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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Page 23
Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 24
Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 25
Suggested Citation:"4 Current Research Applications." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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  4 Current Research Applications Marmosets are proving to be very useful in many different areas of basic and translational research, including neuroscience, aging, infectious disease, and research using transgenes and gene editing tools. NEUROSCIENCE This section is based on the presentations of Afonso Silva, a senior investigator at the National Institute of Neurological Disorders and Stroke (NINDS) and the head of its marmoset research division, and Jan Langermans, chairman of the Animal Science Department of the Biomedical Primate Research Centre (BPRC) in the Netherlands. Investigators at NINDS have, since 1997, been developing marmoset models for Experimental Autoimmune Encephalomyelitis (EAE), a disease that in marmosets approximates the key pathological features of the human disease multiple sclerosis (MS). The NINDS marmoset research division houses about 150 animals and its research focuses on anatomical and functional neuroimaging. BPRC uses marmosets for translational research during late stage, preclinical validation procedures that cannot be mim- icked in other animals (such as rodents) for a variety of conditions, including EAE/MS, Parkinson’s disease, Alzheimer’s disease, and sleep and stress disorders. Langermans presented several studies overseen by Ingrid Phillippens, BPRC’s head of neuropathology. Why Marmosets Translational animal models are beneficial for neuroscience studies because in addition to noninvasive procedures, they can also be used to study new drug testing, electrophysiology, and transcriptomics. Disease- induced animals can also be imaged to enable scientists to view temporal and longitudinal changes in the brain as it progresses from healthy to diseased. Primates, however, have particular characteristics that make them better neuroscience model animals than rodents (Figure 4-1). Silva and Langermans highlighted some of the key advantages of using NHPs in general, and marmosets in particular, for neuroscience research. First, human and NHP brains are similarly organized1, especially in the prefrontal cortex, an area that is both anatomically and functionally central to many neurological disorders (Paxinos et al. 2012). Second, the structural plasticity of the marmoset amyg- dala is similar to that of other NHPs and humans (Marlatt et al. 2011). Third, humans and NHPs have similar sleep patterns and changes to cortisol and melatonin levels, whereas rodents’ sleep patterns and hormonal rhythms levels are very different. Fourth, the marmoset lissencephalic cerebral cortex provides a smooth surface that makes imaging or electrophysiology results much easier to read. Marmosets as Models for EAE/MS NINDS and BPRC researchers both work with EAE marmoset models, which function as a model for MS in humans. In marmosets, the development of brain lesions closely resembles the process in humans, and brain imaging enables researchers to track lesion development and the integrity of the blood brain barrier as the disease progresses. The marmoset EAE model shows different characteristics compared to 1 See Brainmuseum.org. 14 Prepublication Copy

Current Research Applications EAE models in mice or macaques. Importantly, the condition develops more slowly in marmosets, allowing for investigations into early disease processes. Langermans described how BPRC researchers created their EAE model by immunizing animals with recombinant human myelin oligodendrocyte glycoprotein (MOG) to induce MS-like symptoms. One study demonstrated that diet had a large effect on the level of spinal cord demyelination and immunological func- tions (Kap et al. 2018). This result offers important insights on the disease development process and also serves as a warning regarding the importance of consistent animal care, suggesting that if different institu- tions use different diet or housing conditions, these discrepancies can lead to different research results. Silva described how the marmoset model for EAE/MS was developed and is used at NINDS. NINDS scientists have been working with their EAE/MS model for nearly 20 years, performing anatomical and functional magnetic resonance imaging (MRI) to understand brain functions both in normal conditions and disease models (Figure 4-2). Marmoset brains can be imaged while the animals are awake or sedated. Mar- mosets typically undergo sedation (isoflurane is commonly used for anatomic studies) and recover very easily while they remain motionless under sedation. As it is easier to learn about brain functioning when marmosets are awake, NINDS researchers have gone to great lengths to design a comfortable cradle in order to obtain good data without the interference of stress responses (Figure 4-3). Their protocol also includes a 3-week acclimation period in which mar- mosets gradually become familiar with the cradle and the MRI experience. In addition, each marmoset has its own individualized helmet, built from a 3-D model of its head, that keeps the animal comfortable yet effectively restrained. The helmet is also equipped with radiofrequency coils to optimize sensitivity to brain function. FIGURE 4-1 Comparison of brain characteristics of humans, macaques, marmosets, and mice. SOURCE: Yasuhiro Go. Prepublication Copy 15

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research FIGURE 4-2 Anatomical in vivo T1w-MPRAGE 150 µm3 MRI of the normal marmoset brain. SOURCE: Afonso Silva. FIGURE 4-3 Schematic of cradle setup for performing functional MRI (fMRI) on conscious awake marmosets. SOURCE: Afonso Silva. 16 Prepublication Copy

Current Research Applications Silva explained that the researchers use several measures to ensure the marmosets are not stressed while undergoing MRI tests or when training in mock MRI tubes, including cortisol levels as well as ob- servations of eye movement. By these measures, the vast majority of marmosets showed no evidence of elevated stress levels when in the MRI tube compared with animals in their normal enclosures. The few animals that showed agitation during MRI training were removed from the study and the training was dis- continued. Imaging disease models and healthy animals with sophisticated equipment reveals detailed networks of blood vessels in the brain, myelination in the cortex, contrasts between gray and white matter, contrasts between the brain and cerebrospinal fluids, and the brain’s overall water content, data points that provide valuable insights about the progression of EAE-induced lesions in the marmoset brain (Absinta et al. 2016; Maggi et al. 2017). Lesions can also develop in the spinal cord. To image these lesions while avoiding the motion caused by respiration and circulation, NINDS researchers designed an anatomically conforming cradle in which the animal lays in the supine position while lesions are studied at high resolution. From these imaging studies, NINDS scientists have discovered the presence of central veins within each lesion (Gaitán et al. 2014), indicating that the lesions form from the vasculature and then evolve into white matter. Scientists have also learned that changes in blood-brain barrier integrity happen 2-3 weeks before lesions are visible, pointing to a potential therapeutic window when it may be possible to prevent lesions from developing (Maggi et al. 2014). Marmosets as Models for Parkinson’s Disease Langermans described studies of Parkinson’s disease carried out at BPRC. Marmosets make good models for this condition, he explained, because of their small size, the relative ease to monitor their re- sponse to cognition, movement, and disorder tasks; and the opportunity to image specific brain areas at various disease stages. Parkinson’s disease is connected to a genetic variant in only about 5 percent of the human population; most patients have idiopathic Parkinson’s, which can be induced in animals via 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of heroin production (Colpan and Slavin 2010). Parkinson’s is difficult to treat because by the time symptoms are apparent, more than half of the neurons are irrevocably gone. If doctors could intervene at an earlier stage, they might be able to prevent neurons from degenerating. In normal human motor coordination, brain signals follow the striato-thalamocortical pathway, but when Parkinson’s damage begins, this pathway gets blocked and the signals have to take a different route, crossing over the red nucleus, an area present in humans that is active only when learning to crawl during infancy (Appel-Cresswell et al. 2010). When humans or NHPs learn to walk, motion signals switch to the striato-thalamocortical pathway, but this never occurs in rodents, making NHPs better models for Parkin- son’s, Langermans said. When Parkinson’s is induced in marmosets, the red nucleus of high-responding animals enlarges, showing that it is compensating as a new pathway. Parkinson’s progresses very rapidly in most MPTP-induced models. This makes it difficult to learn about the earlier stages of disease development, but is helpful if the research is focused on late-stage inter- ventions. To overcome this problem, BRPC researchers developed a slow-progressing MPTP Parkinson’s model (Philippens 2017). High-responding, early-onset animals showed problems with grooming and mo- bility and also developed apathy. They also presented with significantly larger red nuclei than low-respond- ing animals, indicating blockage in the striato-thalamocortical pathway. At BPRC a battery of tests is used to quantify the progression of Parkinson’s disease in marmosets (Figure 4-4). Marmosets as Models for Alzheimer’s Disease Langermans described BPRC’s use of marmosets to study Alzheimer’s disease, a condition with still many unknown facets including the role of amyloid beta plaques. Scientists remain divided over whether immunology or neurological processes are more important in the development of this disease. To learn Prepublication Copy 17

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research more, BPRC researchers created and are currently studying an amyloid-beta marmoset model. The animals are injected with two different solutions, phosphate buffer saline (PBS) or lipopolysaccharide (LPS), and their plaque progression is measured. So far, more inflammation and plaque has been observed in LPS- injected animals (Philippens et al. 2017). During the discussion, some participants noted that gene editing technologies could be useful in in- ducing amyloid pathology similar to that seen in Alzheimer’s disease in humans, with potential candidates the genes presenilin or Trem2. Of course, it is important to remember that genes that appear to contribute to a disease in humans do not always play the same role in other species, and vice versa. One participant offered an example: a small number of human patients shows the A53D mutation; however, NHPs—except rhesus and great apes—naturally have this mutation without concomitant Parkinson’s disease, so genet- ically inducing that mutation in marmosets is not likely to be useful. This example underscores the im- portance of selecting the right animal model for the research questions being addressed, the participant noted. Using Marmosets to Study Sleep and Stress Langermans noted that BRPC researchers have recently begun using biomarkers to validate their find- ings about early-stage neurological disease. The easiest biomarker is sleep-circadian rhythms. Marmosets are well suited for studying sleep-circadian rhythms because they tend to ignore their monitors, whereas macaques are more likely to remove them, disrupting data collection. In addition, marmoset sleep cycles are comparable to human sleep cycles, while rodent sleep patterns are very different. Marmosets, like hu- mans, also suffer cognitive decline when sleep disorders are induced, Langermans said. FIGURE 4-4 Test methods and biomarkers for the progression of Parkinson’s disease in marmosets. SOURCE: Jan Langermans. 18 Prepublication Copy

Current Research Applications The speaker went on to describe how animal models can also be used to study post-traumatic stress disorder (PTSD), and that NHPs are particularly helpful because of the similarities of their brains, sleep patterns, and hormones to those observed in humans. Retrospective studies of humans with PTSD are dif- ficult and unreliable, but a controlled stress-event animal model approach could advance research and point to potential therapies. One BPRC study, Langermans said, showed that while marmosets do remember adverse stimuli, their memory can be influenced by doses of ketamine, which has therapeutic implications for PTSD treatment in humans. Future Research Directions in Neuroscience In addition to disease imaging, Silva noted that NINDS scientists have successfully used marmosets to study somatosensory systems, auditory systems, and visual processing systems. They have learned that, as in macaques and humans, the high order visual pathway of marmosets can process specific facial information, meaning that marmosets can discriminate faces from other visual stimuli (Hung et al. 2015a, 2015b). Inspired by an incidental finding while collecting marmoset connectome data, researchers at NINDS have also studied the corpus callosum of several animals and discovered interesting abnormalities, Silva said. Some animals have smaller than usual corpus callosums, and researchers plan to examine why, se- quence their genes, and follow future generations to see if the anomaly is passed to offspring. Another direction for future research is comparing resting-state brain functioning in marmosets and humans. Silva described that, in order to study marmosets, researchers first created a detailed 3-D atlas of the marmoset brain, including isotropic views, visible distinctions between white and gray matter, and vis- ible fiber-crossings (Liu et al. 2018). This work has revealed that marmosets and macaques have very sim- ilar brain organization. During the discussion, participants noted how, even though marmosets interact socially with each other in ways that are very different from human interactions, researchers have identified behaviors relevant to human conditions that affect social interaction, like autism spectrum disorder. For example, one partici- pant noted that altering certain neurons in marmosets can lead to changes in eye movements and social interactions, suggesting marmosets altered in this way could offer a model for researching autism or other disorders. One attendee asked whether brain studies had extended into areas that could inform psychopathy re- search. While personality differences can be seen in both wild and captive marmosets, there has been little research into social pathologies. The participant noted that it may be an area worth exploring, though it would be necessary to tease out the effects of an animal’s social group since marmoset behavior is strongly related to the group with which it is interacting (e.g., if they are with their own family group or surrounded by strangers). The breadth and depth of research performed at NINDS and BPRC demonstrates the significant po- tential of working with marmosets to advance neuroscience, as marmosets share many physiological simi- larities to humans and perform well during invasive and non-invasive procedures. AGING Corinna Ross, an associate professor of biology at Texas A&M University, San Antonio, presented on the use of marmosets as models for studying aging. Why Marmosets Marmosets can be better aging models than mice or other rodents. As many speakers mentioned, one key advantage is that marmosets are smaller than other primates. They also reproduce rapidly, mature quickly, and have multiple births, so it takes less time to study developmental programming. When in cap- tivity, researchers can control marmosets’ diet, habitat, and medical exposure at a level that is impossible Prepublication Copy 19

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research to reach with humans. Most importantly for aging researchers, marmosets have a relatively short life span compared to that of other primates. Marmosets as Models for Aging Researchers are comparing aging across species to advance translational animal research and bridge the gap between geriatricians (i.e., clinical scientists who study human aging processes through longitudinal or epidemiological studies) and gerontologists, who study basic science to uncover different pathways that are associated with aging, such as telomere damage or cell loss. Translational animal models allow re- searchers to test interventions, quantify health and life spans, and develop tools that can be used in humans. Ross and her colleagues are especially interested in mobility and cognition during the aging process. They are adapting human and rodent translational phenotyping for use in marmosets, and hope to develop new tools that could be used across species. Most marmosets do not live past six years, and extreme old age (20 or more years) is very rare (Ross and Salmon 2018). Both young and old marmosets die from many causes, such as infection or irritable bowel syndrome (IBS), but older marmosets are more likely to die from cardiac and renal failures, similar to humans (Figure 4-5). To develop marmosets as models for aging, the animals have to survive to old age. Humans typically have a non-linear survivorship curve: a low likelihood of death until old age, when the likelihood of death ramps up rapidly. By contrast, marmosets’ likelihood of death is linear and not related to age. Researchers created the nonlinear, human-like curve they needed in marmosets by raising a new colony under highly controlled conditions. The food was irradiated, the animals were screened for specific viruses, and water was autoclaved, all of which decreased the risk of pathogen exposure and resulted in animals that died of old age instead of non-age related pathologies FIGURE 4-5 Causes of death in marmosets older than 6 years of age and younger than 6 years of age at Southwest National Primate Research Center (SNPRC). Abbreviations: IBD: inflammatory bowel disease. SOURCE: Corinna Ross. Adapted with permission from chapter 21, Marmosets in Aging Research, in The Common Marmoset in Cap- tivity and Biomedical Research, 1st edition, Fox, JG, Marini RP, Wachtman LM, et al., eds; copyright 2018, Elsevier. 20 Prepublication Copy

Current Research Applications How to Measure Aging Ross and colleagues identified five domains for understanding the aging process in marmosets: met- abolic function, homeostatic balance, immune function, mobility, and cognition. To use these domains to assess aging, they developed methods to identify markers for frailty as well as for increased health and longevity. In terms of metabolic function, aging presents itself in several ways. Middle aged animals lose muscle mass and fat faster (Power et al. 2001; Ross et al. 2012) and their maximal oxygen uptake (VO2 max) decreases, but their glucose does not differ from that seen in younger animals. All of these processes are very similar to metabolic functions in aging humans. Homeostatic balance is measured via blood pressure and microbiome assessment. Aging marmosets have higher diastolic and mean arterial pressure measurements, but other blood pressure measures remain stable. Regarding the microbiome, older marmosets have a much lower Shannon diversity index2 than younger ones, similar to aging humans, which may be associated with the risk of infection by C. difficile and other bacteria (although that could also be influenced by the hospital, nursing home, or hospice envi- ronment for humans or the clinical diet for marmosets; see Chapter 5). Immune functions also change with age. Marmosets experience a reduction in serum albumin concen- trations (Ross et al. 2012) and an increase in inflammatory states, with changes in B and T cell function and markers on those cells. Mobility is assessed via cage-side tests. Old and young animals spend the same amount of time in motion and in rest, but older marmosets leap much less than young marmosets and overall have less variety in their movements. Grooming, genital display, and scent marking remain the same, but hanging (i.e., stretching, the suspension from the top of a cage), is more commonly observed in geriatric marmosets and in fact is a high-risk factor for death, which usually occurs in the six months following observation of this behavior. Cognition is assessed through specially designed tests to measure visual and spatial learning, and impulse control—the human “executive functions” that geriatricians associate with disease and aging risk. In one test, younger marmosets learned to control their impulse to choose one treat vs. another faster than older marmosets (although older marmosets did learn impulse control eventually). In a test of “detoured reach,” geriatric marmosets were much less likely to meet the criteria that define success. More techniques are being developed to evaluate cognition in marmosets, e.g., delayed match-to-sample tests and tests using touchscreens. Rapamycin for Increased Longevity In 2009, rapamycin, an immunosuppressant given to humans undergoing organ transplantation, was reported to significantly increase longevity and survivorship in mice (Harrison et al. 2009). The only other intervention known to have the same effect is caloric restriction. Ross and her team determined that low doses of rapamycin do not affect marmoset metabolic function, indicating that it is unlikely to cause diabe- tes in otherwise healthy humans or animals (Ross et al. 2015). Scientists have started rapamycin protocols in middle aged marmosets, and after 12 months, as the animals age, no differences have been noted in activity, locomotion, or metabolism between rapamycin- treated animals and controls (Ross et al. 2015; Sills et al. 2019). As a next step, the researchers are assessing impacts on cognition. 2 The Shannon index is a quantifier of population diversity. Details can be found in Morgan XC & Huttenhower C. 2012. Chapter 12: Human Microbiome Analysis. PLoS Comput Biol, https://doi.org/10.1371/journal.pcbi.1002808. Available at: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002808. Prepublication Copy 21

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research INFECTIOUS DISEASE Jean Patterson, a scientist at the Texas Biomedical Research Institute, works with marmosets in bi- osecurity level-4 (BSL-4) conditions to study their reactions to multiple infectious agents, including West Nile virus, Eastern Equine Encephalitis virus (EEEV), Lassa virus, Ebola/Marburg viruses, GB Virus B, Dengue and Zika viruses. Why Marmosets Deliberately infecting an animal with a disease is only worthwhile if there are specific insights that such an experiment can offer. There are a few compelling reasons to use marmosets as models for studying infectious diseases. Marmosets have similar immune functions to macaques and humans. In addition, mar- mosets are smaller than macaques, and more will fit into the limited space of the BSL-4 labs. And, marmo- sets do not carry the herpes-B virus, which is easily transferred when working with infected animals via needles and can be fatal in humans. This naturally improves the safety conditions of working with marmo- sets. West Nile Virus When infected with West Nile virus, marmosets and macaques show a very similar response to that of infected humans (Verstrepen et al. 2014). West Nile virus may also cause fetal abnormalities; further research on the effects of West Nile, Zika, or other viruses on fetal marmoset development could help our understanding of the effects of those viruses on human fetuses. EEEV EEEV is one of the most severe arboviruses with extremely high morbidity and mortality rates. While North American strains cause such severe illness that attempts have been made to weaponize them, South American strains do not cause disease in humans or marmosets. Both strains are highly virulent in mice, but marmosets respond very similarly to humans (Adams et al. 2008), making marmosets a suitable model for testing potential interventions or vaccines. Lassa Virus Lassa virus causes fatal hemorrhagic fever and is endemic to West African rodents. Infected marmo- sets react very similarly to humans, showing severe morbidity 15 and 20 days post-infection and similar histopathological findings. Marmosets also exhibit immunosuppression patterns similar to those seen in humans, making them a suitable model for Lassa fever (Carrion et al. 2007). Ebola and Marburg Viruses Ebola and Marburg viruses were tested in marmosets before the 2013-2014 Ebola outbreak in West Africa. For both diseases, marmosets suffer similar clinical and morphological features to humans, includ- ing severe morbidity and mortality (Carrion et al. 2011). Many researchers prefer to use macaques to study febrile viruses because of the greater availability of reagents for macaques, but marmosets can also be a good model. Patterson and her colleagues have collab- orated with other researchers to develop an Ebola vaccine using a marmoset model. 22 Prepublication Copy

Current Research Applications GB Virus B GBV-B resembles hepatitis C, a disease for which chimpanzees are the only available animal model. Identifying GBV-B and using marmosets is enabling researchers to discover new countermeasures for hep- atitis C, which can cause serious liver problems, and even death, in humans. Some marmosets are partially resistant to this infection, possibly due to polymorphism in their colonies (Jacob et al. 2004; Weatherford et al. 2009). Dengue Virus Dengue virus infection causes a flu-like illness that occasionally develops into Severe Dengue,3 a potentially lethal complication (Vasconcelos et al. 2016). For years, scientists believed that the second, more lethal form was caused by an antibody-dependent enhancement, i.e., from non-neutralizing antibodies that are activated upon reinfection and can make the disease lethal. Scientists have not yet been able to reproduce this series of events in NHPs, despite multiple attempts. As a new avenue for research, scientists are recreating this antibody enhancement in a marmoset model to induce the re-infection response of hemorrhagic fever. Recent research showed promising indications that some marmosets acquired unexpected morphological changes following two dengue infections. Zika Virus A Zika model is needed because the disease is expensive to treat and its impacts, mostly on children, are so dire. Although Zika was isolated in the 1960s, it was not well known until 2015, when a massive increase in microcephaly in babies was reported in northeastern Brazil. Researchers were able to trace the problem to Zika and create a promising marmoset model fairly quickly. One experiment, where two infected marmosets had spontaneous abortions, showed that the placenta plays a key role as a conduit for fetal infection in both humans and marmosets (Seferovic et al. 2018). Surprisingly, spontaneous abortions were not seen in other NHPs, indicating that marmosets may be espe- cially sensitive to viruses that cause fetal abnormalities in humans. Zika is found in marmoset semen indi- cating that -like in humans- the disease in marmosets is potentially sexually transmitted. Mycobacterium Tuberculosis Marmosets can be used to distinguish between lethal and non-lethal strains of Mycobacterium tuber- culosis. Given one of three strains of this pathogen, marmosets produced disease along a spectrum of pro- gression at rates similar to those observed in humans (Via et al. 2013). Other Uses Marmosets are not an appropriate animal model for every infectious disease. Researchers have been unable to model severe acute respiratory syndrome (SARS) in any NHP model. It is also unclear whether marmosets can be a model for Middle East respiratory syndrome (MERS); experiments have shown con- flicting results, with one finding severe, sometimes fatal pneumonia and another only a mild-to-moderate response. Patterson concluded by stressing that prior to studying animals for infectious diseases, they should be carefully screened for pathogens to avoid any cross-species contamination. For example, in one colony of the New World titi monkeys, researchers discovered the animals were infected with an adenovirus that was killing them and sickening their caretakers. It is also important to follow Koch’s postulates (NASEM 2018) and Rivers’s modifications (Rivers 1937) when testing for viruses or antibodies. 3 Dengue Hemorrhagic Fever: https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue. Prepublication Copy 23

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research GENE EDITING AND TRANSGENIC ANIMALS Erika Sasaki, director of marmoset research for the Central Institute for Experimental Animals (CIEA) in Japan, presented methods for creating genetically modified marmosets. Lentiviral Vectors CIEA first created genetically modified marmosets using lentiviral vectors. Researchers stimulated follicles in marmoset ovaries, collected oocytes that were subsequently fertilized using in vitro fertilization, injected the desired vector/DNA combination into the embryo, kept the embryo in culture conditions for up to six days, and checked the fluorescent protein marker to determine if the gene had been integrated. When the modification was confirmed, the embryo was transferred to a surrogate marmoset who gave birth to the transgenic animal after approximately 145 days. The entire process took roughly six months. Directly injecting a transgene using lentiviral vectors CIEA scientists have been able to produce more than 13 genetically modified transgenic marmoset species, demonstrating germline transmission and nor- mal embryonic development (Sasaki et al. 2009). However, this technique presents with two disadvantages, Sasaki explained. First, it is very difficult to introduce DNA longer than 5 kb in the fertilized embryo. Second, it is difficult to produce targeted knockout animals without available pluripotent chimeric compe- tent stem cells (i.e., allowing chimera formation), which are not available for most mammals, including marmosets. Emerging Gene Editing Techniques To make knockout marmoset models, CIEA scientists explored several recent genome editing tech- niques. After determining that zinc finger nucleases (ZFNs) would be a more effective approach than CRISPR/Cas9, they used ZFNs to knock out the Interleukin 2 receptor to create an immunocompromised X-SCID marmoset (Sato et al. 2016). The interleukin gene, targeted in this proof-of-concept experiment, is important in transplantation immunology and regenerative medicine. Its absence produces a genetic disor- der whose phenotype is usually evident at infancy, so researchers would know the results very early. In addition to ZFNs, CLIA researchers used newer ZFNs, such as HiFi ZFN and eHiFi ZFNs4) and platinum Transcription Activator-Like Effector Nucleases (TALEN) to knock out the IL2 receptor gene in marmoset embryos. They succeeded in maintaining embryos in culture until a later stage of development, and used CEL-1 assays (i.e., an enzymatic mismatch cleavage assay) to determine if the embryos had been successfully modified. Success rates ranged from 33 percent for eHiFi ZFNto 40 percent for HiFi ZFN to 100 percent for platinum TALEN. Through this series of experiments nine immunodeficient animals were born, six of which died within the first 10 days and three that survived for more than 2 years. Genetic analysis of the six who died showed a normal number of B cells but no T or NK cells (similar to an immunocompromised human), no thymus, and undetectable IgG levels. In summation Sasaki pointed to both ZFNs and TALEN as highly effective methods to produce founder knockout marmosets. She reminded the audience that pre-evaluation of the genome editing tools is important and that target gene knock down could be another method to avoid embryonic lethality. DISCUSSION In the discussion, an attendee pointed out that the use of transgenic technologies raises the prospect that genetically modified animals could potentially end up in the wild where they could interbreed with wild populations. In response, it was explained that research funding and oversight mechanisms impose several layers of protections against the release of such animals, making such a scenario extremely unlikely. 4 e: enhanced. 24 Prepublication Copy

Current Research Applications REFERENCES Absinta M, Sati P, Reich DS. 2016. Advanced MRI and staging of multiple sclerosis lesions. Nat Rev Neurol. 12(6):358-368. Adams AP, Aronson JF, Tardif SD, et al. 2008. Common marmosets (Callithrix jacchus) as a nonhuman primate model to assess the virulence of Eastern Equine Encephalitis virus strains. J Virol. 82(18):9035-9042. Available at: https://jvi.asm.org/content/jvi/82/18/9035.full.pdf. Appel-Cresswell S, de la Fuente-Fernandez R, Galley S, et al. 2010. Imaging of compensatory mechanisms in Parkin- son’s disease. Curr Opin Neurol 23(4):407-412. Carrion R Jr, Brasky K, Mansfield K, et al. 2007. Lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues. J Virol 81(12):6482-6490. Available at: https:// www.ncbi.nlm.nih.gov/pmc/articles/PMC1900113. Carrion R Jr, Ro Y, Hoosien K, et al. 2011. A small nonhuman primate model for filovirus-induced disease. Virology 420(2):117-124. Colpan ME, Slavin KV. 2010. Subthalamic and red nucleus volumes in patients with Parkinson’s disease: do they change with disease progression? Parkinsonism Relat Disord 16(6):398-403. Gaitán MI, Maggi P, Wohler J, et al. 2014. Perivenular brain lesions in a primate multiple sclerosis model at 7-tesla magnetic resonance imaging. Mult Scler 20(1):64-71. Harrison DE, Strong R, Sharp ZD, et al. 2009. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460(7253):392-395. Hung CC, Yen CC, Ciuchta JL, et al. 2015a. Functional MRI of visual responses in the awake, behaving marmoset. Neuroimage 120:1-11. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589494. Hung CC, Yen CC, Ciuchta JL, et al. 2015b. Functional mapping of face-selective regions in the extrastriate visual cortex of the marmoset. J Neurosci 35(3):1160-1172. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC4300322. Jacob JR, Lin KC, Tennant BC, et al. 2004. GB virus B infection of the common marmoset (Callithrix jacchus) and associated liver pathology. J Gen Virol 85(Pt9):2525-2533. Kap YS, Bus-Spoor C, van Driel N, et al. 2018. Targeted diet modification reduces multiple sclerosis-like disease in adult marmoset monkeys from an outbred colony. J Immunol 201(11):3229-3243. Liu C, Ye FQ, Yen CC, et al. 2018. A digital 3D atlas of the marmoset brain based on multi-modal MRI. Neuroimage 169:106-116. Maggi P, Macri SM, Gaitán MI, et al. 2014. The formation of inflammatory demyelinated lesions in cerebral white matter. Ann Neurol 76(4):594-608. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4723108/. Maggi P, Sati P, Massacesi L. 2017. Magnetic resonance imaging of experimental autoimmune encephalomyelitis in the common marmoset. J Neuroimmunol 15(304):86-92. Marlatt MW, Philippens IH, Manders E, et al. 2011. Distinct structural plasticity in the hippocampus and amygdala of the middle-aged common marmoset (Callithrix jacchus). Exp Neurol 230(2):291-301. NASEM (National Academies of Sciences, Engineering and Medicine). 2018. Animal Models for Microbiome Re- search: Advancing Basic and Translational Science: Proceedings of a Workshop. Washington, DC: The National Academies Press; p. 20. Available at: https://www.nap.edu/catalog/24858. Paxinos G, Watson C, Petrides M, et al. 2012. The Marmoset Brain in Stereotaxic Coordinates. San Diego, CA: Elsevier Science Publishing. Philippens IH. 2017. Refinement of the MPTP models for Parkinson’s disease in the marmoset. Drug Discov Today 25-26:53-61. Available at: https://www.sciencedirect.com/science/article/pii/S1740675718300100. Philippens IH, Ormel PR, Baarends G, et al. 2017. Acceleration of Amyloidosis by Inflammation in the Amyloid-Beta Marmoset Monkey Model of Alzheimer’s Disease. J Alzheimers Dis 55(1):101-113. Available at: https://www. ncbi.nlm.nih.gov/pmc/articles/PMC5115608. Power RA, Power ML, Layne DG, et al. 2001. Relations among measures of body composition, age, and sex in the common marmoset monkey (Callithrix jacchus). Comp Med 51(3):281-223. Rivers, TM. 1937. Viruses and Koch’s postulates. J. Bacteriol 33(1):1-12. Ross CN, Davis K, Dobek G, et al. 2012. Aging phenotypes of common marmosets (Callithrix jacchus). J Aging Res 2012:567143. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312272. Ross CN, Salmon A. 2018. Aging research using the common marmoset: Focus on aging interventions. Nutr Health Aging 10.3233/NHA-180046. Available at: https://content.iospress.com/articles/nutrition-and-healthy-aging/nh a180046. Prepublication Copy 25

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research Ross CN, Salmon A, Strong R, et al. 2015. Metabolic consequences of long-term rapamycin exposure on common marmoset monkeys (Callithrix jacchus). Aging (Albany NY) 7(11):964-973. Sasaki E, Suemizu H, Shimada A, et al. 2009. Generation of transgenic non-human primates with germline transmis- sion. Nature 459:523-527. Sato K, Oiwa R, Kumita W, et al. 2016. Generation of a nonhuman primate model of severe combined immune- deficiency using highly efficient genome editing. Cell Stem Cell 19(1):127-138. Available at: https://www. cell.com/cell-stem-cell/fulltext/S1934-5909(16)30153-9?_returnURL=https%3A%2F%2Flinkinghub.elsevier. com%2Fretrieve%2Fpii%2FS1934590916301539%3Fshowall%3Dtrue. Seferovic M, Sanchez-San Martin C, Tardif SD, et al. 2018. Experimental Zika virus infection in the pregnant common marmoset induces spontaneous fetal loss and neurodevelopmental abnormalities. Sci Rep 8(1):6851. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931554. Sills AM, Artavia JM, DeRosa BD, et al. 2019. Long-term treatment with the mTOR inhibitor rapamycin has minor effect on clinical laboratory markers in middle-aged marmosets. Am J Primatol 81(2):e22927. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415526. Tardif SD, Mansfield KG, Ratnam R. 2011. The marmoset as a model of aging and age-related diseases. ILAR J 52(1): 54-65. Vasconcelos BC, Vieira JA, Silva GO, et al. 2016. Antibody-enhanced dengue disease generates a marked CNS in- flammatory response in the black-tufted marmoset Callithrix penicillata. Neuropathology 36(1):3-16. Verstrepen BE, Fagrouch Z, van Heteren M, et al. 2014. Experimental infection of rhesus macaques and common marmosets with a European strain of West Nile virus. PLoS Negl Trop Dis 8(4):e2797. Available at: https:// www.ncbi.nlm.nih.gov/pmc/articles/PMC3990483. Via LE, Weiner DM, Schimel D, et al. 2013. Differential virulence and disease progression following Mycobacterium tuberculosis complex infection of the common marmoset (Callithrix jacchus). Infect Immun 81(8):2909-2919. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719573. Weatherford T, Chavez D, Brasky KM, et al. 2009. The marmoset model of GB Virus B infections: Adaptation to host phenotypic variation. J Virol 83(11)5806-5814. Available at: https://jvi.asm.org/content/jvi/83/11/5806. full.pdf. 26 Prepublication Copy

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The marmoset, a type of small monkey native to South America, is a research model of increasing importance for biomedical research in the United States and globally. Marmosets offer a range of advantages as animal models in neuroscience, aging, infectious diseases, and other fields of study. They may be particularly useful for the development of new disease models using genetic engineering and assisted reproductive technologies. However, concerns have been voiced with respect to the development of new marmoset-based models of disease, ethical considerations for their use, the supply of marmosets available for research, and gaps in guidance for their care and management.

To explore and address these concerns, the Roundtable on Science and Welfare in Laboratory Animal Use hosted a public workshop on October 22-23, 2018, in Washington, DC. The workshop focused on the availability of marmosets in the United States and abroad; animal welfare and ethical considerations stemming from the use of wildtype and genetically modified marmosets; and standards of housing and care, dietary needs, and feeding requirements for marmosets in captivity. This publication summarizes the presentations and discussions from the workshop.

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