Generation of in situ CRISPR-mediated primary and metastatic cancer from monkey liver

Liping Zhong,Jian He,Dongni Huang,Chao Tang,Zhikun Zhang,Yu Huo,Junjie Liu,Yun Ma,Yong Huang,Cheng‐Piao Luo ,Zongqiang Lai,Banghao Xu,Tao Lu,Wenlin Gong,Xiyu Liu,Yiqun Luo,Lu Gan,Pan Wu,Nuo Yang,Yongxiang Zhao

doi:10.1038/s41392-021-00799-7

Abstract

Non-human primates (NHPs) represent the most valuable animals for drug discovery. However, the current main challenge remains that the NHP has not yet been used to develop an efficient translational medicine platform simulating human diseases, such as cancer. This study generated an in situ gene-editing approach to induce efficient loss-of-function mutations of Pten and p53 genes for rapid modeling primary and metastatic liver tumors using the CRISPR/Cas9 in the adult cynomolgus monkey. Under ultrasound guidance, the CRISPR/Cas9 was injected into the cynomolgus monkey liver through the intrahepatic portal vein. The results showed that the ultrasound-guided CRISPR/Cas9 resulted in indels of the Pten and p53 genes in seven out of eight monkeys. The best mutation efficiencies for Pten and p53 were up to 74.71% and 74.68%, respectively. Furthermore, the morbidity of primary and extensively metastatic (lung, spleen, lymph nodes) hepatoma in CRISPR-treated monkeys was 87.5%. The ultrasound-guided CRISPR system could have great potential to successfully pursue the desired target genes, thereby reducing possible side effects associated with hitting non-specific off-target genes, and significantly increasing more efficiency as well as higher specificity of in situ gene editing in vivo, which holds promise as a powerful, yet feasible tool, to edit disease genes to build corresponding human disease models in adult NHPs and to greatly accelerate the discovery of new drugs and save economic costs.

Highlights

IntroductionThe main reason for this challenge is that no ideal animal model mimicking human diseases (such as cancer) is available
As well all know, the current success rate of new drug discovery is very low (
The COS-7 cells were transiently transfected with Adeno-Pten-single-guide RNA (sgRNA) and Adeno-cas[9], Adeno-p53-sgRNA, and Adeno-cas[9] for 48 hr, and total genomic DNA was extracted and the Pten and p53 loci were analyzed by the T7E1 assay

Summary

Introduction

The main reason for this challenge is that no ideal animal model mimicking human diseases (such as cancer) is available Experimental animal models, such as rodents and nonhuman primates (NHPs), are essential for both cancer research and pre-clinical assessment of anti-tumor drugs and vaccines.[1,2] Of the two models, rodents are more widely used because of a number of advantages, such as rapid propagation, small size, low cost, clear genetic background, and availability of transgenic techniques. Mouse models of cancer are usually created using gene targeting in embryonic stem cells or somatic cell nuclear transfer methods, through which knock-out, knock-in, and precise modifications of the desired genes are achieved.[7] Despite their apparent advantages, the use of experimental NHPs in cancer research and preclinical evaluation of anti-tumor drugs have been very limited, which is largely due to high costs and serious ethical concerns. Compared to mice and rats, creating loss-of-function or gain-of-function genetic mutations through the breeding of NHPs remains tedious and challenging because of their longer sexual maturation time and slower propagation compared to rodents or other small model organisms.[7,8]

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