CRISPR/Cas9 has been used to genetically modify genomes in a variety of species, including non-human primates. Unfortunately, this new technology does cause mosaic mutations, and we do not yet know whether such mutations can functionally disrupt the targeted gene or cause the pathology seen in human disease. Addressing these issues is necessary if we are to generate large animal models of human diseases using CRISPR/Cas9. Here we used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. Moreover, CRISPR/Cas9 induced mutations in both male and female monkeys, with the markedly depleted dystrophin and muscle degeneration seen in early DMD. Our findings indicate that CRISPR/Cas9 can efficiently generate monkey models of human diseases, regardless of inheritance patterns. The presence of degenerated muscle cells in newborn Cas9-targeted monkeys suggests that therapeutic interventions at the early disease stage may be effective at alleviating the myopathy.

A number of mouse models expressing mutant huntingtin (htt) with an expanded polyglutamine (polyQ) domain are useful for studying the pathogenesis of Huntington's disease (HD) and identifying appropriate therapies. However, these models exhibit neurological phenotypes that differ in their severity and nature. Understanding how transgenic htt leads to variable neuropathology in animal models would shed light on the pathogenesis of HD and help us to choose HD models for investigation. By comparing the expression of mutant htt at the transcriptional and protein levels in transgenic mice expressing N-terminal or full-length mutant htt, we found that the accumulation and aggregation of mutant htt in the brain is determined by htt context. HD mouse models demonstrating more severe phenotypes show earlier accumulation of N-terminal mutant htt fragments, which leads to the formation of htt aggregates that are primarily present in neuronal nuclei and processes, as well as glial cells. Similarly, transgenic monkeys expressing exon-1 htt with a 147-glutamine repeat (147Q) died early and showed abundant neuropil aggregates in swelling neuronal processes. Fractionation of HD150Q knock-in mice brains revealed an age-dependent accumulation of N-terminal mutant htt fragments in the nucleus and synaptosomes, and this accumulation was most pronounced in the striatum due to decreased proteasomal activity. Our findings suggest that the neuropathological phenotypes of HD stem largely from the accumulation of N-terminal mutant htt fragments and that this accumulation is determined by htt context and cell-type-dependent clearance of mutant htt.

Context: Aberrant activation of MAPK has been implicated to play important roles in pathological processes of endometriosis. However, how MAPK are constitutively activated in endometriotic tissues remains largely unknown. microRNA are small noncoding RNA that regulate the stability or translational efficiency of target mRNA by interacting with the 3′ untranslated region. Thus, miRNA are thought to be modulators of the transcriptional response, fine-tuning gene expression. Objective: The aim of this study was to evaluate the functional roles of microRNA-20a (miR20a) in MAPK activation and the pathogenesis of endometriosis. Design: miR20a expression was analyzed in nonpaired (endometrium = 17; endometriosis = 37) and paired (n = 12) endometriotic tissues by quantitative RT-PCR. Overexpression of miR20a in eutopic endometrial stromal cells or inhibition of miR20a in ectopic endometriotic stromal cells was used to evaluate its impact on ERK phosphorylation and subsequently angiogenesis- and proliferation-related gene expression. Results: Levels of miR20a were up-regulated in endometriotic stromal cells. Elevation of miR20a was up-regulated by hypoxia inducible factor-1α. The up-regulation of miR20a causes the down-regulation of dual-specificity phosphatase-2, which leads to prolonged ERK phosphorylation and an increase in the expression of several angiogenic genes. Furthermore, the up-regulation of miR20a enhances the prostaglandin E₂-induced expression of fibroblast growth factor-9, a potent mitogen that stimulates both endothelial and endometrial cell proliferation. Conclusion: Our findings provide the novel mechanism that not only functionally links together hypoxic stress, miR20a expression, aberrant ERK phosphorylation, and angiogenesis but also demonstrates that miR20a is an important modulator in the development of endometriosis.

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that can be caused by genetic mutations in α-synuclein (α-syn) or duplication of wild-type α-syn; PD is characterized by the deposition of α-syn aggregates, indicating a gain of toxicity from accumulation of α-syn. Although the major neuropathologic feature of PD is the degeneration of dopaminergic (DA) neurons in the substantia nigra, non-motor symptoms including anxiety, cognitive defect and sleep disorder precede the onset of motor impairment, and many clinical symptoms of PD are not caused by degeneration of DA neurons. Non-human primate models of PD are important for revealing the early pathology in PD and identifying effective treatments. We established transgenic PD rhesus monkeys that express mutant α-syn (A53T). Six transgenic A53T monkeys were produced via lentiviral vector expressing A53T in fertilized monkey eggs and subsequent embryo transfer to surrogates. Transgenic A53T is expressed in the monkey brain and causes age-dependent non-motor symptoms, including cognitive defects and anxiety phenotype, without detectable sleeping disorders. The transgenic α-syn monkeys demonstrate the specific early symptoms caused by mutant α-syn and provide insight into treatment of early PD.