Genetically modified non-human primate models for research on neurodegenerative diseases

Neurodegenerative diseases(NDs)are a group of debilitating neurological disorders that primarily affect elderly populations and include Alzheimer's disease(AD),Parkinson's disease(PD),Huntington's disease(HD),and amyotrophic lateral sclerosis(ALS).Currently,there are no therapies available that can delay,stop,or reverse the pathological progression of NDs in clinical settings.As the population ages,NDs are imposing a huge burden on public health systems and affected families.Animal models are important tools for preclinical investigations to understand disease pathogenesis and test potential treatments.While numerous rodent models of NDs have been developed to enhance our understanding of disease mechanisms,the limited success of translating findings from animal models to clinical practice suggests that there is still a need to bridge this translation gap.Old World nonhuman primates(NHPs),such as rhesus,cynomolgus,and vervet monkeys,are phylogenetically,physiologically,biochemically,and behaviorally most relevant to humans.This is particularly evident in the similarity of the structure and function of their central nervous systems,rendering such species uniquely valuable for neuroscience research.Recently,the development of several genetically modified NHP models of NDs has successfully recapitulated key pathologies and revealed novel mechanisms.This review focuses on the efficacy of NHPs in modeling NDs and the novel pathological insights gained,as well as the challenges associated with the generation of such models and the complexities involved in their subsequent analysis.

Experimental primates and non-human primate(NHP) models of human diseases in China: current status and progress

Non-human primates （NHPs） are phylogenetically close to humans, with many similarities in terms of physiology, anatomy, immunology, as well as neurology, all of which make them excellent experimental models for biomedical research. Compared with developed countries in America and Europe, China has relatively rich primate resources and has continually aimed to develop NHPs resources. Currently, China is a leading producer and a major supplier of NHPs on the international market. However, there are some deficiencies in feeding and management that have hampered China＇s growth in NHP research and materials. Nonetheless, China has recently established a number of primate animal models for human diseases and achieved marked scientific progress on infectious diseases, cardiovascular diseases, endocrine diseases, reproductive diseases, neurological diseases, and ophthalmic diseases, etc. Advances in these fields via NHP models will undoubtedly further promote the development of China＇s life sciences and pharmaceutical industry, and enhance China＇s position as a leader in NHP research. This review covers the current status of NHPs in China and other areas, highlighting the latest developments in disease models using NHPs, as well as outlining basic problems and proposing effective to better utilize NHP resources and further foster NHP research in China.

Meeting report:the 4^(th) symposium on animal models of non-human primates in Kunming,Yunnan,China

From 2 to 4 November, 2016, the 4th Symposium on Animal Models of Non-Human Primates （NHP） was held in Kunming, Yunnan, China. This meeting was organized by the Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences （CAS） ＆ Yunnan Province Kunming Primate Research Center （KPRC）, Zoological Research, and Kunming Institute of Zoology （KIZ）, CAS.

Large animal models for Huntington's disease research

Huntington'sdisease(HD)isahereditary neurodegenerative disorder for which there is currently no effectivetreatmentavailable.Consequently,the development of appropriate disease models is critical to thoroughly investigate disease progression.The genetic basis of HD involves the abnormal expansion of CAG repeats in the huntingtin(HTT)gene,leading to the expansion of a polyglutamine repeat in the HTT protein.Mutant HTT carrying the expanded polyglutamine repeat undergoes misfolding and forms aggregates in the brain,which precipitate selective neuronal loss in specific brain regions.Animal models play an important role in elucidating the pathogenesis of neurodegenerative disorders such as HD and in identifying potential therapeutic targets.Due to the marked species differences between rodents and larger animals,substantial efforts have been directed toward establishing large animal models for HD research.These models are pivotal for advancing the discovery of novel therapeutic targets,enhancing effective drug delivery methods,and improving treatment outcomes.We have explored the advantages of utilizing large animal models,particularly pigs,in previous reviews.Since then,however,significant progress has been made in developing more sophisticated animal models that faithfully replicate the typical pathology of HD.In the current review,we provide a comprehensive overview of large animal models of HD,incorporating recent findings regarding the establishment of HD knock-in(KI)pigs and their genetic therapy.We also explore the utilization of large animal models in HD research,with a focus on sheep,non-human primates(NHPs),and pigs.Our objective is to provide valuable insights into the application of these large animal models for the investigation and treatment of neurodegenerative disorders.

Defects and asymmetries in the visual pathway of non-human primates with natural strabismus and amblyopia

Strabismus and amblyopia are common ophthalmologic developmental diseases caused by abnormal visual experiences. However, the underlying pathogenesis and visual defects are still not fully understood. Most studies have used experimental interference to establish diseaseassociated animal models, while ignoring the natural pathophysiological mechanisms. This study was designed to investigate whether natural strabismus and amblyopia are associated with abnormal neurological defects. We screened one natural strabismic monkey(Macaca fascicularis) and one natural amblyopic monkey from hundreds of monkeys, and retrospectively analyzed one human strabismus case. Neuroimaging, behavioral,neurophysiological, neurostructural, and genovariation features were systematically evaluated using magnetic resonance imaging(MRI), behavioral tasks, flash visual evoked potentials(FVEP),electroretinogram(ERG), optical coherence tomography(OCT), and whole-genome sequencing(WGS), respectively. Results showed that the strabismic patient and natural strabismic and amblyopic monkeys exhibited similar abnormal asymmetries in brain structure, i.e., ipsilateral impaired right hemisphere. Visual behavior, visual function, retinal structure, and fundus of the monkeys were impaired. Aberrant asymmetry in binocular visual function and structure between the strabismic and amblyopic monkeys was closely related, with greater impairment of the left visual pathway.Several similar known mutant genes for strabismus and amblyopia were also identified. In conclusion,natural strabismus and amblyopia are accompanied by abnormal asymmetries of the visual system,especially visual neurophysiological and neurostructural defects. Our results suggest that future therapeutic and mechanistic studies should consider defects and asymmetries throughout the entire visual system.

Current state of research on non-human primate models of Alzheimer’s disease

With the increasingly serious aging of the global population, dementia has already become a severe clinical challenge on a global scale. Dementia caused by Alzheimer’s disease(AD) is the most common form of dementia observed in the elderly, but its pathogenetic mechanism has still not been fully elucidated. Furthermore, no effective treatment strategy has been developed to date, despite considerable efforts. This can be mainly attributed to the paucity of animal models of AD that are sufficiently similar to humans. Among the presently established animal models, non-human primates share the closest relationship with humans, and their neural anatomy and neurobiology share highly similar characteristics with those of humans. Thus, there is no doubt that these play an irreplaceable role in AD research. Considering this, the present literature on non-human primate models of AD was reviewed to provide a theoretical basis for future research.

Non-human primate models in drug addiction deserve more attention

Dear Editor, The process of relapse involves firm or aberrant memories of environmental cues associated with drug craving or addiction. To date, it is not known where these memories are stored in the brain, what kinds of regulatory biological factors or molecules are involved, nor why it is so difficult to stop addiction psychologically. Currently, rodent animal models, such as the self-administration and conditioning place preference / aversion paradigm are still widely used in the studies of drug withdrawal syndromes or drug-associate memories. However, the differences between humans and rodents--particularly in terms of genetics, and pathology and pharmacology--have significantly limited the application of further studies on this topic. Essentially, rodents lack the longterm or life-time memories humans possess and lose their drug-associated memory only after a few weeks of withdrawal.

Electroencephalography studies of hypoxic ischemia in fetal and neonatal animal models

Alongside clinical achievements,experiments conducted on animal models (including primate or non-primate) have been effective in the understanding of various pathophysiological aspects of perinatal hypoxic/ ischemic encephalopathy (HIE).Due to the reasonably fair degree of flexibility with experiments,most of the research around HIE in the literature has been largely concerned with the neurodevelopmental outcome or how the frequency and duration of HI seizures could relate to the severity of perinatal brain injury,following HI insult.This survey concentrates on how EEG experimental studies using asphyxiated animal models (in rodents,piglets,sheep and non-human primate monkeys) provide a unique opportunity to examine from the exact time of HI event to help gain insights into HIE where human studies become difficult.

Animal models for SARS-CoV-2 and SARS-CoV-1 pathogenesis,transmission and therapeutic evaluation

There is a critical need to develop animal models to alleviate vaccine and drug development difficulties against zoonotic viral infections.The coronavirus family,which includes severe acute respiratory syndrome coronavirus 1 and severe acute respiratory syndrome coronavirus 2,crossed the species barrier and infected humans,causing a global outbreak in the 21st century.Because humans do not have pre-existing immunity against these viral infections and with ethics governing clinical trials,animal models are therefore being used in clinical studies to facilitate drug discovery and testing efficacy of vaccines.The ideal animal models should reflect the viral replication,clinical signs,and pathological responses observed in humans.Different animal species should be tested to establish an appropriate animal model to study the disease pathology,transmission and evaluation of novel vaccine and drug candidates to treat coronavirus disease 2019.In this context,the present review summarizes the recent progress in developing animal models for these two pathogenic viruses and highlights the utility of these models in studying SARS-associated coronavirus diseases.

The 3rd Symposium on Animal Models of Primates——The Application of Non-Human Primates to Basic Research and Translational Medicine

In order to understand the fundamental questions of the biology of life and to duplicate the pathogenesis of human diseases, animal models using different experimental animals, such as rodents, Drosophila, Caenorhabditis elegans, and zebrafish, have been established and used widely for many decades. The controllability of environmental conditions, the high reproducibility, the ease of scale and the comparability of results, as well as the ability to use different standards for ethical protocols, all make an animal model the ideal tool for carrying out studies on human diseases and the development of novel pharmaceuticals and new therapies （Xue et al., 2014）. An ideal animal model should reflect the complete spectra of a specific human disease, with similar features on the following key issues： （1） genetic basis; （2） anatomy and physiology; （3） pathological response（s） and underlying mechanism（s）; （4） phenotypic endpoints as clinical studies; （5） responsiveness to known drugs with clinical efficacy; and （6） prediction of clinical efficacy （McGonigle and Ruggeri, 2014）.

Cell reprogramming therapy for Parkinson’s disease

Parkinson’s disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta.Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson’s disease.The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson’s disease,which could substantially alleviate the symptoms of Parkinson’s disease in clinical practice.However,ethical issues and tumor formation were limitations of its clinical application.Induced pluripotent stem cells can be acquired without sacrificing human embryos,which eliminates the huge ethical barriers of human stem cell therapy.Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons,without the need for intermediate proliferation states,thus avoiding issues of immune rejection and tumor formation.Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson’s disease.However,there are also ethical concerns and the risk of tumor formation that need to be addressed.This review highlights the current application status of cell reprogramming in the treatment of Parkinson’s disease,focusing on the use of induced pluripotent stem cells in cell replacement therapy,including preclinical animal models and progress in clinical research.The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson’s disease,as well as the controversy surrounding in vivo reprogramming.These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson’s disease.

Deep learning-based activity recognition and fine motor identification using 2D skeletons of cynomolgus monkeys

Video-based action recognition is becoming a vital tool in clinical research and neuroscientific study for disorder detection and prediction.However,action recognition currently used in non-human primate(NHP)research relies heavily on intense manual labor and lacks standardized assessment.In this work,we established two standard benchmark datasets of NHPs in the laboratory:Monkeyin Lab(Mi L),which includes 13 categories of actions and postures,and MiL2D,which includes sequences of two-dimensional(2D)skeleton features.Furthermore,based on recent methodological advances in deep learning and skeleton visualization,we introduced the Monkey Monitor Kit(Mon Kit)toolbox for automatic action recognition,posture estimation,and identification of fine motor activity in monkeys.Using the datasets and Mon Kit,we evaluated the daily behaviors of wild-type cynomolgus monkeys within their home cages and experimental environments and compared these observations with the behaviors exhibited by cynomolgus monkeys possessing mutations in the MECP2 gene as a disease model of Rett syndrome(RTT).Mon Kit was used to assess motor function,stereotyped behaviors,and depressive phenotypes,with the outcomes compared with human manual detection.Mon Kit established consistent criteria for identifying behavior in NHPs with high accuracy and efficiency,thus providing a novel and comprehensive tool for assessing phenotypic behavior in monkeys.

Pathological insights from amyotrophic lateral sclerosis animal models:comparisons,limitations,and challenges

In order to dissect amyotrophic lateral sclerosis(ALS),a multigenic,multifactorial,and progressive neurodegenerative disease with heterogeneous clinical presentations,researchers have generated numerous animal models to mimic the genetic defects.Concurrent and comparative analysis of these various models allows identification of the causes and mechanisms of ALS in order to finally obtain effective therapeutics.However,most genetically modified rodent models lack overt pathological features,imposing challenges and limitations in utilizing them to rigorously test the potential mechanisms.Recent studies using large animals,including pigs and non-human primates,have uncovered important events that resemble neurodegeneration in patients’brains but could not be produced in small animals.Here we describe common features as well as discrepancies among these models,highlighting new insights from these models.Furthermore,we will discuss how to make rodent models more capable of recapitulating important pathological features based on the important pathogenic insights from large animal models.

血管内介入方法制备非人灵长类动物局部脑缺血模型的研究进展

由于多项转化研究的失败,卒中治疗专业委员会建议,应该在非人灵长类动物局部脑缺血模型上进行临床前研究。采用血管内介入方法制备局部脑缺血模型与人类卒中发病过程一致,而且还可以进行血管内介入治疗,因此是模拟缺血性脑卒中发病与治疗过程的最佳动物模型。目前研究采用多种血管内介入方法制备模型,结果也不一致,本文将对血管内介入方法制备的非人灵长类动物进行总结,对各种模型的优缺点和潜在应用进行分析。此外,本文还根据不同类型的栓子,不同的闭塞部位,异常的血流动力学和可能的应用范围进行进一步的分析。选择合适的非人灵长类动物模型将有助于推动转化研究开展。

Delayed severe cytokine storm and immune cell infiltration in SARS-CoV-2-infected aged Chinese rhesus macaques

As of June 2020, Coronavirus Disease 2019(COVID-19) has killed an estimated 440 000 people worldwide, 74% of whom were aged ≥65 years,making age the most significant risk factor for death caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infection. To examine the effect of age on death, we established a SARSCoV-2 infection model in Chinese rhesus macaques(Macaca mulatta) of varied ages. Results indicated that infected young macaques manifested impaired respiratory function, active viral replication, severe lung damage, and infiltration of CD11b^+ and CD8^+ cells in lungs at one-week post infection(wpi), but also recovered rapidly at 2 wpi. In contrast, aged macaques demonstrated delayed immune responses with a more severe cytokine storm, increased infiltration of CD11b^+ cells, and persistent infiltration of CD8^+ cells in the lungs at 2 wpi. In addition,peripheral blood T cells from aged macaques showed greater inflammation and chemotaxis, but weaker antiviral functions than that in cells from young macaques. Thus, the delayed but more severe cytokine storm and higher immune cell infiltration may explain the poorer prognosis of older aged patients suffering SARS-CoV-2 infection.

Towards Transgenic Primates:What can we learn from mouse genetics?

Considering the great physiological and behavioral similarities with humans,monkeys represent the ideal models not only for the study of complex cognitive behavior but also for the preclinical research and development of novel therapeutics for treating human diseases.Various powerful genetic tech-nologies initially developed for making mouse models are being explored for generating transgenic primate models.We review the latest genetic engineering technologies and discuss the potentials and limitations for systematic production of transgenic primates.

Recent Advances in Animal Models of Zika Virus Infection

An infection by Zika virus(ZIKV), a mosquito-borne flavivirus, broke out in South American regions in 2015, and recently showed a tendency of spreading to North America and even worldwide. ZIKV was first detected in 1947 and only 14 human infection cases were reported until 2007. This virus was previously observed to cause only mild flu-like symptoms.However, recent ZIKV infections might be responsible for the increasing cases of neurological disorders such as GuillainBarre′ syndrome and congenital defects, including newborn microcephaly. Therefore, researchers have established several animal models to study ZIKV transmission and pathogenesis, and test therapeutic candidates. This review mainly summarizes the reported animal models of ZIKV infection, including mice and non-human primates.

Postnatal therapeutic approaches in genetic neurodevelopmental disorders

Genetic neurodevelopmental disorders are characterized by abnormal neurophysiological and behavioral phenotypes,affecting individuals worldwide.While the subject has been heavily researched,current treatment options relate mostly to alleviating symptoms,rather than targeting the altered genome itself.In this review,we address the neurogenetic basis of neurodevelopmental disorders,genetic tools that are enabling precision research of these disorders in animal models,and postnatal gene-therapy approaches for neurodevelopmental disorders derived from preclinical studies in the laboratory.

Primate stem cells: bridge the translation from basic research to clinic application

A growing body of literature has shown that stem cells are very effective for the treatment of degenerative diseases in rodents but these exciting results have not translated to clinical practice. The difference results from the divergence in genetic, metabolic, and physiological phenotypes between rodents and humans. The high degree of similarity between non-human primates(NHPs) and humans provides the most accurate models for preclinical studies of stem cell therapy. Using a NHP model to understand the following key issues, which cannot be addressed in humans or rodents, will be helpful for extending stem cell applications in the basic science and the clinic. These issues include pluripotency of primate stem cells, the safety and efficiency of stem cell therapy, and transplantation procedures of stem cells suitable for clinical translation. Here we review studies of the above issues in NHPs and current challenges of stem cell applications in both basic science and clinical therapies. We propose that the use of NHP models, in particular combining the serial production and transplantation procedures of stem cells is the most useful for preclinical studies designed to overcome these challenges.

Pathogenic analysis of coxsackievirus A10 in rhesus macaques

Coxsackievirus A10(CV-A10)is one of the etiological agents associated with hand,foot and mouth disease(HFMD)and also causes a variety of illnesses in humans,including pneumonia,and myocarditis.Different people,particularly young children,may have different immunological responses to infection.Current CV-A10 infection animal models provide only a rudimentary understanding of the pathogenesis and effects of this virus.The characteristics of CV-A10 infection,replication,and shedding in humans remain unknown.In this study,rhesus macaques were infected by CV-A10 via respiratory or digestive route to mimic the HFMD in humans.The clinical symptoms,viral shedding,inflammatory response and pathologic changes were investigated in acute infection(1–11 day post infection)and recovery period(12–180 day post infection).All infected rhesus macaques during acute infection showed obvious viremia and clinical symptoms which were comparable to those observed in humans.Substantial inflammatory pathological damages were observed in multi-organs,including the lung,heart,liver,and kidney.During the acute period,all rhesus macaques displayed clinical signs,viral shedding,normalization of serum cytokines,and increased serum neutralizing antibodies,whereas inflammatory factors caused some animals to develop severe hyperglycemia during the recovery period.In addition,there were no significant differences between respiratory and digestive tract infected animals.Overall,all data presented suggest that the rhesus macaques provide the first non-human primate animal model for investigating CV-A10 pathophysiology and assessing the development of potential human therapies.