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.

Taking central nervous system regenerative therapies to the clinic: curing rodents versus nonhuman primates versus humans

The central nervous system is known to have limited regenerative capacity.Not only does this halt the human body’s reparative processes after central nervous system lesions,but it also impedes the establishment of effective and safe therapeutic options for such patients.Despite the high prevalence of stroke and spinal cord injury in the general population,these conditions remain incurable and place a heavy burden on patients’families and on society more broadly.Neuroregeneration and neural engineering are diverse biomedical fields that attempt reparative treatments,utilizing stem cells-based strategies,biologically active molecules,nanotechnology,exosomes and highly tunable biodegradable systems(e.g.,certain hydrogels).Although there are studies demonstrating promising preclinical results,safe clinical translation has not yet been accomplished.A key gap in clinical translation is the absence of an ideal animal or ex vivo model that can perfectly simulate the human microenvironment,and also correspond to all the complex pathophysiological and neuroanatomical factors that affect functional outcomes in humans after central nervous system injury.Such an ideal model does not currently exist,but it seems that the nonhuman primate model is uniquely qualified for this role,given its close resemblance to humans.This review considers some regenerative therapies for central nervous system repair that hold promise for future clinical translation.In addition,it attempts to uncover some of the main reasons why clinical translation might fail without the implementation of nonhuman primate models in the research pipeline.

Nonhuman primate models of focal cerebral ischemia

Rodents have been widely used in the production of cerebral ischemia models. However, successful therapies have been proven on experimental rodent stroke model, and they have often failed to be effective when tested clinically. Therefore, nonhuman primates were recommended as the ideal alternatives, owing to their similarities with the human cerebrovascular system, brain metabolism, grey to white matter ratio and even their rich behavioral repertoire. The present review is a thorough summary of ten methods that establish nonhuman primate models of focal cerebral ischemia; electrocoagulation, endothelin-1-induced occlusion, microvascular clip occlusion, autologous blood clot embolization, balloon inflation, microcatheter embolization, coil embolization, surgical suture embolization, suture, and photochemical induction methods. This review addresses the advantages and disadvantages of each method, as well as precautions for each model, compared nonhuman primates with rodents, different species of nonhuman primates and different modeling methods. Finally it discusses various factors that need to be considered when modelling and the method of evaluation after modelling. These are critical for understanding their respective strengths and weaknesses and underlie the selection of the optimum model.

Local transgene expression and whole-body transgenesis to model brain diseases in nonhuman primate

Animal model is an essential tool in the life sciences research, notably in understanding the pathogenesis of the diseases and for further therapeutic intervention success. Rodents have been the most frequently used animals to model human disease since the establishment of gene manipulation technique. However, they remain inadequate to fully mimic the pathophysiology of human brain disease, partially due to huge differences between rodents and humans in terms of anatomy, brain function, and social behaviors. Nonhuman primates are more suitable in translational perspective. Thus, genetically modified animals have been generated to investigate neurologic and psychiatric disorders. The classical transgenesis technique is not efficient in that model; so, viral vector-mediated transgene delivery and the new genome-editing technologies have been promoted. In this review, we summarize some of the technical progress in the generation of an ad hoc animal model of brain diseases by gene delivery and real transgenic nonhuman primate.

Endovascular middle cerebral arterial occlusion in a nonhuman primate model of chronic stroke

No study has reported the safety, effectiveness, and consistency of endovascular middle cerebral artery occlusion in a chronic cerebral ischemia model. Nor have studies verified the safest and most effective segment, or branch, in the embolic middle cerebral artery. In this experiment, cerebral infarction models were established at M1, and on the upper and lower trunks on the contralateral side of the handedness of rhesus monkeys by using endovascular intervention. The results confirmed a high animal survival rate in stroke models of middle cerebral artery upper trunk occlusion. There was pronounced paralysis at the acute phase, long-term upper extremity dysfunction at the chronic phase, and the models showed good repeatability and consistency. Thus, this study describes a safe and effective model of chronic stroke.

Comparative evaluation of acute phase proteins by C-reactive protein(CRP)and serum amyloid A(SAA)in nonhuman primates and feline carnivores

The feasibility of a commercially available assay for C-reactive protein(CRP,CRP for humans:hCRP,and CRP for dogs:vCRP)and a trial reagent of serum amyloid A(SAA,vSAA for animals)were applied to the measurement of acute phase proteins in zoo animals,particularly in nonhuman primates and feline carnivores was evaluate.Results showed that hCRP and vSAA methods were applicable to measure CRP and SAA in Haplorhini.There was a highly signifcant correlation between both parameters with remarkably high correlation coefcient.A higher proportion of Bonnet macaques in Haplorhini,and the linear regression with good correlation between hCRP and vSAA levels were observed.Reference values in healthy Bonnet macaques were hCRP(46.86±30.97 nmol/L)and vSAA(9.06±1.95μg/mL).Although Ring-tailed lemur,which belonging to Strepsirrhini,showed low vSAA concentrations(reference values:1.08±0.47μg/mL),vSAA in patients was apparently elevated.The vCRP and vSAA methods were applicable to measurements of CRP and SAA in feline carnivores for highly signifcant correlation between both parameters.Theses two methods were also been deteded in lions,tigers and cheetahs.vSAA assays can be applied to measure SAA levels in other carnivores and herbivores.In conclusion,vSAA systems have potential utility as diagnostic tools for health screening and prediction in zoo animals.

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.

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.

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates

BACKGROUND The development of regenerative therapy for human spinal cord injury(SCI)is dramatically restricted by two main challenges:the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing.Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge.The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI.AIM To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells(drNPCs).METHODS Seven non-human primates with verified complete thoracic SCI were divided into two groups:drNPC group(n=4)was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury,and lesion control(n=3)was injected identically with the equivalent volume of vehicle.RESULTS Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways.Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation.Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk,migrating to areas of axon growth cones.CONCLUSION Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI,based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation.The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs.Instead,directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support,thereby further supporting the regeneration processes.

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.

How can we establish animal models of HIV-associated lymphoma?

Human immunodeficiency virus(HIV)infection is strongly associated with a height-ened incidence of lymphomas.To mirror the natural course of human HIV infection,animal models have been developed.These models serve as valuable tools to inves-tigate disease pathobiology,assess antiretroviral and immunomodulatory drugs,ex-plore viral reservoirs,and develop eradication strategies.However,there are currently no validated in vivo models of HIV-associated lymphoma(HAL),hampering progress in this crucial domain,and scant attention has been given to developing animal models dedicated to studying HAL,despite their pivotal role in advancing knowledge.This re-view provides a comprehensive overview of the existing animal models of HAL,which may enhance our understanding of the underlying pathogenesis and approaches for malignancies linked to HIV infection.

Characteristics of a primate model of focal motor cortical seizures suitable for preclinical testing of therapies like DBS

Background and objective: Generating and characterizing primate models of epileptic seizures are important for understanding pathophysiology of diseases and establishing preclinical efficacy of novel therapies like Deep Brain Stimulation. A focal motor epilepsy model is described here. Method: Seizures were obtained after intracortical penicillin injection into the motor strip through a cannula in two awake monkeys and electrocorticograms were recorded by epidural screws. Seizures were analyzed and compared for number, average duration of each seizure and total duration of ictal activity. Pharmaco-resistance for antiepileptic drug was tested by administration of Diazepam during seizures. Results: A motor status with seizures mimicking those seen in Kojevnikov’s syndrome was easily generated several minutes after penicillin injection and lasted 24 h on an average. The model thus characterized appears stable and consistent. There is no significant variation between experiments in individual primate as well as between two specimens. Diazepam though reduced the total duration of seizures, failed to abolish behavioural seizures. Conclusion: This model represents a good alternative model for preclinical research aiming at testing novel therapies because seizures are obtained on demand, last up to 24 h after a single penicillin injection, are stable and resistant to Diazepam.

In vitro and in vivo models of acute alcohol exposure

Alcohol abuse is a global problem due to the financial burden on society and the healthcare system. While the harmful health effects of chronic alcohol abuse are well established, more recent data suggest that acute alcohol consumption also affects human wellbeing. Thus, there is a need for research models in order to fully understand the effect of acute alcohol abuse on different body systems and organs. The present manuscript summarizes the interdisciplinary advantages and disadvantages of currently available human and non-human models of acute alcohol abuse, and identifi es their suitability for biomedical research.

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.

Establishment of AIDS Animal Model with SIVmac239 Infected Chinese Rhesus Monkey

In the present research,two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4+ T lymphocyte in peripheral blood,plasma viral loads,proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection,proviral DNA had been detected in PBMCs,and infectious SIVmac239 virus had been isolated from PBMCs. At the same period,the numbers of CD4+ T lymphocytes were significantly decreased,and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover,antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.

A novel mind-set in primate experimentation:Implications for primate welfare

We emphasize the importance of studying the primate brain in cognitive neuroscience and suggest a new mind-set in primate experimentation within the boundaries of animal welfare regulations.Specifically,we list the advantages of investigating both genes and neural mechanisms and processes in the emergence of behavioral and cognitive functions,and propose the establishment of an open field of primate research.The latter may be conducted by implementing and harmonizing experimental practices with ethical guidelines that regulate(1)management of natural parks with free-moving populations of target nonhuman primates,(2)establishment of indoor-outdoor labs for both system genetics and neuroscience investigations,and(3)hotel space and technologies which remotely collect and dislocate information regarding primates geographically located elsewhere.

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.