Fig 1: 5A2-DOT-X LNPs achieve CRISPR-Cas-based gene editing in therapeutic models.a To restore dystrophin expression, 5A2-DOT-10 LNPs encapsulating Cas9/sgDMD RNPs were injected into TA muscles of DMD exon 44 deletion mice weekly (three injections, 1 mg kg−1 sgDMD, n = 3). Three weeks after the last injection, TA muscles were collected to detect expression of dystrophin protein. b Immunofluorescence images indicated that 5A2-DOT-10 LNPs treatment successfully corrected dystrophin gene and restored the expression of dystrophin proteins in TA muscles. 5A2-DOT-10 LNPs nanoparticle only treatment was used as negative control (NC). Scale bar = 100 μm. Data was repeated two times independently with similar results. c Western blot analysis further confirmed the expression of dystrophin protein in the 5A2-DOT-10 LNPs encapsulating Cas9/sgDMD RNPs treatment group. 4.2% of dystrophin protein was restored. NC: 5A2-DOT-10 LNPs nanoparticle only; WT wild-type group. d To knockout the PCSK9 gene in mouse liver, 5A2-DOT-5 LNPs encapsulating Cas9/sgPCSK9 RNPs were administered to adult C57BL/6 mice via tail vein injection weekly (three injections, 2.5 mg kg−1 sgPCSK9, IV, n = 3). One week after the last injection, mouse serum and livers were collected for analyses. e The relative PCSK9 level in the serum was significantly decreased in 5A2-DOT-5 LNPs encapsulating Cas9/sgPCSK9 RNPs treatment group, detected using PCSK9 Elisa Kit. Data are presented as mean ± s.e.m. (n = 3 biologically independent animals). One-way ANOVA followed by Dunnett’s multiple comparison test was used to determine the significance of data. (*P < 0.05). f T7EI cleavage results from genomic DNA extracted from mice livers confirmed gene editing occurred at the PCSK9 locus. Red arrows indicate cleavage bands. Indel percentages shown under the gel image were measured by Sanger sequencing and TIDE analysis. Source data are in the Source Data file.
Fig 2: LNP-mediated in vivo gene editing.a, b TTR gene editing and serum TTR reduction. LNPs encapsulating Cas9 mRNA/TTR sgRNA (4:1, wt:wt) were i.v. injected into C57BL/6 mice at a total RNA dose of 1 mg/kg. On day 7, DNA was extracted from the liver to determine on-target indel frequency by next-generation sequencing (a, n = 3–4 independent biological replicates), and serum was collected for ELISA analysis of TTR (b, n = 3–4 independent biological replicates). Data are presented as mean. c–e PCSK9 base editing. LNPs encapsulating ABE8.8 mRNA/PCSK9 sgRNA (4:1, wt:wt) were i.v. injected into C57BL/6 mice at a total RNA dose of 0.75 mg/kg (c). On day 7, DNA was extracted from the liver to determine on-target editing frequency by next-generation sequencing (d, n = 4 independent biological replicates), and serum was collected for ELISA analysis of PCSK9 (e, n = 4 independent biological replicates). Data are presented as mean ± SD. Statistical significance was evaluated by a one-way ANOVA with Tukey’s correction. f–h HPD base editing. LNPs encapsulating CBE4max mRNA/HPD sgRNA (4:1, wt:wt) were i.v. injected into FAH–/– mice at a total RNA dose of 0.6 mg/kg (f). Seven days later, DNA was extracted from the liver to determine on-target editing frequency by next-generation sequencing (g, n = 3 independent biological replicates). Data are presented as mean ± SD. Statistical significance was evaluated by a one-way ANOVA with Tukey’s correction. Two weeks later, nitisinone was removed and the survival of FAH–/– mice was monitored (h, n = 5 independent biological replicates). Statistical significance was evaluated using Survival Curve with Log-rank (Mantel-Cox) test.
Fig 3: Epigenomic controllers maintain durable repression in vivo.A In C57BL/6 female mice, a single 3 mg/kg dose of Pcsk9-EC led to a reduction in Pcsk9 serum levels (orange) compared to control mice (black) as measured by ELISA for up to 180 days. Serum was sampled every 14 days post-treatment (n = 10 mice in each cohort, ****P < 0.0001, t test). B Pcsk9 promoter methylation was increased in liver punches from Pcsk9-EC treated mice (orange) compared to control mice (black) up to 180 days post-treatment (n = 10 mice in each cohort; ****P < 0.0001, t test). Source data are provided as a Source Data file.
Fig 4: Hypercholesterolemia exacerbates alterations in cholesterol metabolism. A, The experimental design involved adult mice being fed with HCD for 8 weeks before being injected with MN/MCA1 tumor cells. These mice were maintained on HCD for an additional 23–24 days, after which they were sacrificed for sample collection and analysis. B, Blood levels of total cholesterol, HDL, and LDL in NCD- or HCD-fed MN/MCA1 mice vs. TF mice. Starting from time 0 (T0), mice were maintained on their respective dietary regimens for 8 weeks, injected with MN/MCA1 tumor cells (T1), and then sacrificed at AD stage (T2; n = 9). C and D, Heatmap showing differential mRNA expression of genes involved in hepatic metabolism (n = 5; C) and circulating PCSK9 levels (n = 5; D) in ED or AD MN/MCA1 vs. TF mice fed with either NCD or HCD. E, Immunoblot analysis of LDLR protein in the livers from NCD- or HCD-fed MN/MCA1 mice at AD vs. TF similarly fed (n = 2). F, FACS quantification of BODIPY-Ch ΔMFI of hepatic CD45−CD31− cells from MN/MCA1 mice at ED (n = 3) or AD (n = 5) on NCD or HCD compared with TF mice (n = 3) similarly fed. G and H, FACS quantification of BODIPY-Ch ΔMFI of intratumoral CD45–CD31− cells and CD11b+Ly6Clo/−F4/80+ TAMs (G,) and in BM myeloid progenitors (both CMPs and GMPs) or blood CD11b+Ly6Chi monocytic cells (H) from TF (n = 3), ED (n = 3), or AD (n = 5) MN/MCA1 mice on NCD or HCD. I, Circulating PCSK9 levels in patients with early (I–II) vs. advanced (III–IV) NSCLC (n = 15 vs. n = 21), CRC (n = 16 vs. n = 20), BRC (n = 14 vs. n = 14), PDAC (n = 9 vs. n = 10), BTC (n = 9 vs. n = 10), or PNET (n = 10 vs. n = 10) in comparison with healthy donors (HD, n = 9). J–L, FACS analysis of PCSK9 and LDLR protein expression (J and K) or BODIPY-Ch (L) in Hepa1-6 cells treated with MN/MCA1 TCM, IL6, or IL1β alone or in combination with anti-IL6 (mAb) or anti-IL1β (IL1 receptor antagonist, IL1Ra) agent (n = 3). M, Circulating PCSK9 levels in TF (n = 3) or MN/MCA1 bearing mice in AD (n = 5) on NCD or HCD in the absence (control) or presence of anti-IL6 or anti-IL1β. N, Immunoblot (top) and relative densitometry quantification (bottom, n = 2) of hepatic LDLR protein using protein extracts from mice treated as described above. Data are representative of three (B) or two (E) independent experiments. C, D, and F–N, One experiment. Data are presented as the mean ± SEM (A–H and J–M) or Box-and-whisker min-to-max plots (I). Statistic by two-way ANOVA (B), one-way ANOVA (D–N), or Kruskall–Wallis (I, BRC). BRC, breast cancer; BTC, biliary tract cancer; CRC, colorectal cancer; PDAC; pancreatic ductal adenocarcinoma; PNET, pancreatic neuroendocrine tumor.
Fig 5: Tumor progression alters cholesterol metabolism. A and B, Total HDL and LDL blood cholesterol levels in mice bearing MN/MCA1 fibrosarcomas (n = 6; A), B16-F10 melanomas (n = 11), or LLC lung (n = 5) or MC38 colon (n = 5) cancers (B), at time points corresponding to AD stage. C and D, Total cholesterol, HDL, and LDL blood levels in MN/MCA1 (n = 4; C), B16-F10, LLC, and MC38 mice (D) at time points corresponding to ED and AD stages compared with healthy, age- and sex-matched TF mice (n = 5). E, Total cholesterol (top), HDL (middle), and LDL (bottom) blood levels in patients with early (I–II) vs. advanced (III–IV) NSCLC (n = 76 vs. n = 70), CRC (n = 30 vs. n = 35 for total cholesterol; n = 15 vs. n = 16 for HDL and LDL), BRC (n = 18 vs. n = 18), PDAC (n = 26 vs. n = 32), BTC (n = 20 vs. n = 15), or PNET (n = 20 vs. n = 16) compared with healthy donors (HD, n = 35). F, mRNA expression levels of cholesterol metabolism genes in healthy liver parenchyma from patients with CRC (n = 23) compared with healthy liver parenchyma from patients with benign lesions (n = 6). Data are representative of at least four independent (A–C) or one (D) experiments. Data are presented as the mean ± SEM (A–D) or Box-and-whisker min-to-max plots (E and F). Statistic by t test [A, B, and F (PCSK9, CYP27A1, and ABCG8)], Mann–Whitney (F), two-way ANOVA (C and D), and one-way ANOVA or Kruskal–Wallis (E). BRC, breast cancer; BTC, biliary tract cancer; Ch, cholesterol; CRC, colorectal cancer; CTRL, control; PDAC, pancreatic ductal adenocarcinoma; PNET, pancreatic neuroendocrine tumor.
Supplier Page from Abcam for Mouse PCSK9 ELISA Kit