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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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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 mimicked 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.

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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Why Marmosets

Translational animal models are beneficial for neuroscience studies because in addition to non-invasive 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 (see 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 organized,1 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 amygdala 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 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 functions (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 institutions 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

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1 See Brainmuseum.org.

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.
×
Image
FIGURE 4-1 Comparison of brain characteristics of humans, macaques, marmosets, and mice.
NOTE: Mya = million years ago.
SOURCE: Yasuhiro Go.

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 (see Figure 4-2). Marmoset brains can be imaged while the animals are awake or sedated. Marmosets 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 (see Figure 4-3). Their protocol also includes a 3-week acclimation period in which marmosets 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.

Silva explained that the researchers use several measures to ensure the marmosets are not stressed while undergoing MRI tests or when training

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.
×
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.
×

in mock MRI tubes, including cortisol levels as well as observations 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 discontinued.

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 response 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

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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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 Parkinson’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 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 interventions. 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 mobility and also developed apathy. They also presented with significantly larger red nuclei than low-responding 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 (see Figure 4-4).

Image
FIGURE 4-4 Test methods and biomarkers for the progression of Parkinson’s disease in marmosets.
NOTE: TH = tyrosine hydroxylase.
SOURCE: Jan Langermans.
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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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 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 inducing 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 genetically inducing that mutation in marmosets is not likely to be useful. This example underscores the importance 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 findings 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 humans, also suffer cognitive decline when sleep disorders are induced, Langermans said.

The speaker went on to describe how animal models can also be used to study posttraumatic 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 difficult 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

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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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, sequence 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 visible fiber-crossings (Liu et al. 2018). This work has revealed that marmosets and macaques have very similar 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 participant 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 research. 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 because 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 potential of working with marmosets to advance neuroscience, as marmosets share many physiological similarities to humans and perform well during invasive and non-invasive procedures.

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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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 captivity, researchers can control marmosets’ diet, habitat, and medical exposure at a level that is impossible to reach with humans. Most importantly for aging researchers, marmosets have a relatively short lifespan 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 researchers to test interventions, quantify health and lifespans, 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 6 years, and extreme old age (20 or more years) is 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 (see 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 non-linear, 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.

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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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). Abbreviation: IBD = inflammatory bowel disease.
SOURCE: Corinna Ross. Adapted with permission from Chapter 21, Marmosets in Aging Research, in The Common Marmoset in Captivity and Biomedical Research, 1st edition, Fox, JG, Marini RP, Wachtman LM, et al. (Eds.); copyright 2018, Elsevier.

How to Measure Aging

Ross and colleagues identified five domains for understanding the aging process in marmosets: metabolic 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

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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. Available at: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002808.

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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hospice environment for humans or the clinical diet for marmosets; see Chapter 5).

Immune functions also change with age. Marmosets experience a reduction in serum albumin concentrations (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 6 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 versus 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 diabetes 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 rapamycintreated animals and controls (Ross et al. 2015; Sills et al. 2019). As a next step, the researchers are assessing impacts on cognition.

INFECTIOUS DISEASE

Jean Patterson, a scientist at the Texas Biomedical Research Institute, works with marmosets in biosecurity level-4 (BSL-4) conditions to study

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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their reactions to multiple infectious agents, including West Nile virus, Eastern equine encephalitis virus (EEEV), Lassa virus, Ebola and Marburg viruses, GB virus B, Dengue virus, and Zika virus

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, marmosets are smaller than macaques, and more will fit into the limited space of the BSL-4 labs. And, marmosets 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 marmosets.

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 marmosets 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).

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.
×

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, including 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 collaborated with other researchers to develop an Ebola vaccine using a marmoset model.

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 hepatitis 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 re-creating this antibody enhancement in a marmoset model to induce the reinfection 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

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3 Dengue hemorrhagic fever, available at: https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue.

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.
×

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 especially sensitive to viruses that cause fetal abnormalities in humans. Zika is found in marmoset semen indicating 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 tuberculosis. Given one of three strains of this pathogen, marmosets produced disease along a spectrum of progression 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 conflicting 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.

GENE EDITING AND TRANSGENIC ANIMALS

Erika Sasaki, the director of marmoset research for the Central Institute for Experimental Animals (CIEA) in Japan, presented methods for creating genetically modified marmosets.

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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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 6 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 6 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 normal embryonic development (Sasaki et al. 2009). However, this technique presents two disadvantages, Sasaki explained. First, it is 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 competent 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 techniques. 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 disorder whose phenotype is usually evident at infancy, so researchers would know the results very early.

In addition to ZFNs, CIEA 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 ZFN to 40 percent for HiFi ZFN to 100 percent for platinum TALEN.

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4 e: enhanced.

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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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.

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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.
×
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.
×
Page 26
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 27
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 28
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 29
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 30
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 31
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 32
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 33
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 34
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 35
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 36
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 37
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 38
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 39
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 40
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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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