Durable and efficient gene silencing in vivo by hit-and-run epigenome editing

Molecular cloning and mRNA manufacturing

ETRs have been transiently delivered as both plasmid DNA (in cell traces) or mRNA (in cell traces, main cells and in vivo). To this finish, ETRs have been cloned in an expression vector containing: (i) an upstream CMV promoter; (ii) an upstream T7 promoter for mRNA in vitro transcription (IVT); (iii) a downstream WPRE sign; (iv) a 3′-terminal stretch of 64 adenines (64A); and (v) a SpeI plasmid linearization web site for mRNA IVT (Prolonged Information Fig. 1b). The coding sequences of the dCas9-based ETRs have been beforehand described¹⁵. For plasmid-mediated gRNA expression, the crRNA sequences have been cloned right into a beforehand described expression vector containing a U6 promoter and the sequence of the Staphylococcus pyogenes Cas9 trRNA³⁹. gRNAs concentrating on the CGI of the mouse Pcsk9 have been designed utilizing Chop Chop⁴⁰ (https://chopchop.cbu.uib.no/) and chosen based on excessive simulated exercise and specificity. ZFPs and TALEs have been designed and synthesized by Merck and Thermo Fisher Scientific, respectively, and subcloned into the mammalian expression plasmid instead of the dCas9 sequence. mRNAs have been produced by IVT utilizing the T7 Megascript Package (Thermo Fisher Scientific, AMB1334-5) based on the producer’s directions. For the in vitro experiments, partially modified mRNAs have been produced by IVT, together with the next modifications to the usual protocol: (i) inclusion of the anti-reverse cap analogue 3´-O-Me-m7G(5′)ppp(5′)G (NEB, M0251) at a last focus of 8 mM; and (ii) discount of the GTP focus from 7.5 to 2.5 mM. For the in vivo experiments, closely modified mRNAs have been produced by IVT, together with the next modifications to the usual protocol: (i) inclusion of CleanCap-AG (Trilink BioTechnologies, N-7113) at a last focus of 4 mM; and (ii) substitution of UTP with N1-Met-ψ-Uridine (Trilink BioTechnologies, N-1081) at a last focus of seven.5 mM. mRNAs have been then purified utilizing the RNeasy Mini Package (Qiagen, 74134). The standard and integrity of the mRNAs have been assessed with a 4200 TapeStation System, and portions have been measured by a NanoDrop 8000. sgRNAs have been synthetized by Axolab based on the a beforehand described nucleotide-modification scheme⁴¹. For in vitro or in vivo research, the Cas9 mRNA was in vitro transcribed as described above or bought from Trilink BioTechnologies (L-7606), respectively. Sequences of the ETRs and gRNAs used on this examine are listed in Supplementary Desk 6. The plasmids used on this examine can be found upon signing of a cloth switch settlement.

Cell tradition, therapy and engineering

Hepa 1-6 cells (CRL-1830, ATCC) have been cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning, 10-013-CV) supplemented with 10% fetal bovine serum (FBS; EuroClone), 1% l-glutamine (EuroClone, ECB3000D) and 1% penicillin–streptomycin (Euroclone, ECB3001D) at 37 °C in a 5% CO₂ humidified incubator. The Hepa 1-6 Pcsk9^tdTomato cell line was generated by nucleofecting 3 × 10⁵ Hepa 1-6 cells with: (i) an HDR donor plasmid containing the 2A-tdTomato-polyA cassette inside homology arms to exon 12 of Pcsk9; (ii) a Cas9-expression plasmid; and (iii) a plasmid expressing a gRNA concentrating on the final exon of Pcsk9 (ref. ⁴²). tdTomato-positive cells have been than sorted at single-cell degree and amplified. The Hepa 1-6 Pcsk9^tdTomato cell line is obtainable upon signing of a cloth switch settlement. Main mouse hepatocytes from C57BL/6 male mice have been bought from Biopredic Worldwide as adherent monolayers on collagen-coated 96-well plates and maintained based on the producer’s directions. Supernatants of handled and management cells have been collected at totally different time factors and saved as one-time-use aliquots at −20 °C.

Gene-delivery procedures

For the in vitro experiments within the Hepa 1-6 Pcsk9^tdTomato cells, 3 × 10⁵ cells have been transfected with both RNAs or plasmid DNAs utilizing the 4D-Nucleofector X System (Lonza) in SF Cell Line answer (Lonza, V4XC-2032) and with the CM-137 pulse program. For the in vitro and in vivo experiments with LNPs A, B, C, D and E, these research-grade reagents have been formulated by Precision NanoSystem (PNI) combining lipid mixes and RNA, the latter dissolved in a PNI proprietary formulation buffer. The lipid mixes are made of 4 totally different elements dissolved in ethanol-based answer: an ionizable lipid, a helper lipid, ldl cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG). The chemical nature of the helper lipid differs among the many formulations: (i) 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) for LNP A; (ii) 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine (DSPC) for LNP B; (iii) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) for LNP C; and (iv) 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholin (DOPC) for LNPs D and E. The ionizable lipid in addition to the molar ratio at which the 4 elements have been blended are proprietary info of PNI. RNAs and lipids have been blended into the NanoAssemblr Ignite instrument (PNI) utilizing microfluidic cartridges (Ignite NxGen Cartridge; PNI) with a complete circulation price (TFR) of 12 ml min⁻¹ and a circulation price ratio (FRR) of three:1 (RNAs:lipids). Nitrogen-to-phosphate (NP) ratios of 6 and 9 have been used within the preliminary LNP screening experiments, and this ratio was set to six for the remaining in vivo experiments. The lipid combine LNP D is obtainable upon request from Precision NanoSystems (PNI) utilizing the code iL00V77. LNP D formulated with the specified RNA could be straight bought from PNI by signing an settlement. The estimated turnaround time is 1–2 months. Alternatively, the LNP D and the formulation machine could be bought from PNI. On this case, PNI will technically assist the investigator within the setting of the formulation protocol. LNPs produced with the GenVoy-ILM reagent (NWW0042, 25 mM) have been formulated following the producer’s directions with the NanoAssemblr Ignite instrument (PNI) and utilizing microfluidic cartridges (Ignite NxGen Cartridge; PNI). Formulation parameters have been set as follows: (i) TFR of 12 ml min⁻¹; (ii) FRR of three:1; and (iii) NP ratio of 4. All LNPs have been concentrated utilizing an Amicon Centrifugal Filter (MWCO 30 kDa), the ethanol was eliminated by a 3:1 dilution in 1× PBS (pH7-7.3, Mg2⁺/Ca2⁺– free) and LNPs have been lastly filtered manually by way of a 0.22-µm syringe. Particle sizes and their polydispersity index (PDI) have been analysed utilizing dynamic gentle scattering (DLS), and the RNA encapsulation effectivity and concentrations have been decided utilizing a RiboGreen plate-based assay. The outcomes of the DLS analyses and RNA quantification of the LNPs used on this examine are reported in Supplementary Desk 7.

Move cytometry and cell sorting

Move cytometry was carried out utilizing CytoFLEX S (Beckman Coulter) and uncooked information have been analysed utilizing FCS Specific v.7 (DeNovo Software program) to extract the proportion of Pcsk9^tdTomato–unfavourable cells. When indicated, tdTomato-positive or -negative cells have been sorted with a FACSAria Fusion Cell Sorter (BD Biosciences) as both bulk populations or on the single-cell degree. The gating technique for each the circulation cytometry and the cell sorting procedures is reported in Supplementary Fig. 1.

RNA sequencing

Complete RNA was extracted from 6 × 10⁶ Pcsk9-silenced cells utilizing the RNeasy Mini Package (Qiagen, 74134) and quantified utilizing the Qubit 2.0 Fluorimetric Assay (Thermo Fisher Scientific). Unstranded libraries have been ready with the NEBNext Extremely RNA Library Prep Package for Illumina after rRNA depletion, and sequencing was carried out utilizing an Illumina NovaSeq 6000 platform (NovaSeq Management Software program v.1.7) to acquire at the very least 30 million of 150bp-long paired-end reads per pattern. Learn high quality was managed with Fastqc v.0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and low-quality reads and the adapters have been eliminated utilizing Trim Galore v.0.6.6 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) based on the next parameters: –quality 20, –length 25, –paired. Excessive-quality remaining reads have been aligned to the mouse reference genome GRCm38 utilizing STAR v2.7.6a (ref. ⁴³) with default parameters. Gene counts have been quantified utilizing the featureCounts operate from the Subread package deal v.2.0.1 (ref. ⁴⁴) and Gencode M25 because the gene mannequin. Uncooked counts have been corrected for biases because of totally different library preparations, if current, utilizing the ComBat_seq operate from the R Bioconductor package deal sva v.3.38.0 (ref. ⁴⁵). Learn distribution was estimated utilizing the unfavourable binomial generalized log-linear mannequin carried out within the R Bioconductor package deal DESeq2 v.1.30.0 (ref. ⁴⁶). Differential gene expression was examined utilizing the nbinomWaldTest operate and P values have been corrected utilizing the Benjamini–Hochberg method.

WGMS

Genomic DNA was extracted from 6 × 10⁶ Pcsk9^tdTomato-silenced cells utilizing the Maxwell RSC Cultured Cell extraction package (AS1620) and quantified utilizing a NanoDrop 8000. Libraries have been ready utilizing an enzymatic method for cytosine conversion with NEBNext DNA Extremely II Reagents and sequencing was carried out utilizing an Illumina HiSeq (HiSeq Management Software program v.3.4) platform to provide at the very least 250 million 150-bp-long paired-end reads per pattern. Learn high quality was managed with Fastqc v.0.11.9, and low-quality reads and the adapters have been eliminated utilizing Trim Galore v0.6.6. with the next parameters: –quality 20, –length 25, –paired, –clip_R2 5. Excessive-quality remaining reads have been analysed utilizing the Bismark learn mapper methylation caller device v.0.23.0. Intimately, reads have been aligned to each the transformed and the unconverted genomes (GRCm38) utilizing Bismark v.0.23.0 with default parameters. Duplicates have been then eliminated utilizing the deduplicate_bismark script and the methylation standing was obtained utilizing the bismark_methylation_extractor script. Then, the methylation name was loaded into the R setting and processed utilizing the R Bioconductor package deal MethylKit v.1.16.1. Imported information have been filtered utilizing the filterByCoverage operate (low rely filter equal to 1 and excessive percentile equal to 99.9) and normalized utilizing the normalizeCoverage operate. Data from the totally different samples was merged utilizing the unite operate contemplating the positions coated in all replicates. The share of methylation was calculated with the percMethylation operate and the correlation among the many samples was decided making use of the cor operate (default Pearson methodology). Differential methylation evaluation was carried out utilizing the R Bioconductor packages bsseq v.1.26.0 (ref. ⁴⁷) and DSS v.2.44.0 (ref. ⁴⁸). First, the article was created utilizing the makeBSseqData operate ranging from the Bismark output. The DMLtest operate was used for the normalization step and the differential evaluation with the next parameters: smoothing = TRUE and smoothing.span = 500. Then, the callDML operate was utilized to find out the differential methylated loci (DML) setting as thresholds delta = 0.4 and p.threshold = 1 × 10⁻³. To exclude any confounding DMRs not related to off-target methylation, the delta methylation threshold was set at 0.4; that’s, the minimal worth not calling any DMR in Cas9-treated cells, a unfavourable management having no direct methylation exercise. The DMRs have been outlined making use of the callDRM operate with the identical thresholds. The DMRs recognized have been annotated utilizing the annotatePeakInBatch operate from the R Bioconductor package deal ChIPpeakAnno v.3.24.2 (ref. ⁴⁹) utilizing the Gencode M25 annotation and the next parameters: PeakLocForDistance = “center”, FeatureLocForDistance = “TSS”, output = “each” and a number of = TRUE. DMRs of all handled samples have been computed utilizing as reference the identical mock-treated controls.

Mouse dealing with and coverings

Eight-week-old C57BL/6N feminine mice have been bought from Charles River Laboratories. Procedures involving animal dealing with and care adopted nationwide and worldwide regulation and insurance policies and have been permitted by the Institutional Animal Care and Use Committee (authorization numbers 604/2020-PR and 233/2022-PR, offered by the Italian Ministry of Well being). Housing temperature and relative humidity have been 22 °C (±2 °C) and 55% (±5%), respectively. A 12-h gentle–12-h gentle cycle was used and all doable efforts have been made to attenuate the variety of mice used and their struggling. For in vivo administration of both Cas9 or ETRs, mRNA-LNP options have been diluted in PBS with out calcium and magnesium (Corning, 21-031-CV). Subsequently, mice have been randomly assigned to a therapy group and heated with an infrared lamp to acquire vasodilatation. Lastly, 250 µl of LNP answer or PBS (herein outlined as car) have been intravenously injected into the tail vein. For plasma analyses (see subsequent part), round 200 µl of blood was collected from the retro-orbital plexus of every experimental mouse by utilizing a non-heparinized micro-haematocrit capillary tube (Kimble Chase, CSX40A502), after which moved into an EDTA-sprayed blood assortment tube (Sarstedt, 20.1288.100). Blood was then centrifuged for 10 min at 2,000g at room temperature. Purified plasma was lastly collected from the supernatant and saved as one-time-use aliquots at −20 °C. For experiment termination and organ assortment, mice have been euthanized by CO₂ inhalation and tissues (liver, spleen, lungs and kidney) have been eliminated and snap-frozen for additional molecular analyses. For partial hepatectomy, mice have been anaesthetized by 2% isoflurane steady inhalation. Earlier than hepatectomy, mice have been fasted for 4 h. Surgical procedure was carried out based on the Higgins protocol⁵⁰. Briefly, the stomach pores and skin was shaved, and a 2-cm higher midline incision was made starting from the xyphoid. After opening the peritoneum, the liver was gently mobilized and uncovered. The left lateral lobe was lifted, tied up and resected by way of 3.0 silk sutures (Ethicon, EH6823H) distal to the utilized ligatures. Muscle and pores and skin have been closed in two layers with 4.0 Vicryl (Ethicon, V994H) and an autoclip wound-closing system, respectively. For postoperative analgesia, carprofene (5 mg per kg) was utilized by subcutaneous injection into the neck fats pad. Liver tissue and blood have been collected throughout hepatectomy and at necropsy for molecular evaluation and Pcsk9 plasma quantification. For isolation of hepatocytes, mice have been first anaesthetized with isoflurane and the liver was uncovered and perfused (32 ml per min) by way of the inferior vena cava with HBSS-HEPES 0.03% collagenase IV (Sigma). The digested mouse liver was collected, handed by way of a 100-μm cell strainer (BD Biosciences) and processed right into a single-cell suspension. Cells have been spun down and washed 3 times with successive centrifugations at totally different speeds (30g, 25g and 20g) for 3 min every at room temperature to acquire hepatocytes.

Plasma evaluation

To quantify PCSK9, plasma from handled mice and supernatants from main mouse hepatocytes have been thawed and diluted 1:200 and 1:2, respectively. Dilutions have been then loaded on a industrial pre-spotted ELISA package based on the producer’s directions (R&D Techniques, MPC900). Equally, absorbance assays have been used to quantify the degrees of LDL-C (P/N 00018256040, Werfen), ALT (P/N 00018257440, Werfen), AST (P/N 00018257540, Werfen), LDH (P/N 00018258240, Werfen) and albumin (P/N 00 18250040), following the producer’s directions.

In vivo molecular analyses

Genomic DNA was extracted from snap-frozen tissues (round 30 mg) utilizing the Maxwell 48 Promega RSC Tissue DNA Purification Package (AS1610) based on the producer’s directions. The place indicated, enhancing and epi-editing efficiencies have been quantified from purified hepatocytes (see ‘Mouse dealing with and coverings’). In these circumstances, genomic DNA was extracted utilizing the Maxwell 48 RSC Tissue DNA Purification Package (AS1610) from 1 × 10⁶ cells based on the producer’s directions. Gene-editing efficiencies on the Pcsk9 locus have been quantified utilizing the T7 assay or focused deep sequencing. For the T7 assay, a 765-bp genomic area encompassing the CRISPR–Cas9 binding web site was PCR-amplified utilizing the primers listed in Supplementary Desk 8. PCRs have been then processed utilizing the Alt-R Genome Modifying Detection Package (IDT, 1075932), run on the Agilent ScreenTape System, and the proportion of enhancing was quantified based on the producer’s directions. For focused deep sequencing, the promoter area or exon 1 of Pcsk9 have been PCR-amplified utilizing the primers listed in Supplementary Desk 8. Libraries have been then ready utilizing the NEBNext Extremely II DNA Library Prep Package for Illumina for the Pcsk9 promoter or utilizing the NEBNext Extremely DNA Library Prep Package for Illumina for Pcsk9 exon 1, and sequenced utilizing the Illumina MiSeq platform (MiSeq Management Software program v.2.6). Sequencing information have been analysed with CRISPResso2 v.2.8 (ref. ⁵¹) to detect nucleotide insertions and/or deletions. Reads have been aligned to the boundary sequence across the putative slicing web site (400 bp centred on the sgRNA complementary web site for Cas9-treated samples or 300 bp centred on the ZFP-8 recognition web site for EvoETR-8-treated samples) utilizing bowtie2 v.2.2.5 (refs. ^52,53) in paired mode and default parameters. After that, unique fastq recordsdata have been subset to retain solely the reads mapping to the area of curiosity utilizing the filterbyname module of the BBMap aligner v.39.01 contained within the BBTools suite (https://sourceforge.net/projects/bbmap/). The remaining reads have been analysed with CRISPResso2 in paired-end mode setting the choices for Trimmomatic software program v.0.39 (ref. ⁵⁴; http://www.usadellab.org/cms/?page=trimmomatic) to take away low-quality positions (rating < 30) and Illumina adapters (–trim_sequences –trimmomatic_command trimmomatic –trimmomatic_options_string ‘ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 MINLEN:100’). Then, every couple of paired-end reads was merged utilizing FLASH v.1.2.11 (ref. ⁵⁵) to provide a single contig, which was mapped to the enter amplicon reference (promoter area or first exon, relying on the experiment). The sgRNA complementary web site and the ZFP-8 goal sequence have been offered to focus the evaluation on the goal area, and the quantification window was set to twenty bp per aspect across the reduce web site (Cas9 samples) or the ZFP-8 center level (EvoETR-8 samples). Recognized alleles have been quantified by measuring the variety of reads and their relative abundance on the premise of complete learn counts contemplating solely insertions and deletions. The share of CpG methylation on the Pcsk9 promoter or on the in-vitro-identified DMRs was quantified utilizing focused bisulfite deep sequencing (focused BS-seq). Particularly, purified genomic DNA was transformed with the EpiTect Quick Bisulfite Conversion Package (Qiagen, 59104) based on the producer’s directions. Then, the promoter area of Pcsk9 and unintended DMRs have been PCR-amplified utilizing the primers listed in Supplementary Desk 8. For the Pcsk9 promoter, libraries have been ready utilizing the NEBNext Extremely DNA Library Prep Package for Illumina. For the opposite DMRs, the NEBNext Extremely II DNA Library Prep Package for Illumina was used. Libraries have been sequenced utilizing an Illumina MiSeq platform in paired-end mode (MiSeq Management Software program v.2.6). Learn high quality was managed with Fastqc v.0.11.9, and low-quality reads and adapters have been eliminated utilizing Trim_Galore v0.6.6. with the next parameters: –quality 20, –length 25, –paired, –rrbs. Excessive-quality remaining reads have been analysed utilizing the Bismark learn mapper methylation caller device v.0.23.0 (ref. ⁵⁶). Intimately, reads have been aligned to each unconverted and transformed genomes (GRCm38) utilizing Bismark with the –local parameter and the methylation standing was obtained utilizing bismark_methylation_extractor script. The methylation calls have been loaded into the R setting and processed utilizing the R Bioconductor package deal MethylKit v.1.16.1 (ref. ⁵⁷). Imported information have been filtered utilizing the filterByCoverage operate (low rely filter equal to 10 and excessive percentile equal to 99.9) and normalized utilizing the normalizeCoverage operate. The info from the totally different samples have been merged utilizing the unite operate contemplating the positions coated in at the very least one replicate per situation. The share of methylation at every CpG was calculated utilizing the percMethylation operate.

Statistics and reproducibility

Information have been plotted and analysed utilizing GraphPad Prism v.9 (GraphPad Software program). When indicated within the determine legends, statistical significance was evaluated by utilizing GraphPad Prism v.9 (GraphPad Software program) and making use of the described checks. The entire in vitro experiments have been carried out with technical replicates (n ≥ 2) and the precise variety of replicates is indicated within the respective legend. In vivo experiments have been designed together with a number of mice (n ≥ 3). The precise variety of handled mice in any experimental group for any experiment is indicated within the determine legend.

Reporting abstract

Additional info on analysis design is obtainable within the Nature Portfolio Reporting Summary linked to this text.