Profound cellular defects attribute to muscular pathogenesis in the rhesus monkey model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutations in the DMD gene. Muscle fibers rely on the coordination of multiple cell types for repair and regenerative capacity. To elucidate the cellular and molecular changes in these cell types under pathologic conditions, we generated a rhesus monkey model for DMD that displays progressive muscle deterioration and impaired motor function, mirroring human conditions. By leveraging these DMD monkeys, we analyzed freshly isolated muscle tissues using single-cell RNA sequencing (scRNA-seq). Our analysis revealed changes in immune cell landscape, a reversion of lineage progressing directions in fibrotic fibro-adipogenic progenitors (FAPs), and TGF-ß resistance in FAPs and muscle stem cells (MuSCs). Furthermore, MuSCs displayed cell-intrinsic defects, leading to differentiation deficiencies. Our study provides important insights into the pathogenesis of DMD, offering a valuable model and dataset for further exploration of the underlying mechanisms, and serves as a suitable platform for developing and evaluating therapeutic interventions.

Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Discovery and characterization of sgRNA-sequence-independent DNA cleavage from CRISPR/Cas9 in mouse embryos.

The CRISPR/Cas9 system can induce off-target effects in programmed gene editing, but there have been few reports on cleavage detection and their affection in embryo development. To study these events, sgRNAs with different off-target rates were designed and compared after micro-injected into mouse zygotes, and γH2AX was used for DNA cleavage sites analysis by immunostaining and CUT&Tag. Although the low off-target sgRNA were usually selected for production gene editing animals, γH2AX immunofluorescence indicated that there was a relative DSB peak at 15 h after Cas9 system injection, and the number of γH2AX foci at the peak was significantly higher in the low off-target sgRNA-injected group than in the control group. Further, the result of CUT&Tag sequencing analysis showed more double-strand breaks (DSBs) related sequences were detected in low off-target sgRNA-injected group than control and the distribution of DSB related sequences had no chromosome specificity. Gene Ontology (GO) annotation analysis of the DSB related sequences showed that these sequences were mainly concentrated at genes associated with some important biological processes, molecular functions, and cell components. In a conclusion, there are many sgRNA-sequence-independent DSBs in early mouse embryos when the Cas9 system is used for gene editing and the DSB related sequence could be detected and characterized in the genome. These results and method should also be considered in using or optimizing the Cas9 system.

No off-target mutations in functional genome regions of a CRISPR/Cas9-generated monkey model of muscular dystrophy.

CRISPR/Cas9 is now widely used in biomedical research and has great potential for clinical applications. However, the safety and efficacy of this gene-editing technique are significant issues. Recent reports on mouse models and human cells have raised concerns that off-target mutations could hamper applying the CRISPR technology in patients. The high similarities of nonhuman primates to humans in genome content and organization, genetic diversity, physiology, and cognitive abilities have made these animals ideal experimental models for understanding human diseases and developing therapeutics. Off-target mutations of CRISPR/Cas9 have been analyzed in previous studies of nonhuman primates, but no report has investigated genome-wide off-target effects in living monkeys. Here, we used rhesus monkeys in which a genetic disorder mimicking Duchenne muscular dystrophy had previously been produced with CRISPR/Cas9. Using whole-genome sequencing to comprehensively assess on- and off-target mutations in these animals, we found that CRISPR/Cas9-based gene editing is active on the expected genomic sites without producing off-target modifications in other functional regions of the genome. These findings suggest that the CRISPR/Cas9 technique could be relatively safe and effective in modeling genetic disease in nonhuman primates and in future therapeutic research of human diseases.

Phenotypes and genetic etiology of spontaneous polycystic kidney and liver disease in cynomolgus monkey.

Introduction: Polycystic kidney disease (PKD) is a common autosomal dominant or recessive genetic disease, often accompanied by polycystic liver disease (PLD). Many cases of PKD in animals have been reported. However, little is known about the genes that cause PKD in animals. Methods: In this study, we evaluated the clinical phenotypes of PKD in two spontaneously aged cynomolgus monkeys and explored the genetic etiology using whole-genome sequencing (WGS). Ultrasonic and histological consequences were further investigated in PKD- and PLD-affected monkeys. Results: The results indicated that the kidneys of the two monkeys had varying degrees of cystic changes, and the renal cortex was thinned and accompanied by fluid accumulation. As for hepatopathy, inflammatory cell infiltration, cystic effusion, steatosis of hepatocytes, and pseudo-lobular were found. Based on WGS results, the variants of PKD1:(XM_015442355: c.1144G>C p. E382Q) and GANAB: (NM_001285075.1: c.2708T>C/p. V903A) are predicted to be likely pathogenic heterozygous mutations in PKD- and PLD-affected monkeys. Discussion: Our study suggests that the cynomolgus monkey PKD and PLD phenotypes are very similar to those in humans, and are probably caused by pathogenic genes homologous to humans. The results indicate that cynomolgus monkeys can be used as the most appropriate animal model for human PKD pathogenesis research and therapeutic drug screening.

Efficient Modulation of Exon Skipping via Antisense Circular RNAs.

Splice-switching antisense oligonucleotides (ASOs) and engineered U7 small nuclear ribonucleoprotein (U7 Sm OPT) are the most commonly used methods for exon skipping. However, challenges remain, such as limited organ delivery and repeated dosing for ASOs and unknown risks of by-products produced by U7 Sm OPT. Here, we showed that antisense circular RNAs (AS-circRNAs) can effectively mediate exon skipping in both minigene and endogenous transcripts. We also showed a relatively higher exon skipping efficiency at the tested Dmd minigene than U7 Sm OPT. AS-circRNA specifically targets the precursor mRNA splicing without off-target effects. Moreover, AS-circRNAs with adeno-associated virus (AAV) delivery corrected the open reading frame and restored the dystrophin expression in a mouse model of Duchenne muscular dystrophy. In conclusion, we develop an alternative method for regulating RNA splicing, which might be served as a novel tool for genetic disease treatment.

Interaction of p53 and ASPPs regulates rhesus monkey embryonic stem cells conversion to neural fate concomitant with apoptosis.

The tumor suppressor p53 is a key regulator of cell apoptosis and cell cycle arrest. Recent studies show that the delicate balance of p53 expression is important for neural tube defects, neuronal degeneration, embryonic lethality, as well as differentiation and dedifferentiation. Moreover, p53 showed different regulatory patterns between rodent and primate embryonic stem cells (ESCs). However, the role of p53 and apoptosis stimulating protein of p53 (ASPP) during neural differentiation (ND) from primate ESCs is still unknown. In this study, using an FGF-2 and/or HGF selectively containing ND culture systems for rhesus monkey ESCs (rESCs), the changes of p53 and ASPPs, and p53 targets, i.e. BAX and p21, were analyzed. Our results showed that the expression patterns of ASPP1/ASPP2 and iASPP were opposite in rESCs but similar in differentiated cells, and the expression of p53 was approximately consistent with BAX, but not p21. These findings indicate that the strong expression of iASPP in ESCs and weak expression of ASPP1/ASPP2 maintain the stability of stemness; and in ND niche, unimpaired iASPP may decrease its inhibition of ASPP1/ASPP2 expression, the interaction of p53 and ASPPs causing rESCs to convert towards a neural fate concomitant with apoptosis, but not to cell cycle arrest.