A Lactose-Derived CRISPR/Cas9 Delivery System for Efficient Genome Editing In Vivo to Treat Orthotopic Hepatocellular Carcinoma

Abstract

Gene editing is a crucial and effective strategy to treat genetic diseases. Safe and effective delivery vectors are specially required for efficient gene editing in vivo of CRISPR/Cas9 system. Interestingly, lactose, a natural saccharide, can specifically bind to asialoglycoprotein receptors, highly expressed on the surface of hepatocellular carcinoma (HCC) cells. Herein, a lactose-derived branched cationic biopolymer (LBP) with plentiful reducible disulfide linkages and hydroxyl groups is proposed as a potential delivery vector of CRISPR/Cas9 system for efficient genome editing in vivo to treat orthotopic HCC. LBP is synthesized via a facile one-pot ring-opening reaction. LBP possesses excellent compacting ability, degradability, biocompatibility, gene transfection performances, and HCC-targeting ability. LBP-mediated delivery of classical pCas9-survivin, which can target and knockout survivin oncogene, produces efficient gene editing performances, and superb anti-cancer activities in orthotopic HCC mouse models. This study provides an attractive and safe strategy for the rational design of CRISPR/Cas9 delivery system.

Keywords: CRISPR/Cas9; biopolymers; delivery vectors; lactose; orthotopic hepatocellular carcinoma.

Figure 1

Schematic illustration of the preparation of lactose‐derived branched biopolymer (LBP) and the resultant delivery and gene editing processes with pCas9‐survivin to treat orthotopic hepatocellular carcinoma (HCC). A lactose‐derived branched cationic biopolymer (LBP) with plentiful bio‐reducible disulfide linkages and hydroxyl groups was first synthesized via a facile one‐pot ring‐opening reaction, and the LBP‐mediated delivery of pCas9‐survivin, which could target and knockout survivin oncogene, showed effective gene editing and anti‐cancer activities in orthotopic HCC mouse model.

Figure 2

a) GPC characterizations of LBP⁴ in the presence of DTT (10 mm) at different time points. b) AFM images of LBP⁴/pDNA complex at the mass ratio of 40 in the absence (−) and presence (+) of DTT. c) Cytotoxicity of LBP/pDNA and PEI/pDNA complexes in BEL7402 cell lines at various mass ratios (mean ± SD, n = 3). d) In vitro gene transfection efficiencies of LBP/pDNA complexes at mass ratios from 10 to 60 in BEL7402 cell lines in comparison with those mediated by PEI (M _w ≈ 25 kDa) at its optimal N/P ratio of 15 (mean ± SD, n = 3). e) CLSM images and flow cytometry of BEL7402 cells treated with LBP²/pDNA or LBP⁴/pDNA at the mass ratio of 40 and PEI/pDNA at the N/P ratio of 15 for 4 h in the absence (−) and presence (+) of lactose, where YOYO‐1‐labeled pDNA was shown in green, and DAPI‐labeled nuclei were shown in blue. f) Representative fluorescence images of livers in different treatment groups (n = 3).

Figure 3

a) Structure of pCas9‐survivn. b) Representative images of GFP expression mediated by the LBP⁴/pCas9 and PEI/pCas9 complexes in BEL7402 cells at the 24th hour after transfection, and the percentages of positive cells were determined by flow cytometry analyses. c) Agarose gel electrophoreses of enzyme digestion PCR products amplified from the survivin locus and off‐target sites in BEL7402 cells in the absence (−) and presence (+) of T7EI enzyme. d) Sanger sequencing analysis and T‐A cloning sequencing results of PCR amplicons of the targeted sites in control and LBP⁴/pCas9 groups. e) Western blot of three transfections with LBP⁴/pCas9 and their controls, and corresponding statistical analyses of relative content of survivin protein expression in control and LBP⁴/pCas9 at the 48th hour after transfection (mean ± SD, n = 3, *p < 0.05).

Figure 4

a) Flow cytometry analyses of the percentages of apoptotic cells at the 72nd hour after transfection in different treatment groups. b) Representative cloning formation images in different treatment groups. c) Representative images of BEL7402 cells in different treatment groups on the surface of lower chambers of Matrigel transwell at the 48th hour after transfection. d) Representative images of wound‐healing assays of BEL7402 cells in different treatment groups from 0 to 72 h.

Figure 5

a) Representative bioluminescence, images of each treatment group at 0th and 35th day (n = 5). b) Liver images of each treatment group, where the orthotopic tumor nodules were highlighted with blue solid circles (n = 5). c) Sanger sequencing analyses and T‐A cloning sequencing results of PCR amplicons of the targeted sites in the control and LBP⁴/pCas9 groups. d) Inmmunohistochemical analyses of Cas9 protein expression in heart, liver, spleen, lung, and kidney of the control and LBP⁴/pCas9 groups. e) Inmmunohistochemical analyses of survivin and ki‐67 proteins expressions in tumor tissues after different treatments.