofilament (NF; green) and Iba1 (red) 28 d after intraganglionic injection of AAV5-CCL2. Scale bars, 50 m. E, Represen- tative images of neurons cultured from L5 DRGs freshly dissected from animals subjected 28 d previously to intraganglionic injection of AAV5-GFP or AAV5-CCL2. The culture period was 15 h and DRG neurons and their neurites were visualized by immunofluorescence staining for III tubulin. Scale bars, 100 m. F, A quantification graph comparing the mean neurite length. ***p 0.001 by unpaired t test.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Injection, Staining, Cell Culture, Immunofluorescence

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure9. CCL2overexpressionpromotesaxonregenerationinaspinalcordinjurymodel.A–D,RepresentativeimagesofCTB-labeledaxons(red)andGFAP-immunostainedspinalcordsections (green)fromanimalsinjectedwithAAV5-GFP7dbeforeinjury(A),withAAV5-CCL27dbeforeinjury(B,7D),orwithAAV5-CCL21dafterinjury(C,1D),andthosesubjectedtopreconditioning SNIbeforecreatingthespinallesion(D).DashedlinesindicatecaudallesionbordersasdeterminedbyGFAPimmunostaining.TheboxedregionsinA–Daremagnifiedinthecenterpanels.Scalebars, 100 m. A’–D’, Axons regenerating beyond the caudal lesion border were reconstructed using Photoshop software from six consecutive parasagittal sections collected at a 60-m intersection interval.Thevirtualsectionimageusesdifferentcolorstodistinguishthedifferentsections.Scalebars,100m.E,Aquantificationgraphcomparingthemeantotalaxonlengthbeyondthecaudal lesion border measured in the composite images. ***p 0.001 compared with the AAV5-GFP control value by one-way ANOVA followed by Tukey’s post hoc analysis. F, A quantification graph of themeanlongestdistanceofaxonregenerationbeyondthecaudallesionborder.***p0.001comparedwiththeAAV5-GFPcontrolvaluebyone-wayANOVAfollowedbyTukey’sposthocanalysis. G,AquantificationgraphcomparingthenumberofCTB-positiveaxonsatdifferentdistancesfromthelesionepicenter.Negativevaluesfordistanceindicateareascaudalfromtheepicenter.*p 0.05and***p0.001betweenanimalsinjectedwithAAV5-GFPandthosewithAAV5-CCL27dbeforethelesion(7D);##p0.01and###p0.001betweenanimalswithAAV5-GFPandthose withAAV5-CCL21dafterthelesion(1D);§§p0.01and§§§p0.001betweenanimalswithAAV5-GFPandthosesubjectedtoSNI7dbeforethespinallesion,byone-wayANOVAfollowed by Tukey’s post hoc analysis. N 8, 8, 9, and 6 animals for the AAV5-GFP, AAV5-CCL2 (7D), AAV5-CCL2 (1D), and SNI groups respectively.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Software, Control

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure 10. Overexpression of CCL2 did not induce neuropathic pain. A, Representative images of CGRP-immunostained lumbar spinal cord sections obtained from animals subjected to intraganglionic injection of AAV5-GFP or AAV5-CCL2 7 d before or 1 d after dorsal hemisection spinal cord injury. Scale bars, 200 m. B, Graphs showing the withdrawal response latency (in seconds) to nociceptive heat stimulation 14 and 28 d after spinal cord injury in animals with intraganglionic injection of AAV5-GFP or AAV5-CCL2 7 d before or 1 d after the dorsal hemisection spinal injury. C, Graphs showing the flexor reflex withdrawal threshold (in grams) after stimulation of the plantar surface with a series of Von Frey hairs in the same animals. N 8, 8, and 9 animals for the AAV5-GFP, AAV5-CCL2 (7D), and AAV5-CCL2 (1D) groups, respectively.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Over Expression, Injection

Journal: Journal of Neuroscience

Article Title: CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury

doi: 10.1523/jneurosci.1924-15.2015

Figure Lengend Snippet: Figure 11. A diagram of the proposed model for the role of CCL2-mediated macrophage activation following preconditioning SNI. A, An axotomy at the sciatic nerve produces injury signals that are retrogradely transmitted to neurons in the DRGs. B, The injury signals induce initial activation (dotted black arrows) of multiple RAGs. Activation of a particular RAG (RAGX) may be specifically linked to the production of CCL2 by DRG neurons (dotted blue arrows). C, Activatedmacrophages(M2-polarized)expressingCCR2provideproregenerativefactorsresult- ing in the amplification and maintenance (thick red arrows) of RAGs. Changes in the tip mor- phology of central axons reflect the enhanced regenerative capacity of DRG neurons.

Article Snippet: The concentration of CCL2 in the culture media was measured using an ELISA kit for rat CCL2 (R&D Systems), according to the manufacturer’s protocol.

Techniques: Activation Assay, Amplification

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: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

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: Top 5 gRNA selected based on their activity and durability in HeLa cells (from Fig. ), 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 .

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Activity Assay, Incubation, Transfection, 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 . 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).

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: CpG Methylation Assay, Transgenic Assay, Formulation

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 . 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 , log 2 FC > 1 or log 2 FC < −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 log 2 FC 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 log 2 FC > 1 or < −1. DEG, differentially expressed gene.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Activity Assay, Formulation, Isolation, RNA Sequencing, Control, Quantitative Proteomics, Methylation, Sequencing, CpG Methylation Assay, Genome Wide, Comparison

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 . 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. 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.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: CpG Methylation Assay, Expressing, RNA Sequencing, Methylation, Sequencing, Gene Expression, Comparison

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: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

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: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

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: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Formulation, 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 , 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. IC 50 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 (c PCSK9 ) 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; IC 50 , half-maximal inhibitory concentration.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: In Silico, Labeling, Sequencing, Activity Assay, Formulation, Cell Culture, Saline, Clinical Proteomics, CpG Methylation Assay, Methylation, Concentration Assay

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: 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.

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Control, Clinical Proteomics

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: 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).

Article Snippet: During the studies, the plasma of animals was collected periodically for analysis of circulating PCSK9 protein using the Simple Plex Human PCSK9 assay kit on Ella instrument (R&D Systems).

Techniques: Formulation, Saline

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cytokine expression in subjects with Mycobacterium avium ssp. paratuberculosis positive blood cultures and a meta-analysis of cytokine expression in Crohn’s disease

doi: 10.3389/fcimb.2024.1327969

Figure Lengend Snippet: Multiple boxplots of three cytokines, IFNg, IL-17A, and TNFa that are statistically significant between CD positive and negative subjects. Cytokine values were transformed by log (x+1).

Article Snippet: Cytokine IL-22 in participants’ plasma samples was measured using custom made Simple Plex Cytokine cartridges (SPCKE-PS-001529, ProteinSimple) with the Ella Next Generation ELISA system (ProteinSimple).

Techniques: Transformation Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cytokine expression in subjects with Mycobacterium avium ssp. paratuberculosis positive blood cultures and a meta-analysis of cytokine expression in Crohn’s disease

doi: 10.3389/fcimb.2024.1327969

Figure Lengend Snippet: Meta-analysis results for the cytokine variables with log (x+1) transformation.

Article Snippet: Cytokine IL-22 in participants’ plasma samples was measured using custom made Simple Plex Cytokine cartridges (SPCKE-PS-001529, ProteinSimple) with the Ella Next Generation ELISA system (ProteinSimple).

Techniques: Transformation Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cytokine expression in subjects with Mycobacterium avium ssp. paratuberculosis positive blood cultures and a meta-analysis of cytokine expression in Crohn’s disease

doi: 10.3389/fcimb.2024.1327969

Figure Lengend Snippet: Meta-analysis of plasma cytokine differences between CD patients and controls in three studies. (A) Forest plot of log (IL-1β); (B) Forest plot of log (IL-2); (C) Forest plot of log (IL-4); (D) Forest plot of log (IL-5); (E) Forest plot of log (IL-10); (F) Forest plot of log (GM-CSF); (G) Forest plot of log (IFNγ); (H) Forest plot of log (TNFα).

Article Snippet: Cytokine IL-22 in participants’ plasma samples was measured using custom made Simple Plex Cytokine cartridges (SPCKE-PS-001529, ProteinSimple) with the Ella Next Generation ELISA system (ProteinSimple).

Techniques: Clinical Proteomics