Brain initiatives in different countries are taking on different, but complementary, approaches to brain mapping, noted Joshua Gordon. In Japan, the Brain/MINDS (Mapping by Integrated Neurotechnologies for Disease Studies) project is focused on mapping the marmoset brain; China has taken on mapping and cataloging the cell types in the macaque brain; and the United States has launched an initiative to discover and catalog human cell types. Likewise, he said, both the U.S. BRAIN Initiative and the European Brain Initiative include significant theory components. Mu-Ming Poo, director of the China Brain Project, said that within the next 10 to 15 years, they hope to map the cell-type-specific connectome of important brain regions in the macaque as an intermediate step toward mapping all the connections in the human brain using platforms for neuromodulation, neural information processing, and transgenic nonhuman primate development. They are also working on technologies for single-neuron gene expression profiling and labeling, circuit tracing, mesoscopic and microscopic brain imaging, and in vivo electrophysiological and electrochemical recording (Poo et al., 2016).
PRIMATE RESEARCH CENTERS
As noted earlier, advancing primate research will require breeding and husbandry facilities for large numbers of animals. The United States has seven National Primate Research Centers (NPRCs)1 supported, in part, by National Institutes of Health (NIH) base grants, said John Morrison. Each center houses approximately 4,000 to 5,000 monkeys, participates in 80 to
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1 To learn more about the National Primate Research Centers, go to https://nprcresearch.org/primate (accessed December 22, 2018).
100 experimental protocols, and forms collaborative partnerships with affiliates, he added. Several centers have large outdoor corrals that provide a naturalistic setting excellent for breeding. In addition, they enable scientists to work with monkeys—including those that have been genetically manipulated—across the life span, from embryos through to geriatric colonies. The centers also provide imaging facilities and animal husbandry teams, said Morrison. Having highly skilled husbandry professionals is particularly important when working with monkey models of schizophrenia or other conditions with complex phenotypes, he said.
Morrison provided an example of how the California NPRC advanced translational vision research moved quickly and efficiently. After the Center’s animal health technicians noticed that some of the animals were avoiding sunlight, the vision scientist group tested the animals and found they had no cone function. They went on to identify the gene associated with this phenotype and have now obtained a grant to breed more of these animals and move forward with developing gene therapy.
This is just one example of how the large teams at NPRCs facilitate convergent research in nonhuman primates, said Morrison. When there are very complex phenotypes, it is not enough to record from one part of the frontal cortex; there is also a need to study metabolism and cardiovascular and respiratory function, to get a full phenotype, and to find biomarkers, he said.
According to Karen Parker, the NPRC P51 base grants have not increased, or have modestly increased, in the past 5–10 years. While all NPRCs have undergone extensive construction and renovation projects in the past decade, she added that some facilities within the NPRCs have not been updated since the 1960s or 1970s. Jon Levine, director of the Wisconsin NPRC, noted that the lack of increased funding means a decrease in real dollars over this time. These resource limitations will make it difficult for the Centers to add emerging technologies, including genome editing, said Parker; they also will be unable to expand to meet user demand.
Parker called for a national investment to support and advance nonhuman primate models of complex brain disorders, including scaling emerging genetic technologies and sophisticated behavioral tools for use at NPRCs. She suggested that to advance transgenic work, this may mean distributing efforts among the seven NPRCs or relying on academic institutions to provide the financial backstops for this research. Levine offered some good news regarding funding for nonhuman primate research. He said the NIH’s Office of Research Infrastructure Programs recently completed Phase I of a nonhuman primate needs assessment and is in the midst
of preparing a report from a Phase II expert panel discussion. This report will identify the desperate need to have a national strategy to develop marmosets as a resource and to support the development of these tools, he said.
The German Primate Center in Goettingen is the only publicly funded center in Germany doing both research and breeding for the academic community, said Stefan Treue. In Europe, nonhuman primates comprise only about 0.05 percent of all animals used in research, he said, yet their small proportion belies their scientific importance. Only about 10 percent of these are used for basic research studies; most are used for toxicology studies and other aspects of product development and safety assessment. As in the United States, noncommercial, nonhuman primate research in Europe is distributed across national primate centers and academic laboratories, said Treue. He suggested that such a dual approach is critical to optimizing the role of nonhuman primate research.
Developing Nonhuman Primate Resources: To Centralize or Not?
Research involving primates has been slowed by limitations in the number of animals that can be generated in a reasonable time frame, said Yoland Smith. What is needed, he said, is a resource that provides scientists with access to nonhuman primates, but that does not require each lab or each academic institution to establish its own breeding facility. Guoping Feng concurred, noting that it is not feasible or cost-effective for every university to house a transgenic primate facility. He advocated a centralized facility shared by several universities. Gordon commented that a large-scale, Jackson Lab-type approach (which breeds genetically modified mice for labs around the world) to maintaining and sharing nonhuman primate lines might not be feasible, though it would be important to develop infrastructure and policies to promote sharing. While centralized breeding facilities offer many advantages, Bettina Buhring of the National Institute of Mental Health (NIMH) also encouraged individual laboratories to continue working on animal models that are nearly ready. It would be unethical, she said, to withhold such tools simply because the infrastructure is not yet ready.
Steven Hyman noted that vigilance will be needed to keep these animals healthy. He added that this will require animals to be outbred, which necessitates a different way of interpreting results than is used with inbred mouse strains where there is a totally isogenic background. A chip that
enables fast whole genome sequencing of marmosets and macaques might be a useful tool, he said.
Hideyuki Okano reiterated the importance of sharing animals around the world, but noted that most airlines will not transport monkeys. A solution, he said, is to transport frozen sperm. Sperm from genetically modified marmosets is available from his institute, the RIKEN Center for Brain Science, he said. Feng added that once the first male founder is generated, in vitro fertilization can be done quickly. Another potential solution mentioned by Gordon is to develop the infrastructure for observatories where scientists from various institutions could conduct their research.
John Spiro agreed that coordinated efforts are needed to move forward, adding that this will require a cultural acceptance that these models must be shared among other groups. However, he added that in the case of disorders like autism where even people with the same driver mutation may look very different, a large number of monkeys may be needed to accurately model the human condition.
Nonhuman primate research centers could also facilitate breeding of animals when researchers identify unique pathologies mimicking human diseases, said Jean Bennett. For example, macular degeneration, which is responsible for about 8 million cases of blindness in the United States, would be a great target for gene therapy if there were good models, she said. After performing ophthalmoscopy on more than 100 nonhuman primates over the past 20 years, her team found one animal with white spots on the retina that resembled drusen, the hallmark pathology seen in people with macular degeneration. Genetic analysis of this animal showed biallelic mutations in the gene for an inherited form of macular degeneration called Stargardt disease, suggesting a possible path toward breeding an animal model of macular degeneration. She said it would be important for the field to consider strategies about what could be done when such unique animals are identified so others can study them.
SHARING INFORMATION AND DATA
Reproducibility of results has dogged many areas of biomedical research, said Mark Frasier, senior vice president of research programs at The Michael J. Fox Foundation (MJFF) for Parkinson’s Research. To address this need, MJFF funds academic investigators to bring their expertise, knowledge, and sophisticated tools to characterizing models in a
standardized way, and then to make those models accessible through different vendors for both academic and industry research. Although MJFF has not done this with nonhuman primate models yet, the model has been successfully implemented with other tools such as viral vectors, he said. Frasier added that they are also supporting more natural history work to understand disease at the human level in a multidimensional way, and that the data from these studies are shared across the globe.
Sharing of other information is also critical to minimize duplications, said Marina Emborg. Frances Jensen agreed, adding that sharing of negative as well as positive data is needed within the nonhuman primate research community, especially data from unique transgenic animals. Given the limited resources available for nonhuman primate work, Frasier wondered how to balance innovation in developing new methodologies with the need for standardization. Parker suggested that funders might make sharing of resources an expectation of all grant recipients.
TRAINING AND EDUCATING THE NEXT GENERATION OF RESEARCHERS
One of the lessons learned from efforts to develop genetic models in mice is that going from a gene mutation to a mechanistic causal pathway and then to a druggable target is difficult, according to Robert Desimone, director of the McGovern Institute and the Doris and Don Berkey Professor of Brain and Cognitive Sciences at the Massachusetts Institute of Technology. Moreover, manipulating genes in the brain markedly increases the difficulty, he said. These issues will not be solved by any one lab, one center, or even one country, he said; many laboratories working on many different aspects of the problem will be needed. The challenges involve money, facilities, and people. Given the impracticality of every institution having its own primate facility, he advocated finding ways to share genetic material and establish centralized breeding colonies that can make animals available to investigators at many universities. To accomplish all of this, he said, more trained researchers will be needed.
Poo said the China Brain Project envisions a national brain health training and education center to promote sustainable research in primate biology. As part of this effort, they plan to establish a permanent summer program to train students who are interested in working with nonhuman primates, said Poo. A few workshop speakers added that other changes will also be needed in academic institutions to incentivize young scientists
to pursue careers that involve working with nonhuman primates. For example, Hyman pointed to the need for career development pathways that credit scientists for their participation in large consortia.
ORGANIZING EFFORTS TO MEET INFRASTRUCTURE AND SCIENTIFIC NEEDS
Feng agreed about the urgency of crafting a strategy to develop genetically modified nonhuman primate models. In 2 or 3 years, he said, many genetically modified lines will be available, yet the infrastructure to breed and distribute them has not kept up. Gordon said NIMH (mostly through the U.S. BRAIN Initiative, but also the NIH Blueprint for Neuroscience Research) has identified the two highest priorities: (1) obtaining and expanding marmoset breeding colonies in the United States, and (2) establishing centers or expanding existing centers that will continuously breed marmosets and make them available. Morrison said there are currently only two primate centers, Wisconsin and Southwest, that breed marmosets. The expertise, resources, and space are available to establish a breeding colony at the California NPRC, he said; however, funds are lacking. Gordon said there is agreement among the neuroscience institutes that collaborations and pooling of resources across multiple NIH institutes should be supported.
Given the precious nature of these resources and the ethical pressure to limit their use, Susan Amara, director of the Division of Intramural Research Programs at NIMH, wondered if a larger, global consortium representing industry, academia, federal and nonprofit funders, and other stakeholder groups could coordinate efforts on generating transgenic nonhuman primate models to ensure that resources are well used, and to minimize the potential for duplication of efforts. Morrison agreed, and suggested that in setting resources and priorities, the research community should be guided not by what has been done in rodent models, but by what the human literature suggests could be gained by research in monkeys. Hyman added that the Foundation for the National Institutes of Health might be the entity that could bring industry partners onboard.
Lisa Stanek and Morrison noted that collaborations between industry and academia are becoming more common, but they suggested that more could be done. Stanek said that many academics may not appreciate that industry partners are ready, willing, and waiting to work with them. She said that a collaboration between Sanofi and Ben Deverman’s laboratory in developing novel adeno-associated virus (AAV) capsids for nonhuman
primate research was a perfect marriage of his skills in the academic setting and their infrastructure that enabled them to execute nonhuman primate studies relatively rapidly.
However, Frasier countered that a large consortium can be effective if there is a shared common goal, but it may lack the agility to answer some disease-focused questions. For example, he said MJFF has partnered with other organizations, including NIH and other nonprofit funders, to focus on a common but more targeted question related to Parkinson’s disease. Stanek also pointed to a model advanced by the CHDI Foundation, a nonprofit, private, disease-specific foundation focused on Huntington’s disease that brings researchers or organizations together to address specific questions and avoid duplicating efforts.
Story Landis, director emeritus of the National Institute of Neurological Disorders and Stroke and co-chair of the Forum on Neuroscience and Nervous System Disorders, suggested that it might also be useful to engage organizations such as the Allen Brain Institute that already have a robust pipeline of gene expression and single-cell technologies. She said they have ways of mapping connectivity that would take years for another institution to put together. All they need are the marmosets, she said. Hyman suggested that a neutral convener might be able to bring these organizations into the discussion. He added that because many of the important tools for nonhuman primate research are being developed in China and Japan, there may be value in convening some of these meetings in Asia.
FINAL REMARKS
In closing, Steven Hyman highlighted some of the important points that emerged during the workshop. One of the key messages, he said, is that nonhuman primate research complements studies in humans and other organisms. Researchers have the opportunity to select a model system—ranging from cell culture to drosophila, zebrafish, mice, and now nonhuman primates—that is most likely to be able to answer the research questions at hand. The workshop made clear that translational research involves a new set of constraints that does not exist in basic research, he said, especially the need for conservation across all components that contribute to an outcome: molecular targets, cell synapses, and circuits. Indeed, it is that need for conservation that makes selecting the right model so important, said Hyman.
As an example, he noted that in humans, anxiety disorders and addictions are as much the result of top-down control failures as with the bottom-up functions of reward and fear circuits. Nonhuman primates enable scientists both to study failures of top-down control in realistic contexts as well as to flag reward circuits and possibly treat addiction, he said. Depending on the question being asked, genetic modification with either transgenesis or viral vectors may add further utility to nonhuman primate models, said Hyman. For example, as described in Chapter 3 by Hideyuki Okano, a transgenic marmoset model of Parkinson’s disease (PD) carrying an α-synuclein mutation enabled his team to model sleep disturbances that are a common complaint of patients with PD, but that were not seen in the mouse MPTP model that had been so useful in developing deep-brain stimulation as a treatment for the motor symptoms of PD. Hyman added that antisense oligonucleotides and gene therapy have taken the value of these nonhuman primate models another step further.
Hyman predicted that in the case of autism, gene-modified monkeys will be needed for every 1 of the 100 genes identified as strong drivers of autism that John Spiro mentioned in Chapter 3. Each of these models will enable different questions to be asked and answered, including questions related to intervention, off-target effects, biomarkers, and safety, he said.
To address the scientific and practical challenges identified throughout the workshop, Guoping Feng highlighted the urgency of crafting a strategy to develop genetically modified nonhuman primate models and the needed infrastructure to support this promising area of research.
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