Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys
On May 18th, 2017, the world's top journal "Cell" published the Kunming University of Science and Technology, Yunnan Key Laboratory of Primate Biomedical Research (LPBR), Ji Weizhi, Chen Yongchang team and Sun Yi team of Tongji University, using TALEN technology to carry out Rettsyndrome(RTT) Crab Monkey model.
Rett syndrome (RTT) is a progressive neurodevelopmental disorder that mostly manifests in girls with a morbidity rate of 1:10,000–1:15,000. Almost 95% of RTT is believed to be caused by mutations of an X-linked gene methyl-CpG-binding protein 2 (MECP2) . MECP2 mutations are most often embryonic lethal for boys, except for very few, who are born with severe encephalopathy leading to death before 2 years of age. RTT girls seem to have normal development for up to 6–18 months but manifest a series of symptoms associated with intellectual disability, loss of acquired language, and compromised cognitive, social, and motor skills, etc. As RTT is a monogenic disorder, genetic modification technologies have made it possible to develop animal models for further study. RTT animal models were first generated in mice and recently in rats.Gene-edited nonhuman primates (NHPs),such as rhesus and cynomolgus monkeys,share high levels of genetic, physiological, social behavioral, and CNS developmental similarities with humans, making them suitable for the study of neurodevelopmental disorders. In order to establish the RTT monkey model, researchers from Kunming University of Science and Technology, Yunnan Key Laboratory of Primate Biomedical Research (LPBR) and Tongji University, after more than one year of research, first reportedthat they successfully developed a monkey model of the MECP2 gene using TALEN, a targeted gene editing method , published in the "Cell stem cell" magazine.
This report in "Cell" is different from the rodent model,Mecp2 knockout rodents show "limb-clasping" phenotype, but this phenotype is shared by neurodegeneration conditions in mice, which is not characteristic for RTT. In contrast, monkey models showed complicated behavioral features, such as fragmented sleep, increased stereotypy, reduced active avoidance of noisy or heat stimuli, and reduced environmental exploration, all of which resemble symptoms of patients. These activities are not widely set up for detection in mouse models. This study showed that a valid monkey model might offer robust phenotype or endophenotype, which could be implemented as outcome measures in human clinical trials in the future to facilitate drug development.
Generation of Cynomolgus Monkey Chimeric Fetuses using Embryonic Stem Cells
By improving the culture conditions of stem cells, Yunnan Key Laboratory of Primate Biomedical Research (LPBR) first isolated and established Cynomolgus monkey ESCs which capable of producing chimeric monkeys in the world. Cynomolgus monkey ESCs (cESCs) grown in adjusted culture conditions are able to incorporate into host embryos and develop into chimeras with contribution in all three germ layers and in germ cell progenitors. Under the optimized culture conditions, which are based on an approach developed previously for naive human ESCs, the cESCs displayed altered growth properties, gene expression profiles, and self-renewal signaling pathways, suggestive of an altered naivelike cell state. The results of this study are published in Cell Stem Cell (Cell stem cell. 2014; 14(3): 323-328).
The generation of monkey ESCs with the ability to produce same-species chimeras has important implications for stem cell research, clinical regenerative medicine, and non-human primate models of human disease. First, this approach can be an important platform upon which to test the pluripotency of primate ESCs or iPSCs. Second, this type of approach could pave the way for the use of chimerism to generate functional organs, such as pancreas and kidney, from PSCs by injection of monkey PSCs into pancreatogenesis- or nephrogenesis-disabled monkey early embryos as demonstrated by previous reports . Third, monkey chimeras generated by ESCs could be used to produce human disease models in non-human primates for the study of mechanisms of diseases by gene editing.
Generation of Gene-Modified Cynomolgus Monkey
In 2014, Yunnan Key Laboratory of Primate Biomedical Research (LPBR) cooperated with domestic research groups,first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, successfully achieve precise gene targeting in cynomolgus monkeys,and successfully obtained the first Cas9-mediated gene knockout cynomolgus monkeys.
The results of the study were published in the first unit in Cell (Cell. 2014; 156(4): 836-843), which attracted widespread attention. More than a dozen foreign media have reported this, and Nature has referred to this result as "Milestone for targeted gene-editing technology promises better models for human diseases." At the end of 2014, the study was selected by Nature as one of the successful research events in 2014.
(http://www.nature.com/news/365-days-2014-in-science-1.16573). The article was named one of the 10 best research papers from Cell in 2014 (http://onlinedigeditions.com/publication/frame.php?i=236801&p=&pn=&ver=flex). In the subsequent studies, we further confirmed that CRISPR/Cas9-mediated genetic modification was performed on the germ cells of the experimental animals, and the results were published in Cell Research (Cell research. 2015; 25(2): 262-265).