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.

Advances in spinal cord injury:insights from non-human primates

Spinal cord injury results in significant sensorimotor deficits,currently,there is no curative treatment for the symptoms induced by spinal cord injury.Basic and pre-clinical research on spinal cord injury relies on the development and characterization of appropriate animal models.These models should replicate the symptoms observed in human,allowing for the exploration of functional deficits and investigation into various aspects of physiopathology of spinal cord injury.Non-human primates,due to their close phylogenetic association with humans,share more neuroanatomical,genetic,and physiological similarities with humans than rodents.Therefore,the responses to spinal cord injury in nonhuman primates most likely resemble the responses to traumatism in humans.In this review,we will discuss nonhuman primate models of spinal cord injury,focusing on in vivo assessments,including behavioral tests,magnetic resonance imaging,and electrical activity recordings,as well as ex vivo histological analyses.Additionally,we will present therapeutic strategies developed in non-human primates and discuss the unique specificities of non-human primate models of spinal cord injury.

Optical-neural Stimulation in Non-human Primates:Modulating Brain Function and Behavior

Optical-neural stimulation,which encompasses cutting-edge techniques such as optogenetics and infrared neurostimulation,employs distinct mechanisms to modulate brain function and behavior.These advanced neuromodulation techniques offer accurate manipulation of targeted areas,even selectively modulating specific neurons,in the brain.This makes it possible to investigate the cause-and-effect connections between neural activity and behavior,allowing for a better comprehension of the intricate brain dynamics towards complex environments.Non-human primates serve as an essential animal model for investigating these complex functions in brain research,bridging the gap between the basic research and clinical applications.One of the earliest optical studies utilizing optogenetic neuromodulation in monkeys was conducted in 2009.Since then,the optical-neural stimulations have been effectively applied in non-human primates.This review summarises recent research that employed optogenetics or infrared neurostimulation techniques to regulate brain function and behavior in non-human primates.The current state of optical-neural stimulations discussed here demonstrates their efficacy in advancing the understanding of brain systems.Nevertheless,there are still challenges that need to be addressed before they can fully achieve their potential.

Treatment of spinal cord injury with biomaterials and stem cell therapy in non-human primates and humans

Spinal cord injury results in the loss of sensory,motor,and autonomic functions,which almost always produces permanent physical disability.Thus,in the search for more effective treatments than those already applied for years,which are not entirely efficient,researches have been able to demonstrate the potential of biological strategies using biomaterials to tissue manufacturing through bioengineering and stem cell therapy as a neuroregenerative approach,seeking to promote neuronal recovery after spinal cord injury.Each of these strategies has been developed and meticulously evaluated in several animal models with the aim of analyzing the potential of interventions for neuronal repair and,consequently,boosting functional recovery.Although the majority of experimental research has been conducted in rodents,there is increasing recognition of the importance,and need,of evaluating the safety and efficacy of these interventions in non-human primates before moving to clinical trials involving therapies potentially promising in humans.This article is a literature review from databases(PubMed,Science Direct,Elsevier,Scielo,Redalyc,Cochrane,and NCBI)from 10 years ago to date,using keywords(spinal cord injury,cell therapy,non-human primates,humans,and bioengineering in spinal cord injury).From 110 retrieved articles,after two selection rounds based on inclusion and exclusion criteria,21 articles were analyzed.Thus,this review arises from the need to recognize the experimental therapeutic advances applied in non-human primates and even humans,aimed at deepening these strategies and identifying the advantages and influence of the results on extrapolation for clinical applicability in humans.

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.

Progress of non-human primate animal models of cancers

Cancer is the second leading disease causing human death.Pre-clinical in vivo studies are essential for translating in vitro laboratory research results into the clinic.Rodents,including the mouse and rat,have been widely used for pre-clinical studies due to their small size,clear genetic backgrounds,rapid propagation,and mature transgenic technologies.However,because rodents are evolutionarily distinct from humans,many pre-clinical research results using rodent models cannot be reproduced in the clinic.Non-human primates（NHPs） may be better animal models than rodents for human cancer research because NHPs and humans share greater similarity in regards to their genetic evolution,immune system,physiology and metabolism.This article reviews the latest progress of cancer research in NHPs by focusing on the carcinogenesis of different NHPs induced by chemical and biological carcinogens.Finally,future research directions for the use of NHPs in cancer research are discussed.

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.

Stem cell therapy for Parkinson’s disease using non-human primate models

Stem cell therapy (SCT) for Parkinson’s disease (PD) has received considerable attention in recent years. Non-human primate (NHP) models of PD have played an instrumental role in the safety and efficacy of emerging PD therapies and facilitated the translation of initiatives for human patients. NHP models of PD include primates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism, who are responsive to dopamine replacement therapies, similar to human PD patients. Extensive research in SCT has been conducted to better treat the progressive dopaminergic neurodegeneration that underlies PD. For effective application of SCT in PD, however, a number of basic parameters still need to be tested and optimized in NHP models, including preparation and storage of cells for engraftment, methods of transplantation, choice of target sites, and timelines for recovery. In this review, we discuss the current status of NHP models of PD in stem cell research. We also analyze the advances and remaining challenges for successful clinical translation of SCT for this persistent disease.

Application of the genome editing tool CRISPR/Cas9 in non-human primates

In the past three years, RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats （CRISPR） adaptive immune system has been used to facilitate efficient genome editing in many model and non-model animals. However, its application in nonhuman primates is still at the early stage, though in view of the similarities in anatomy, physiology, behavior and genetics, closely related nonhuman primates serve as optimal models for human biology and disease studies. In this review, we summarize the current proceedings of gene editing using CRISPR/Cas9 in nonhuman primates.

Treatment of Pneumocystis jirovecii pneumonia in non-human immunodeficiency virus-infected patients using a combination of trimethoprim-sulfamethoxazole and caspofungin

BACKGROUND Pneumocystis jirovecii pneumonia(PJP)is an infectious disease common in immunocompromised hosts.However,the currently,the clinical characteristics of non-HIV patients with PJP infection have not been fully elucidated.AIM To explore efficacy of trimethoprim–sulfamethoxazole(TMP-SMX)and caspofungin for treatment of non-human immunodeficiency virus(HIV)-infected PJP patients.METHODS A retrospective study enrolled 22 patients with non-HIV-infected PJP treated with TMP-SMX and caspofungin from 2019 to 2021.Clinical manifestations,treatment and prognosis of the patients were analyzed.RESULTS Five patients presented with comorbidity of autoimmune diseases,seven with lung cancer,four with lymphoma,two with organ transplantation and four with membranous nephropathy associated with use of immunosuppressive agents.The main clinical manifestations of patients were fever,dry cough,and progressive dyspnea.All patients presented with acute onset and respiratory failure.The most common imaging manifestation was ground glass opacity around the hilar,mainly in the upper lobe.All patients were diagnosed using next-generation sequencing,and were treated with a combination of TMP-SMX and caspofungin.Among them,17 patients received short-term adjuvant glucocorticoid therapy.All patients recovered well and were discharged from hospital.CONCLUSION Non-HIV-infected PJP have rapid disease progression,high risk of respiratory failure,and high mortality.Combination of TMP-SMX and caspofungin can effectively treat severe non-HIVinfected PJP patients with respiratory failure.

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.

Future of liver transplantation: Non-human primates for patient-specific organs from induced pluripotent stem cells

Strategies to fill the huge gap in supply versus demand of human organs include bioartificial organs, growing humanized organs in animals, cell therapy, and implantable bioengineered constructs. Reproducing the complex relations between different cell types, generation of adequate vasculature, and immunological complications are road blocks in generation of bioengineered organs, while immunological complications limit the use of humanized organs produced in animals. Recent developments in induced pluripotent stem cell (iPSC) biology offer a possibility of generating human, patient-specific organs in non-human primates (NHP) using patient-derived iPSC and NHP-derived iPSC lacking the critical developmental genes for the organ of interest complementing a NHP tetraploid embryo. The organ derived in this way will have the same human leukocyte antigen (HLA) profile as the patient. This approach can be curative in genetic disorders as this offers the possibility of gene manipulation and correction of the patient's genome at the iPSC stage before tetraploid complementation. The process of generation of patient-specific organs such as the liver in this way has the great advantage of making use of the natural signaling cascades in the natural milieu probably resulting in organs of great quality for transplantation. However, the inexorable scientific developments in this direction involve several social issues and hence we need to educate and prepare society in advance to accept the revolutionary consequences, good, bad and ugly.

Consequences of early adverse rearing experience(EARE) on development： insights from non-human primate studies

Early rearing experiences are important in one＇s whole life, whereas early adverse rearing experience（EARE） is usually related to various physical and mental disorders in later life. Although there were many studies on human and animals, regarding the effect of EARE on brain development, neuroendocrine systems, as well as the consequential mental disorders and behavioral abnormalities, the underlying mechanisms remain unclear. Due to the close genetic relationship and similarity in social organizations with humans, non-human primate（NHP） studies were performed for over 60 years. Various EARE models were developed to disrupt the early normal interactions between infants and mothers or peers. Those studies provided important insights of EARE induced effects on the physiological and behavioral systems of NHPs across life span, such as social behaviors（including disturbance behavior, social deficiency, sexual behavior, etc）, learning and memory ability, brain structural and functional developments（including influences on neurons and glia cells, neuroendocrine systems, e.g., hypothalamic-pituitary-adrenal（HPA） axis, etc）. In this review, the effects of EARE and the underlying epigenetic mechanisms were comprehensively summarized and the possibility of rehabilitation was discussed.

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.

Xenotransplantation of embryonic pig pancreas for treatment of diabetes mellitus in non-human primates

Transplantation therapy for diabetes in humans is limited by the low availability of human donor whole pancreas or islets. Outcomes are complicated by immunosuppressive drug toxicity. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce transplant immunogenicity. Pig insulin is biologically active in humans. In that regard the pig is an appropriate xenogeneic organ donor. Insulin-producing cells originating from embryonic pig pancreas obtained very early following pancreatic primordium formation [embryonic day 28 (E28)] engraft long-term in rhesus macaques. Endocrine cells originating from embryonic pig pancreas transplanted in host mesentery migrate to mesenteric lymph nodes, engraft, differentiate and improve glucose tolerance in rhesus macaques without the need for immune suppression. Transplantation of embryonic pig pancreas is a novel approach towards beta cell replacement therapy that could be applicable to humans.

Molecular identification of hemoplasmas in free ranging non-human primates in Thailand

Objective: To survey hemoplasmas infection in free ranging non-human primates from 8 provinces in Thailand. Methods: DNA from ethylenediaminetetraacetic acid blood of 262 free ranging non-human primates were identified as hemoplasmas using PCR and phylogenetic analysis based on 16 S r RNA and rnp B genes. Results: A total of 148 non-human primates(56.49%) were determined positive for Candidatus Mycoplasma haemomacaque, including 125 Macaca fascicularis and 23 Macaca mulatta. Hemoplasmas can cause anemia in monkey but all positive samples were healthy. The positive rates in male and female non-human primates were not significantly different. Conclusions: Candidatus Mycoplasma infection is prevalent in free ranging Macaca fascicularis and Macaca mulatta in Thailand.

Non-human primate pluripotent stem cells for the preclinical testing of regenerative therapies

Non-human primates play a key role in the preclinical validation of pluripotent stem cell-based cell replacement therapies.Pluripotent stem cells used as advanced therapy medical products boost the possibility to regenerate tissues and organs affected by degenerative diseases.Therefore,the methods to derive human induced pluripotent stem cell and embryonic stem cell lines following clinical standards have quickly developed in the last 15 years.For the preclinical validation of cell replacement therapies in non-human primates,it is necessary to generate non-human primate pluripotent stem cell with a homologous quality to their human counterparts.However,pluripotent stem cell technologies have developed at a slower pace in non-human primates in comparison with human cell systems.In recent years,however,relevant progress has also been made with non-human primate pluripotent stem cells.This review provides a systematic overview of the progress and remaining challenges for the generation of non-human primate induced pluripotent stem cells/embryonic stem cells for the preclinical testing and validation of cell replacement therapies.We focus on the critical domains of(1)reprogramming and embryonic stem cell line derivation,(2)cell line maintenance and characterization and,(3)application of non-human primate pluripotent stem cells in the context of selected preclinical studies to treat cardiovascular and neurodegenerative disorders performed in non-human primates.

Soybean Hull and Its Effect on Atherosclerosis in Non-Human Primates (Macaca fascicularis)

Twenty five monkeys were used in this experiment. They were divided into 5 groups with 5 animals as the replicates in each group and were adapted for two weeks to the environment before the data were collected. The animals were subjected to 5 experimental diets, i.e. T1 (Basal diet); T2 (Basal diet + palm oil); T3 (Basal diet + palm oil + soybean hull); T4 (Basal diet + cholesterol) and T5 (Basal diet + cholesterol + soybean hull). The diets were given for a period of 8 months and water were given ad lib. Blood serum was taken before and during the experiment. The cholesterol, triglycerides, LDL and HDL were measured using the spectrophotometric method. At the end of the experiment thorax surgery was performed on the animals under general anesthesia. The aorta was removed surgicalIy for histopathological observation stained with hematoxylin and eosine.The results showed that the soybean hull decreased the serum cholesterol level in the groups given palm oil (T2 vs T3) and the groups given cholesterol (T4 vs T5) i.e.: 163.4 vs 124.7 mg/dl and 359 vs 288.5 mg/dl respectively. The soybean hull did not significantly affect the serum triglyceride nor the LDL level when palm oil was given in the diet, but it significantly decreased the two parameters where cholesterol was given in the diet (102.5 vs 98.6 mg/dl triglyceride) and (231 .9 vs 183 mg/dl LDL). The soybean hull did not seem to affect the HDL level.Histopathological observation of the aorta indicated that given T1, T2, T3, T4 and T5 caused 45%, 41 .67%, 31.25%, 86.25% and 53.38% lesion (Atheroma arteriale) resPectively.It was concluded that the soybean hull given in the diet has the ability to prevent the development of atherosclerosis in the aorta of the experimental 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.

Research progress in molecular epidemiology of Cryptosporidium in non-human primates

Cryptosporidium is an emerging single-cell zoonotic pathogen.By invading human and animal small intestinal epithelial cells,the host produces a variety of clinical symptoms,mainly diarrhea.Spores of Cryptosporidium can be transmitted through water-borne,food-borne,and mutual transmission between hosts,which has important public health significance.Studies have shown that non-human primates can be infected with multiple Cryptosporidium genotypes.Moreover,this species has a high genetic similarity with humans,so it needs to be taken seriously.This article reviews the infection rates,genotypes,and zoonotic risk of Cryptosporidium in non-human primates.