Several workshop participants noted that the advantages that nonhuman primates offer to study and develop treatments for nervous system disorders will require adapting a range of research approaches—including transgenesis, chimera, viral vectors, and gene therapy—many of which have been successfully applied in rodent models, and some even in humans. According to Ben Deverman, director of the vector engineering research group at the Stanley Center for Psychiatric Research at the Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard University, tools such as different types of sensors, actuators, optogenetic tools, and gene editors enable scientists to study how specific cells and circuits affect behavior and learning. They are especially powerful, he said, because they are genetically encoded, which enables scientists to perturb or modulate specific cell types and circuits. When combined with genetic modification—either through transgenesis, viral gene delivery, genome editing, or cloning—scientists have a wide array of tools with which to study how gene disruptions affect brain function and explore potential interventions.
Deverman added that in mouse models, the large number of transgenic lines has facilitated the development of these tools by restricting the expression of transgenes to specific cell types and brain regions. However, the Cre technology typically used to generate transgenic mice carries with it some risk (e.g., potential toxicities) when applied to nonhuman primates, he said.
Even without genetic modification, several workshop participants said that nonhuman primate models have been valuable in studying human disease. Joshua Gordon, director of the National Institute of Mental Health (NIMH), noted that primates are needed to study cognitive processes and other complex behaviors. For mental health disorders, the case is even stronger, he said, because at both neurobiological and genetic levels, structures in the brain do not exist in rodents and other lower species. For example, Angela Roberts and colleagues have used a marmoset model to explore the variable sensitivity to selective serotonin reuptake inhibitors (SSRIs) seen in humans with affective disorders such as anxiety and depression. Genetic sequencing of the marmoset genome showed that like humans, marmosets express a range of polymorphisms in the serotonin transporter gene and that a certain polymorphism, similar in function to one seen in humans, is associated with changes in expression of the transporter gene, changes in the anxiety response to a human intruder, and variable responses to SSRIs (Santangelo et al., 2016).
Roberts and colleagues have also used marmosets to test the hypothesis that in people with depression, overactivity in an area of the brain called the subgenual anterior cingulate cortex causes anhedonia, a symptom characterized by loss of pleasure that is commonly associated with depression, schizophrenia, and Parkinson’s disease (PD) (Alexander et al., 2018). They showed that overactivation of this brain region in marmosets blunted cardiovascular and behavioral anticipatory arousal and their ability to work for a reward.
Throughout the workshop, several participants discussed the state of science of genetically modified nonhuman primate models and their potential usefulness to enhance understanding of nervous system disorders and advance therapeutic development for PD, Huntington’s disease (HD), autism spectrum disorder (ASD), and circadian disorders. Workshop participants also explored some of the technological advances that might further enhance development of these types of models, including cloning and gene modification through vector delivery.
Transgenic Marmoset Models to Study Parkinson’s Disease
Gene transfer (Sasaki et al., 2009) and genome editing (Sato et al., 2016) have both been used successfully to develop neurodegenerative and neurodevelopmental disease models, said Hideyuki Okano. Among neurodegenerative diseases, nonhuman primate models have been especially helpful in PD to understand the cells and circuits involved and to evaluate potential new therapies (Emborg, 2007). PD is a highly heterogeneous and complex disorder with both motor and non-motor features. It is associated with the degeneration of dopaminergic neurons and other pathologies resulting from a combination of genetic and environmental factors (Kalia and Lang, 2015). Okano and colleagues generated a transgenic marmoset model of PD that overexpresses the A30P mutation in the α-synuclein gene. A30P is one of many mutations associated with familial forms of PD (Kruger et al., 2001). Using a wireless system that simultaneously measures electroencephalography and electromyography in the marmoset, they showed that REM [rapid eye movement] sleep without atonia—which is seen in humans with PD—was also present in the sleep stage in the PD
transgenic marmosets. They also examined longitudinal changes of dopamine neurons using positron-emission tomography imaging with a radioligand that specifically binds the dopamine transporter. This allowed them to track the association of motor and sleep dysregulation symptoms with loss of dopamine neurons. Okano said they also noticed tremors and gait disturbances in the transgenic marmosets similar to what is seen in humans with PD. Using dopamine as a rescue treatment, they showed that the gait symptoms are likely due to dopamine deficit.
Using diffusion imaging in this marmoset model, Okano and colleagues also demonstrated a reduction in the number of nigro-striatal fibers projecting into the striatum. In the future, Okano hopes to use this model to determine which neuronal circuits are damaged at different clinical stages of disease and whether there is a relationship between clinical symptoms and pathogenic protein accumulation. Another group in Japan is currently developing an α-synuclein positron-emission tomography (PET) probe, which Okano and colleagues are evaluating in the PD transgenic marmosets.
Transgenic Rhesus Macaque Models of Huntington’s Disease
Anthony Chan and colleagues at the Yerkes National Primate Research Center and the Emory University School of Medicine developed a transgenic rhesus macaque primate model of HD, a complex autosomal dominant neurodegenerative disorder that affects about 40,000 people in North America with motor, cognitive, and psychiatric manifestations (Yang et al., 2008). HD is caused by an expansion of trinucleotide repeats (cytosine-adenine-guanine or CAG) within the huntingtin gene (HTT). Most people have fewer than 26 CAG repeats. The disease manifests when the number of repeats exceeds 36, with disease onset highly correlated with an increasing number of repeats.
According to Yoland Smith, Chan’s model was created by transfecting mature rhesus oocytes with a lentivirus carrying a mutant form of the HTT gene, followed by in vitro fertilization of these oocytes and implantation into surrogates. Chan, Smith, and colleagues now have a group of transgenic rhesus monkeys modeling HD that they have been following longitudinally with the same kinds of cognitive, behavioral, and imaging assessments that are used clinically in humans. Smith said these studies indicate that monkeys modeling HD develop slowly progressive motor, cognitive, and pathological changes that closely resemble what is seen in
HD patients. As was hypothesized, the monkeys show progressive worsening on tests of object retrieval at 16 months of age and visuospatial orientation at 36 months of age, said Smith. At 5 years of age, the monkeys also showed increased anxiety, irritability, and aggression in response to an acute stressor (the human intruder task) and they also exhibited elevated levels of pro-inflammatory cytokines and increased induction of immune pathway genes (Chan et al., 2015; Raper et al., 2016). Imaging studies show reductions in brain volume and alterations in white matter connectivity as well as microstructural changes that parallel progressive motor and cognitive decline (Meng et al., 2017).
At necropsy, the brains of the transgenic monkeys also appeared similar to the brains of humans with HD, with widespread deposition of mutant huntingtin aggregates in the striatum and cerebral cortex and significant neuronal loss in the caudate nucleus and putamen (Chan et al., 2015). Smith added that more recent studies indicate other similarities between the HD monkeys and humans with HD, including relative sparing of GABAergic and cholinergic interneurons in the striatum.
Nonhuman Primate Models of Autism Spectrum Disorder
ASD is a neurodevelopmental disorder characterized by persistent deficits in social communication and interaction and the presence of restricted, repetitive patterns of behaviors (APA, 2013). It is also thought to be etiologically heterogeneous. A literature review conducted in 2011 concluded that defects in more than 100 genes have been implicated in ASD (Sanders et al., 2015). John Spiro, deputy scientific director of the Simons Foundation Autism Research Initiative, said that between 65 and 100 highly penetrant genes are known to be major risk factors for this disorder. Given this large number of genes, nonhuman primate models are likely to play an increasingly important role in autism research. For example, in developing gene therapy approaches, Spiro suggested that a nonhuman primate model might be a critical step before moving to human trials. Gene therapy is an extremely appealing route to pursue because in theory it could allow investigators to bypass decades of basic research needed to understand the biology associated with a particular gene and to proceed directly to seeing if modifying a gene might alter the disorder.
Among the genes associated with ASD are MECP2, SHANK3, and CHD8. MECP2 encodes a regulator of neural development called MethylCpG-binding protein-2 (Wen et al., 2017). Mu-Ming Poo, director of the Institute of Neuroscience of the Chinese Academy of Sciences (CAS) and
CAS Center for Excellence in Brain Science and Intelligence Technology, and colleagues at the Institute of Neuroscience at CAS generated lentivirus-based transgenic cynomolgus macaque monkeys that express the human MECP2 gene in the brain, show germline transmission of the transgene, and exhibit behaviors similar to those seen in humans with ASD (Liu et al., 2016a). For example, the transgenic monkeys display repeated circular movements that mimic the stereotypical movements of individuals with autism, said Poo. Like humans with ASD, the monkeys also display anxiety responses, reduce social interactions, and show differences in cognitive function, he added.
Poo and colleagues wanted to produce a second generation of transgenic monkeys, but the relatively slow sexual maturation of macaques meant that it would be 5 or 6 years before these animals would reproduce. To circumvent this problem, they turned to a technique that had been successful in mice, in which the testis tissues from a young monkey were incubated subcutaneously in nude mice for 11 months to speed up sperm maturation. Mature sperm were then injected into oocytes to produce embryos that were transferred to female surrogate monkeys, resulting in eight pregnancies, seven births, and six healthy monkeys, including five that carried the MECP2 transgene (Liu et al., 2016b). The problem with this approach, said Poo, is that the monkeys are all different because multiple copies of transgenes are randomly inserted in the genome, thus limiting reproducibility.
SHANK3 is a gene that is critical for synaptic development, said Guoping Feng, noting that single-gene mutations in SHANK3 lead to a severe neurodevelopmental disorder with autism spectrum features in humans (Durand et al., 2007). Feng’s lab at MIT has collaborated with a group in China to develop viable cynomolgus macaque monkeys with both homozygous and heterozygous mutations, and has shown that these animals show reduced motor activity, dramatic sleep disruption similar to what is seen in humans, and impaired social interaction, including lack of reciprocal play and vocalizations. Feng and colleagues are examining resting state local connectivity in these animals to try to understand the circuit defects that lead to these problems. The hope, he said, is that they will be able to modulate the affected circuits, correct the problems, and then translate this approach into humans. These studies may also lead to the identification of resting state function biomarkers that will be applicable to humans, said Feng.
Working with collaborators in Japan and at the National Institutes of Health, Feng’s group has also established a marmoset genetic engineering
platform and produced two SHANK3 knock-out marmosets. Next, they plan to generate knock-in models, including models that insert Cre into PV neurons, he said. Because marmosets are more social than macaques, Feng believes they will provide a superior model for studying disorders associated with social dysfunction, although analyzing marmoset behavior is challenging because they move in three dimensions: walking, running, and jumping and leaping. Working with the MIT Computer Science and Artificial Intelligence Lab, Feng and colleagues have also developed software to automatically track marmoset movements and interactions, and they are working to develop tools that will assess sophisticated cognitive functions.
Ablation Model of Circadian Disorders
Poo and colleagues have also been developing a macaque monkey model to investigate the genetics of circadian rhythm disorders, which have been linked to chronic and degenerative diseases of aging. The circadian clock is regulated by multiple genes that regulate metabolism, including Bmal1 (Bass and Takahashi, 2010). According to Poo, Bmal1 along with other genes such as CLOCK, PER, and CRY1 drives rhythmic gene expression in all cells of the body. Using CRISPR technology, Poo and colleagues have generated complete and partial Bmal1 knock-out macaque models and are using these to assess how the loss of this gene affects the cycling of enzyme and hormone levels and the behavior phenotype.
Cloning Macaque Monkeys
Poo noted that even though CRISPR avoids the random insertion of multiple gene copies seen with transgenics, the production of these knockout and knock-in models is inefficient and subject to off-target effects and mosaicism, where progeny have a mixture of edited and unedited cells. The problems and difficulties associated with embryonic gene editing via viral expression of transgenes or CRISPR editing led Poo and colleagues to explore cloning as an alternative. This approach has the additional benefit of producing animals with uniform genetic backgrounds, said Poo. His group is using somatic cell nuclear transfer, where they fuse fetal fibroblast cells with enucleated oocytes to produce embryos expressing the genome of the animal from which the fibroblasts were obtained. These embryos are then implanted into surrogates to produce genetically uniform
offspring (Liu et al., 2018). Poo added that he hopes that future manipulations, screening, and gene editing can be carried out in primary cultures before performing somatic cell nuclear transfer.
Gene Modification via Vector Delivery
Gene modification in primates and other animal models can also be accomplished through delivery of genes with viral or other types of vectors (Nayerossadat et al., 2012). Deverman’s lab has been developing vector tools in mice that they hope can be applied to other nontransgenic organisms using viral vectors alone. They developed a method called CREATE (Cre recombination-based adeno-associated virus [AAV] targeted evolution) to select variants of the naturally occurring AAV9, which crosses the blood–brain barrier and transduces various neuron populations in the adult mouse brain with high efficiency after intravenous injection (Deverman et al., 2016). While the variant they identified—AAV-PHP.B—transfers genes 40 times more efficiently than AAV9, large doses of vector are required. For even more efficient transduction, they used CREATE to evolve the virus further and ended up finding a virus they call PHP.eB that, with a 24-fold lower dose, transduced more than 50 percent of the cells in the striatum and nearly 70 percent in the cerebellum (Chan et al., 2017).
Deverman noted that this technology is useful not only for disease modeling or developing potential gene therapies, but also has some relevance to circuit studies. In collaboration with David Anderson’s lab at the California Institute of Technology, his group created a Cre-dependent vector that expresses a neuropeptide called Tac2, which is upregulated under stressful conditions in mice. Using this vector, they showed that the symptoms resulting from chronic isolation stress, such as aggressive behaviors and prolonged fear responses, could be mimicked through expression of Tac2. In other words, Tac2 actually drives the response to stress.
Whether these approaches will work in nonhuman primates remains an unanswered question, said Deverman. The viruses seem to work well in rats, but not in marmosets, and there have been mixed studies in macaques, he said. Because of this, his team is trying to develop new vectors that work across species. They are also developing approaches that achieve cell-type specificity in the absence of transgenics. For example, they have developed CREATE 2.0, which uses deep sequencing of the viruses combined with negative selection in silico to find sequences that are broadly transducing and specific for various cell types. Another approach they are
investigating is inserting cell-type specific promoters in their vectors. Using this approach with different promoters, they were able to target dopaminergic, serotonergic, and GABAergic neurons with high efficiency (Chan et al., 2017). They are also exploring techniques to reduce off-target expression as well as intersectional strategies that enable targeting of specific neurons by injecting one virus at axon terminals and another at the soma.
Nonhuman primate models have proven valuable not only as models to advance understanding of human diseases and discover new therapeutic approaches, but also to ensure that treatments can be delivered safely to humans (Friedman et al., 2017). Lisa Stanek, senior principal scientist at Sanofi Genzyme, said that when it comes to gene therapy, a large complex primate brain is needed to understand the delivery and distribution of vectors in the brain. In addition, she said, the genome should be similar to that of humans because some of the off-target effects might not be seen in a rodent model. Viral vector gene therapy also sometimes relies on connections within the brain in order to transport the vectors, said Stanek. Nonhuman primate models would therefore be useful to study whether those connections exist even in the diseased brain, she said. Nonhuman primate models may also be useful for biomarker development, added Stanek.
A recent example of the potential of gene therapy was realized in December 2017, when the Food and Drug Administration approved Luxturna® (voretigene) as the first directly administered gene therapy that targets a specific disease-causing gene mutation, according to Jean Bennett, F. M. Kirby Professor of Ophthalmology at the Perelman School of Medicine, University of Pennsylvania. The therapy is now available to patients with a rare form of blindness caused by mutations in the RPE65 gene, which encodes a protein that converts light to an electrical signal in the retina. Mutations in the gene result in gradual loss of vision and eventually complete blindness. Bennett developed the reagent that is now known as Luxturna® in collaboration with her colleagues at the University of Pennsylvania.
Proof of concept for the treatment was performed in a naturally occurring animal model, said Bennett. Briards are herding dogs that develop a form of progressive retinal degeneration caused by mutations in RPE65. In 2001, Bennett and colleagues at the University of Pennsylvania, Cornell
University, and the University of Florida, Gainesville, published a paper showing that gene therapy with a recombinant AAV restored vision in blind Briards within a few months of receiving a single injection (Acland et al., 2001). Bennett said the first dog treated showed robust improvements on electroretinogram assessments with waveforms similar to what is seen in normal dogs, and that these improvements persisted over his life.
To gain assurance that the treatment was safe and effective in humans, Bennett and colleagues next tested the therapy in nonhuman primates. It was not only because the structure and vasculature of the human eye are different from the canine eye, but also because among species other than human, only primates have a macula, the region in the eye that provides humans with fine visual acuity, said Bennett (Kostic and Arsenijevic, 2016). The monkey studies indicated that the treatment was safe and effective, providing the evidence they needed to move forward into human studies. Bennett showed videos of one child who received the treatment. Before the treatment he walked with a blind cane and needed assistance to get around. A year and a half later, he was able to ride his bike to a friend’s house, see the blackboard at school, play sports, and do puzzles. Ten years later, he reports that he is hunting and hitting targets, said Bennett.
Bennett said the question from all subjects enrolled in the clinical trial is “When can I have my second eye injected?” Readministration raises concerns about immune responses, and according to Bennett, efficacy after readministration of AAV in humans has been described in only one other clinical trial. However, in a study in dogs and subsequently in nonhuman primates, readministration in the contralateral eye was shown to be safe and effective (Amado et al., 2010). These results led to U.S. and European Union approval of the reagent as a drug, said Bennett.
Bennett and colleagues have tried to develop treatments for other eye diseases, such as choroideremia—an X-linked degenerative retinal disease that leads to loss of night vision, peripheral vision, and eventually blindness. Lacking a naturally occurring animal model of this disease, her team turned to induced pluripotent stem cells for proof-of-concept studies (Vasireddy et al., 2013), then demonstrated that the treatment was safe in nonhuman primates before proceeding to a clinical trial, which has also shown a high degree of safety.
While the nonhuman primate models have been predictive of safety, Bennett noted that few retinal diseases occur naturally in nonhuman primates, perhaps because they are selected against in the wild. One exception is that male squirrel monkeys have an incidence of color blindness similar to that of the X-linked form of red–green color blindness seen in
human men. A gene therapy approach to treating red–green color blindness has been developed in the monkeys (Mancuso et al., 2009), which may lead to clinical trials in humans, said Bennett.
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