FINAL REPORT

Eleventh Comparative Medicine Resource Directors Meeting

Enabling Biomedical Research: From Fundamental Science to Precision Medicine

August 9 and 10, 2016

DoubleTree, Bethesda, MD

  

Eleventh Comparative Medicine Resource Directors (CMRD) Meeting Enabling Biomedical Research: From Fundamental Science to Precision Medicine August 9 and 10, 2016 – DoubleTree Inn, Bethesda, MD

Introduction “Enabling Biomedical Research: From Fundamental Science to Precision Medicine,” the Eleventh Comparative Medicine Resource Directors Meeting, was held August 9-10, 2016, in Bethesda, Maryland. All Resource Directors funded by the Office of Research Infrastructure Programs (ORIP)/Division of Comparative Medicine (DCM) were invited to attend. This biennial meeting offers a forum to provide useful information to DCM-funded Resource Directors and for developing synergistic working groups, interactions, and collaborations. A primary objective was to generate broad interests in Resources, discuss “lessons learned,” and to convey a sense of what’s been done and what can be done in the future. Another important objective was to allow Resource Directors the opportunity to discuss optimization of administrative processes. ORIP Program Officers and others from various NIH Institutes, Centers, and Offices (ICOs) contributed to scientific discussions to further enhance national use of DCM-funded resources. There were 62 Resource Directors and personnel from 46 DCM‐funded resources from 21 states and Puerto Rico as well as 38 NIH staff representing 10 ICOs and the Office of the Director (OD) at the meeting. The meeting also included 3 outside speakers from the USDA, Institute of EthnoMedicine, and the J. Craig Venter Institute. The attendees included the Principal Investigators of DCM‐supported centers funded by contracts, P40, U24 or U42 grant mechanisms, as well as some grantees that have Resource‐related projects funded via the R24 mechanism. There were 7 sessions with 30 presentations and a separate poster session with 41 posters that covered aspects of “Generating, Managing, and Integrating Phenotypic Data Across Animal Models; “Administrative Practices at NIH‐Supported Resources;” “Animal Models and Precision Medicine;” “Promoting a Resource’s Animals and Services;” “Genome Editing: Technical and Community Lessons Learned;” “Microbiota and Beyond: Opportunities and Concerns for Animal Resources;” and “New NIH Policies and Good Resource Practices.”

Stephanie Murphy (Director, DCM, ORIP) Introduction and Welcome Dr. Murphy welcomed all participants and provided opening remarks. She indicated that the goals of this meeting were threefold: (1) to provide a forum for exchange of new information, advances and ideas; (2) to facilitate development and continuation of synergistic working groups, interactions and collaborations among resources and between resources and the various NIH ICOs; and (3) to offer opportunities for sharing experiences, strategies and best practices so as to optimize access, use, and administration of the valuable resources we all represent and support. Dr. Murphy also encouraged the audience to visit the ORIP twitter page “@NIH_ORIP for up to date information and to participate by using the hashtag: #CMRD2016.

James Anderson (Director, DPCPSI) Valid Research Requires Valid Antibodies Antibodies are among the most common reagents in biomedical research; unfortunately, many commercially available antibodies may be unreliable. Shortcomings include lack of specificity, vendor-to-vendor variability and batch-to-batch variability. Additionally, antibodies developed for one experimental application may be used in another experimental application, but because antibody to target binding is optimal only in the former, false positive, false negative or errors in quantification can occur in the latter.

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Dr. Anderson explained that although the biomedical community has long recognized that antibodies can be problematic, researchers seldom check the validity of the antibody chosen for a particular experiment. This has led to an unknown number (but an estimated large percentage) of publically funded research projects that simply cannot be replicated. Dr. Anderson emphasized that NIH wishes to improve the reproducibility of biomedical research, including the vast amount of biomedical research that relies on antibodies. He acknowledged, however, that no universally recommended and accepted guidelines for antibody validation currently exist. The NIH strongly encourages the scientific community to establish consensus standards and best practices that can be cited by applicants. Most NIH grant applications now require applicants to include new information that addresses the integrity and reproducibility potential of key resources and reagents used in experiments, including antibodies:

• Applicants will be required to briefly describe the methods they plan to use to authenticate key resources based on their scientific experience and judgment, referencing relevant standards where applicable.

• If key resources (e.g., antibodies, cell lines, specialty chemicals, other biologics) have been purchased or obtained from an outside source that provided data on prior authentication, the investigator is still expected to provide their own authentication plans for these key resources.

• Actual data demonstrating that authenticated resources are available for the proposed research do not need to be included in the plan.

Dr. Anderson also described the NIH Protein Capture Reagents Program which is supported by the Common Fund and which was developed to provide standard operating procedures (SOP) to quantitatively assess the selectivity and specificity of antibodies for immunoprecipitation experiments using mass spectrometry. To date, there are greater than 1700 reagents (e.g., monoclonal or recombinant antibodies) to over 640 human transcription factor (hTF) antigens and hTF-associated proteins. SOP performance was validated in multiple independent laboratories and the project generated readily accessible quantitative data on antibody quality. Dr. Anderson provided an example that included the name of the antibody, its product description, its physical characteristics (e.g., culture supernatant or purified), host isotype, clonality, reactivity (if available) and handling notes. Importantly, the information provided includes “tested research applications” (e.g., Western Immunoblotting) and Quality Assurance information. In Dr. Anderson’s example, the Anti-human ANAPC2 monoclonal antibody was tested (via SOP) for Western Immunoblotting but is explicitly ‘not recommended’ for immunoprecipitation experiments.

Dr. Anderson also noted the upcoming conference, “Antibody Validation: Standards, Policies, and Practice” (September 25-27, 2016) that will bring together ‘stakeholders from academia, antibody producers, pharma, funders, and journals to share perspectives and contribute to tangible solutions for validating antibodies. He closed his talk by emphasizing the importance of reproducibility and transparency in biomedical research and asked the audience, “How do you validate your resources?”

Paul Alan Cox (The Institute for EthnoMedicine) Keynote Address Replicating a Puzzling Paralytic Disease of Pacific Islanders in a Nonhuman Primate Model Dr. Cox is the Director of the Institute of Ethnomedicine (Jackson Hole, WY). His current research efforts are focused on neurodegenerative illnesses and possible environmental toxins that may

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contribute to their etiologies. Dr. Cox has worked since the late 1990s on the link between beta-methylamino-L-alanine (BMMA), a non-proteinogenic amino acid, and Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) disease.

U.S. Army physicians first described what became known as ALS/PDC in adults living in the Chamorro villages of Guam. ALS/PDC, as the name suggests, shares symptoms and other features with Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig’s Disease; e.g., muscle weakness, paralysis, death), Parkinson’s Disease (PD; e.g., tremors) and to a lesser extent, Alzheimer’s Disease (AD; e.g., dementia, neurofibrillary tangles). Dr. Cox explained that although the disease was found to run in some families, there were no clear patterns of inheritance. The number of villagers who developed the disease, however, was staggering: In one village alone, one-quarter to one-third of all deaths between 1944 and 1953 could be attributed to the disease. Outsiders who joined the Chamorro community and adopted their way of life, including their diet, were at an increased risk for ALS/PDC. Taken together, Dr. Cox and others believed that something in the environment was causing or triggering the disease.

Evidence that the neurotoxin BMMA may be responsible for ALS/PDC in Chamorro villagers in Guam is circumstantial, but is accumulating. BMMA is produced by symbiotic cyanobacteria within specialized roots of the cycad tree. Protein-bound BMMA accumulates to very high levels (i.e., biomagnified) in cycad seeds, flour made from ground cycad seeds, and in animals (e.g., flying foxes, deer, pigs) that forage on the seeds, all of which were staples in the Chamorro diet. Elevated levels of protein-bound BMAA were found in the brains of Chamorro villagers but not in the brains of unaffected controls. Additional studies have found BMAA in the brains of North American patients who had died of Alzheimer’s disease, and in other patients with Parkinson’s Disease, ALS and Alzheimer’s Disease. BMAA was not found in control brains nor was it found in the brains of patients suffering from the autosomal dominant disorder, Huntington’s disease. The question posed by Dr. Cox at this point was where BMAA was coming from in populations not living on Guam.

The answer may lie in the ability of cyanobacteria to live not only as symbionts but to also live alone in fresh or marine water. Cyanobacteria, once known as blue-green algae, has the capacity to expand in the form of large ‘blooms’ if conditions in water are optimal: in cases of fertilizer run-off, at the edge of desert crusts after a rain, and potentially in warming waters as the climate changes. The vast majority of cyanobacteria tested can produce BMAA and Dr. Cox noted that many fish, shellfish and other aquatic life feed on cyanobacteria blooms. Dr. Cox and others have looked for associations between large cyanobacteria blooms and greater numbers of people suffering from ALS, PD or AD. The idea is that people living near water that has historically supported blooms of cyanobacteria may have accumulated BMAA over time through ingestion of aquatic life (e.g., shellfish, etc.) and be at higher risk of developing progressive neurodegenerative diseases. This possibility is being investigated, but as of now is still only a hypothesis.

Dr. Cox described experiments that shed light on possible mechanisms of action of BMAA in cells and in the brains of affected individuals. Chronic dietary exposure to BMAA produces tangles and amyloid plaques in the brain. BMAA is a non-proteinogenic amino acid, meaning that it is not found in the genetic code of any organism, and it can cross the blood-brain barrier where it can be trapped in proteins by taking the place of serine during mRNA translation. BMAA can bind to tRNA, displace serine and cause protein misfolding, aggregation and apoptosis. High

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concentrations of competing L-serine have been shown to prevent or lessen misincorporation of BMAA and apoptosis in neuronal cell cultures.

Recently, Dr. Cox and collaborators conducted an experiment in nonhuman primate vervets with chronic exposure to BMAA to determine if their brain tissues contained features of ALS/PDC pathology. While the exposure to BMAA accumulates over years or decades in the Chamorro villagers, the vervets were exposed to BMAA for 140 days with or without equal amounts of L-serine. A second experiment was conducted in vervets with 10-fold less BMAA. Results clearly demonstrated that beta-amyloid deposits occur in vervets with chronic exposure to BMAA and BMAA increases the likelihood of developing beta-amyloid deposits. The addition of serine to the group receiving the high dose of BMAA had lower densities of neurofibrillary tangles, indicating a potential neuroprotective role for L-serine. Dr. Cox posed the next question, “Could L-serine be neuroprotective in human beings?”

Dr. Cox is hopeful that the dietary amino acid L-serine can prevent or delay onset of Alzheimer’s disease pathology and delay onset of cognitive impairment in individuals with evidence of pathology. He described the remarkable longevity and cognitive attributes of certain elderly populations who eat diets rich in serine. A Phase I clinical trial for ALS involved 20 patients and was designed as a double-blind, randomized trial that provided 5-30 grams per day of oral L-serine. Results demonstrated that up to 30 grams of L-serine were well tolerated by patients. Efforts are underway to conduct a Phase II clinical trial for 66 ALS patients to see if L-serine can work to improve cognition and improve survival from the disease. As Dr. Cox emphasized, more research is needed to definitively answer the question of whether BMMA combines with genetic predisposition to cause ALS/PDC and other neurodegenerative diseases in humans.

Session 1: Generating, Managing, and Integrating Phenotypic Data across Animal Models Moderators: Harold Watson (ORIP) and Brian Oliver (NIDDK)

Calum MacRae (Brigham and Women’s Hospital, Harvard Medical School) Co-Clinical Modeling to Generate New Translatable Phenotypes Dr. MacRae is the Principal Investigator of the Community Zebrafish Resource for Modeling Genome Wide Association Studies (Brigham and Women’s Hospital). Dr. MacRae is broadly interested in cardiovascular development, function, and health. He and his collaborators use zebrafish as a model organism to identify phenotypes associated with heart development and function. Zebrafish possess highly perturbed transcripts that are also found in human heart cells and tissues and that can be genetically modified to study multiple disease alleles. Dr. MacRae described the process of automated, high throughput (HTP) screens of functional genomics that can be carried out in 96- or 384- well plates containing zebrafish embryos. These screens can detect new phenotypic abnormalities and potential biomarkers of disease. The process can also be used to screen large chemical libraries for agents that can reverse or prevent heart-related disease phenotypes. Potential therapeutics identified in the zebrafish high throughput screening protocol can be validated in mammalian models and cultured human heart cells before clinical trial testing.

Dr. MacRae described experiments that screened a library of bioactive compounds in a zebrafish-based model of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). ARVC is an inherited condition caused by mutations in one or more genes. It is a significant cause of sudden

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death, particularly in young individuals. Zebrafish expressing a mutation in the plakoglobin gene in cardiac monocytes exhibit enlargement of the heart, thinning of heart walls, wasting and higher mortality than controls. Larval mutant fish raised in 96-well plates exhibit clear disease phenotypes within the heart as soon as 48 hours after fertilization. By screening over 4000 compounds, one in particular, SB2, an annotated GSK3β inhibitor of Wnt pathway signaling, was able to suppress and even reverse disease phenotypes, including myocardial apoptosis, fibrosis, and left ventricular dysfunction. Dr. MacRae summarized by stating that zebrafish are one example of co-clinical modeling that generates translatable phenotypes and methodologies to identify potential therapeutics.

Anita Bandrowski (CRBS, University of California, San Diego) Persistent Unique Identifiers, Their Role in Grouping Information The central theme in Dr. Bandrowski’s talk was the importance of accurate and accessible research resource identifiers in peer-reviewed scientific publications to improve research reproducibility. Far too often, she pointed out, scientific papers poorly identify the materials used to perform experiments or the resource is only known to one of the authors. Dr. Bandrowski and collaborators identified 25 major biomedical journals (primarily in neuroscience) to approach with the idea of performing a pilot study to determine the feasibility of requiring authors to include Research Resource Identifiers (RRIDs) in a standard format, such as a citation, in their manuscripts. Three resource types were prioritized: antibodies, model organisms, and tools used for analysis (including statistical software, databases etc.). To be useful, the RRIDs had to be accessible to researchers and computer-based search algorithms and had to be uniform across all participating journals and publishers. Additionally, traditional journals participating in the pilot study were asked to include the RRIDs in the keyword field, to allow indexing in PubMed and identification for other researchers without requiring a paid subscription. To assist authors in locating appropriate RRIDs, a central web-based portal, scicrunch.org/resources, was created that aggregates RRIDs from authoritative community databases and displays the results in a common format. Searches can be performed using a resource name, catalog number, animal strain, etc. scicrunch.org/resources includes stock center data ranging from the Ambystoma Genetic Stock Center to Zebrafish International Stock Center. Most stock centers possess a link between the genotype of animals and the stock center animal identifier.

Dr. Bandrowski also discussed the importance of reporting RRIDs only for reagents and resources used to conduct the published study. One of the goals of the project was to provide a means by which researchers could search (e.g., PubMed or Google Scholar) for papers using a particular research resource by its RRID and gain a sense of how many published journal articles used that resource, as opposed to a mention in the introduction or discussion showing awareness but not use. After the first 100 papers, the project obtained over 97% accuracy in RRID citations from authors, and at the 1000 paper time point, that accuracy continues to improve.

The pilot project was deemed a success and today there are over 150 biomedical journals participating in some way, mainly encouraging voluntary participation, but more journals are now requiring RRIDs, the most stringent criterion. Improvements may include hyperlinked resources that take readers to the repository or an article overlay that displays the model organism and repository. It has been recognized, however, that challenges exist with respect to inclusion of RRIDs in papers. Dr. Bandrowski asked audience members to help increase awareness of RRIDs

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and to ask for assistance in RRID incorporation into their own research manuscripts. Organizations that have spearheaded or significantly participated in these efforts acknowledged by Dr. Bandrowski include the Neuroscience Information Framework, the Oregon Health and Science University Library, the Future of Research Communication and e-Scholarship (FORCE11), and NIH.

Mike Tyers (IRIC, University of Montreal)

Curation of Biological Interaction Data for Interpretation of Phenotypes across Model Organisms

Biocuration is the process by which biological information is reviewed, annotated and incorporated into data repositories that can be accessed for computational analysis. The recent advent of readily available HTP screening and other technologies has greatly increased the amount of “omics” raw data published in the literature. Human physiology and disease is a manifestation of genetic and protein interactions that form highly complex networks. Biocuration needs new tools to augment text mining and manual annotation efforts to keep up with the wealth of new information and to provide the scientific community with one or more searchable databases that can be used to understand and predict gene/phenotype functional relationships and analyze the dynamic network of interactions.

The Biological General Repository for Interaction Datasets (BioGRID) is an open access, searchable database developed by Dr. Tyers and colleagues containing curated datasets from the primary biomedical literature of all major model organism species and humans. The BioGRID is freely accessible via a web-based portal and is downloadable. It is updated and archived on a monthly basis, and any alterations to the dataset are noted and traceable providing investigators with the most accurate data possible. Projects using BioGRID undertaken by Dr. Tyers and colleagues include the curation of the available literature related to the ubiquitin-proteasome system (UPS). These efforts resulted in the annotation of 1276 human UPS genes that contribute to the stability and activities of the proteome. Greater than 86,000 interactions were identified and over 400,000 ubiquinated sites were also annotated. This information can be used, for example, to view the conservation of the UPS network from yeast to human and other species and to study the function of the ubiquitin proteasome pathway in disease and drug discovery. Other projects described by Dr. Tyers that use the power of the BioGRID resource include investigations examining the relationship between the UPS network and Parkinson’s disease and separately, the curation of a glioblastoma network.

Dr. Tyers warned of the impending data deluge contained within the burgeoning literature related to CRISPR/Cas9-based gene editing in humans and model organisms. CRISPR-Cas9 screens can reveal the mechanisms of action of some chemical compounds as they relate to chemical-genetic networks.

Session 2: Administrative Practices at NIH-supported Resources

Moderators: Stephanie Murphy (Director, DCM, ORIP) and Sheri Hild (ORIP)

Oleg Mirochnitchenko (ORIP) and Manuel Moro (ORIP)

Updates on Workshops for Phenomics and One Health

Symposium Update - Linking Disease Model Phenotypes to Human Conditions

The emerging field of phenomics, which identifies how physical and biochemical traits change in response to alterations in the genome and environment, is poised to improve patient-based medicine if

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certain challenges can be overcome. Participants at The Tenth Comparative Medicine Resources Directors Meeting (August 2014) recognized the potential of phenomics and suggested that a future colloquium be held in which the opportunities and challenges of this field could be evaluated in depth.

In response to this recommendation, the DCM/ORIP sponsored the symposium “Linking Disease Model Phenotypes to Human Conditions” in September 2015. Symposium sessions focused on the following topics:

Current status of human clinical phenotype ontology and terminology and associated data annotation and use

Cross-species phenotype analysis and ontology Large-scale, high-throughput analysis of disease model phenotyping data and annotation of

gene function Linkage of disease-relevant phenotypes with physiologically relevant molecular pathways

and networks Clinical and experimental biology data integration and positioning of molecular phenotypes

in the emerging field of precision medicine Availability of resources for submission, representation, analysis, and sharing of

phenotypic and genomic information; development of resource identification and tracking to improve reproducibility, and tracking of resource utilization and trends

Speaker presentations and discussions at the symposium identified urgent needs in phenomics, which included:

Better availability of model organisms Standardized methodologies to organize, annotate, and analyze phenotypic data. Development of new approaches to visualize and measure genomic/phenomic relationships

in animal models at subcellular-, cellular-, tissue-, organ- and organism-based levels and relate these data to human health

At the conclusion of the symposium, attendees requested that similar, follow-up symposia be held regularly to evaluate progress in certain areas and to provide a platform for in-depth discussions related to informatics. Other proposals included the development of new NIH program announcements that would encourage institutes and centers to deepen their engagement in comparative analyses of human disease and model organism phenotypes. Underscoring all suggestions was the ongoing mandate to develop new or improve existing mechanisms in biomedical phenomics while ensuring better reproducibility, rigor and transparency.

One Health Workshop Update - Integrating the Veterinarian Scientist into the Biomedical Research Enterprise The One Health Initiative recognizes that the health of humans, animals and the environment affect one another in profound ways. Humans and animals suffer many of the same diseases (e.g., cancer and arthritis) and it is well understood that changes in the environment can increase or decrease the potential for disease transmission. The goal of the One Health Initiative is to improve the health of all species by enhancing cooperation and collaboration between health care professionals, biomedical, and environmental scientists through education, training, leadership and management resources.

In April 2015, a workshop was held on the NIH Bethesda, MD campus that included representatives from federal and non-federal research agencies, scientists, and individuals from the private sector interested in how the One Health Concept can advance the NIH mission. Session topics of the workshop included:

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Case studies of animal and human subjects-based research from multidisciplinary teams of scientists studying an array of disease conditions

One Health panel perspectives from various centers, federal agencies and biopharma Panel discussions regarding One Health-based NIH training programs

Workshop participants expressed support for a broadening of the One Health program at NIH to include clinical studies of naturally occurring animal diseases and studies that examine the impact of human-animal interactions on human health. Additional information can be found at: https://dpcpsi.nih.gov/sites/default/files/ORIP%20One%20Health%20Workshop%20Final%20Rep ort%206%2011%202015.pdf.

Stacia Fleisher (NCATS)

NIH Grants Policy: Some Things Have Changed, Others Remain the Same

Ms. Fleisher, Deputy Director for the NCATS Office of Grants Management, pointed out that NIH-supported Resource Directors should understand that the NIH grants policy is akin to a pendulum: overall there are only a small number of changes, but any one or all of the changes can have a significant impact on the conduct and funding of biomedical research. In 2016, NIH updated application forms in response to policy changes that require additional data collection and greater detail in reporting requirements. These changes are highlighted in each grant application and have been published by NIH (available online, e.g., NOT-OD-16-004). A few areas that have undergone change in grant applications include:

Rigor and transparency in research Vertebrate animals Inclusion reporting Data safety monitoring Research training and training tables Assignment request form Font requirements Biosketch clarifications

For example, in the use of vertebrate animals section, the means of euthanasia should be consistent with American Veterinary Medical Association (AVMA) guidelines. Applications not consistent with AVMA guidelines will undergo secondary review, causing a potential delay in funding. Ms. Fleisher noted that it is important for the primary applicant institution to make sure that collaborators appearing on subawards use the appropriate forms, obtain and maintain appropriate assurances, and adhere to the grant application instructions. Failure to check that subaward applicants are compliant can result in the return of a grant application un-reviewed.

Ms. Fleisher also noted that as of December 2016, changes in U.S. labor law overtime rules will be put in place, and these changes will affect postdoctoral stipends. NIH, she assured the audience, is aware that these changes may affect financial and staffing operations at grantee institutions.

Looking to changes that will go into effect in the near future (potentially before the end of 2016), NIH anticipates the release of an updated set of research terms and conditions for use in research grants that recognize and define new and emerging technologies and terminologies (e.g., personalized medicine; precision medicine). Additionally, NIH will soon require grantees to make publically available (via RePORTER or other similar mechanism) a concise summary of the cumulative outcomes or findings of each NIH-supported research project.

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https://dpcpsi.nih.gov/sites/default/files/ORIP%20One%20Health%20Workshop%20Final%20Rep

  

As part of uniform guidance clarifications on NIH policy, grantees are expected to use program income prior to requesting cash advance payments from the grant award. Several questions were asked on this topic. Ms. Fleisher indicated the degree which this would be documented and enforced by grant management personnel is still under discussion with leadership. Initial thoughts were quarterly updates may be sufficient to address the proper use of program income.

Session 3: Animal Models and Precision Medicine

Moderators: Oleg Mirochnitchenko (ORIP) and Colin Fletcher (NHGRI)

Dr. Oleg Mirochnitchenko introduced the three Pilot Centers that currently receive support through a U54 cooperative agreement funding mechanism (PAR 14-280, “Pilot Centers for Precision Disease Modeling”). The Pilot Centers provide leadership, pre-clinical/co-clinical coordination, and informatics support to investigators that are performing preclinical studies in new generation precision animal models that are most likely to inform subsequent clinical trials in individual patients (personalized medicine) and groups of patients (precision medicine). Each Center will engage in three (or more) focused research projects, but are also expected to build core disease model systems that can be applied to a broad spectrum of diseases.

Ideally, collaborative studies supported by these U54 grants are expected to produce:

Better understanding of the relationship between genotype and phenotype • Stratification of diseases into subtypes according to their underlying biological mechanisms • New tests of complex genetic variations in relevant biological systems • Improvements in the disease simulation process; recapitulation of molecular mechanisms • Clinical trial “like” animal model testing and clinical/model iterations • Rigorous evaluation of animal model predictability and validity

Scott Lowe (Sloan-Kettering Institute for Cancer Research) Memorial Sloan-Kettering Cancer Center (MSKCC) Pilot Center for Precision Disease Modeling

The MSKCC Pilot Center for Precision Disease Modeling in New York City consists of two Cores: a Preclinical/Co-Clinical Core, which includes Genomic, Mouse Modeling and Mouse Hospital Units, and a separate Bioinformatics Core. Three additional Disease Modeling Units are investigating sensitivity and resistance to molecularly targeted leukemia therapies (Project 1); RTEL1 mutations in Hoyeraal Hreidarsson Syndrome (Project 2); and genetic determinants of colorectal cancer initiation and maintenance (Project 3).

To support this project, Dr. Lowe highlighted attributes of the MSKCC Pilot Center for this endeavor, which include:

A history of producing animal models of human disease A mouse hospital capable of performing preclinical studies of putative drug protocols

similar to human clinical studies A strong computational and genomics infrastructure to inform and interpret modeling

efforts Available gene sequence information from the Integrated Mutation Profiling of Actionable

Cancer Targets Cohort (MSK-IMPACT™) project, which generates DNA sequence data from approximately 10,000 patients at Memorial Sloan-Kettering per year

The capability to conduct high throughput/low cost DNA sequencing of key cancer genes in animal models corresponding to human MSK-IMPACT™

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Currently, the MSKCC Pilot Center is creating a pipeline for patient-derived xenograft (PDX) production within the Mouse Hospital Unit. The idea is to implant and propagate patient tumor tissue-derived organoids in immunodeficient mice and then test single- and multi-tumor therapy protocols in a manner similar to clinical trials in patients. The Bioinformatics Core will maintain PDX tumor model-derived data and patient data (e.g., MSK-IMPACT™) in a dedicated database. This database will be searchable using tumor type/subtype, genomic profile, and treatment history. Close attention to toxicities arising from activation or inactivation of the tumor targets in normal tissue will additionally provide information about potential deleterious side effects.

The Center is also using embryonic stem cell - genetically engineered mouse models (ESC-GEMMs) to investigate the effects of activating and inhibiting gene expression temporally and spatially on target tissues and organs. Dr. Lowe discussed results from ongoing projects at the Pilot Center in which ESC-GEMMs were created using Cre-lox technology and the CRISPR/Cas9 system to provide a means by which candidate genes can be turned on or turned off at will, activating or inactivating mechanisms that initiate, encourage or suppress tumor growth. He also discussed the potential of small hairpin RNA (shRNA) to provide researchers with a means to track abnormal cell growth in histological sections using fluorescence.

Ross Cagan (ICAHN School of Medicine at Mount Sinai) A New Disease Platform Leveraging Complex Drosophila and Mammalian Models

The overall goal of the Center for Personalized Cancer Therapeutics (CPCT) is to develop therapeutics using polypharmacology that can cure or bring a disease to remission without deleterious side effects. The idea is to obtain histological sections of a patient’s cancerous tumor, sequence genes within the tumor and use that information to create thousands of ‘fruit fly avatars’ that carry multiple cancer mutations identical to those of the patient. The avatars are exposed to an extensive collection of FDA approved drugs to identify one drug or a cocktail of drugs that can mitigate the effects of oncogenes and signaling pathways that drive tumorigenesis. Drugs that exhibit efficacy may be refined before testing in more complex, murine models of cancer. Ultimately, it is hoped that the functional network platform at the CPCT can provide relevant and timely information to the patient’s health care providers about existing drug protocols or new therapies which are specifically tailored to the patient’s own disease profile.

Dr. Cagan provided a few examples of current projects supported by the U54 award (e.g., Ras pathway mutations/RASopathy; colorectal cancer) and explained that promising results in fruit fly avatars have sometimes failed in other models, which is a concern. Modeling tumor heterogeneity in parallel, however, in simple model systems is an emerging cost effective means by which biomarkers can be identified and candidate treatments can be tested.

Robert Burgess (The Jackson Laboratory) The Jackson Laboratory Center for Precision Genetics: From New Models to Novel Therapeutics The Jackson Laboratory Center for Precision Genetics is supported to generate new models of human disease for use in preclinical studies and to hasten translation of these studies into clinical trials. Dr. Burgess described their vision to develop a comprehensive, “ultimate” precision disease-modeling center that applies patient-derived molecular, phenotypic and pharmacokinetic data to precision non-mammalian models of disease that are capable of high-throughput genetic and drug

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screening approaches. High-throughput screening would yield insights into genotype/phenotype relationships and provide targets for preclinical testing in optimized mammalian models. Funding from the U54 will provide, in part, the means to generate ‘humanized’ mouse models of a variety of human diseases. In one project, humanized mouse models are being developed to test personalized gene therapy vectors targeting variants of GARS, the gene causing autosomal dominant Charcot-Marie-Tooth neuropathy type 2D. Other projects supported by the U54 include development of new models and therapies for epileptic encephalopathy, ALS, age-related chronic kidney disease and age-related macular degeneration. One additional project seeks to delete major histocompatibility complex genes in the mouse and replace them with genes encoding human leukocyte antigens (HLA). The goal is to provide researchers with a better model of diabetes and insulitis. In total, more precise genetic models for preclinical studies should improve the predictive validity of these studies and their translation to human clinical trials.

Session 4 Promoting a Resource’s Animals and Services Moderators: Miguel Contreras (ORIP) and Bruce Fuchs (ORIP)

Christopher Tuggle (Iowa State University)

Expanding the Utility of Severe Combined Immune-Deficient Pig

Dr. Tuggle presented a short video that highlighted the SCID Pig Facility at Iowa State University. The video introduced the naturally occurring Severe Combined Immuno-deficient (SCID) pig that lacks an adaptive immune system and possesses organs with similar size, physiology and genetics as humans. The SCID pig has been proposed as an ideal model for human regenerative medicine, vaccine development, stem cell-based therapies and cancer drug development. SCID pigs must be maintained in specialized, sterile biocontainment facilities immediately upon birth and fed specialized diets free of disease-causing organisms. To minimize stress, which could affect research results, specially clothed technicians and scientists interact with pigs by touching, petting, and provide training with clickers and food rewards. To date, the SCID pig has been successfully engrafted with human cancer cell lines without rejection, and plans to engraft human cells into the bone marrow of these animals are underway in order to produce a pig with a humanized immune system. This unique animal model has potential to make a significant impact and researchers have been contacted by many collaborators interested in the use of the SCID pig in future translational research.

Skip Virgin (Washington University) Primate Infectious Disease Resource (PIDR) Dr. Virgin introduced the need for disease diagnosis in nonhuman primates within the U.S. National Primate Research Center network and the emerging capability to quickly and accurately identify infectious disease pathogens and variations in the microbiome, particularly within the gastrointestinal tract. In partnership with the Tulane National Primate Research Center, Dr. Virgin and colleagues have established the Primate Infectious Disease Resource and are currently establishing metagenomic profiles from specific pathogen-free, non-human primate breeding colonies and others to determine how changes in the microbiome lead to viral, bacterial, fungal and other potential pathogen-mediated gastrointestinal diseases. For example, Dr. Virgin reported that 30 new viral genomes were identified in their first study, and particular attention was paid to RNA-type viruses. Follow-up studies of these pathogens will include development of new methods of detection to aid veterinary professionals in diagnosis and treatment.

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Elinor Karlsson (Broad Institute, Inc.)

A Comprehensive Canine Genetic Resource Including Gene and Variation Annotation

The presentation emphasized that dogs are an ideal genetic model because many of the over 400 acknowledged dog breeds suffer similar diseases as humans, including heart disease, cancer, arthritis, obsessive compulsive disorder and others. Dogs are often treated with the same or highly similar drugs as humans. Complete genome sequencing of purebred breeds of dogs may reveal genotype/phenotype relationships in disease that are shared with humans. The basis of the Canine Genetic Resource is RNA sequencing data and 11 different primary cell cultures from 30 tissue samples of six dogs, euthanized for unrelated, humane medical reasons. These data will provide an extensive transcriptome resource of selected canine tissues. The goal is to provide the scientific community with an improved dog gene annotation resource and a catalyst for future comparative studies of disease in humans and dogs.

Steven Britton (University of Michigan)

Aerobic Rat Models to Resolve the Exercise-Health-Aging Connection

Dr. Britton noted the association between morbidity and mortality with low exercise capacity, and described an ongoing breeding program in the rat that produced animals with an approximate 8-fold difference in treadmill exercise capacity. Animals in the low capacity treadmill group exhibited increased body mass, while their high capacity treadmill counterparts demonstrated lower body mass. Animals in the former group also had higher risk factors for complex disease than the latter. Experiments with this rat resource have revealed differences in DNA repair between the groups and reduced AMPK activity in low capacity treadmill runners when compared to high capacity treadmill runners. This rat model continues to be developed for additional breeding generations for further genotype/phenotype analysis and new hypothesis driven research.

Harvey Blackburn (USDA, National Center for Genetic Resources Preservation) Resource Population Security with the National Germplasm Program. Dr. Blackburn described the facilities at the National Center for Genetic Resources at Fort Collins, CO that acquires, evaluates and preserves genetic materials from plants, animals, microbes, aquatic organisms and insects. In case of national emergency, the Center employs cryogenic preservation to safeguard the world’s largest genetic collection of animal genetic resources, including 900,000 samples from 35,000 animals. Development of the resource was started in 1999 but some samples were originally collected about 58 years ago; the resource is used every day by researchers. Extreme care is taken to ensure the integrity of all collections and material transfer agreements must traverse bureaucratic procedures before access to any part of a collection is granted. When asked, Dr. Blackburn stated that the U.S. has by far the largest collection of species followed by Canada, the Netherlands, and France. With respect to experimental genetic model organisms, Dr. Blackburn noted that C. elegans was one of the first to be added. To ensure preservation of all samples, Fort Collins, CO is ideal for this resource, as it has the ability to withstand forces of nature and has the capacity to securely hold up to 20 million samples. Databases that catalog the source, identity and location of each sample are also housed in the facility. Dr. Blackburn invited attendees interested in preserving genetic research resources to contact him.

Randal Voss (University of Kentucky) Ambystoma Genetic Stock Center Dr. Voss presented a short video tour of the Ambystoma Genetic Stock Center at the University of

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Kentucky. The Center provides researchers with embryos, larvae and adult axolotls, also known at the Mexican salamander (Ambystoma mexicanum) that possess unrivaled regenerative capacity of limbs and other critical tissues. The axolotl thrives in captivity and unlike other salamanders, breeds multiple times a year. This unique animal exhibits neoteny - it retains gills and other aquatic structures into adulthood. The colony housed in the Ambystoma Genetic Stock Center originated in the 1930’s and is genetically homogenous. Strict quality control procedures ensure that the health of the collections is maintained at all times.

Session 5: Genome Editing: Technical and Community Lessons Learned Moderators: Sige Zou (ORIP) and Eugene Koonin (NLM)

Eugene Koonin (NLM)

From Bacterial Adaptive Immunity to a New Generation of Genome Editing Tools Dr. Koonin reviewed the mechanisms of Type II CRISPR/Cas systems found in bacteria and archaea. The CRISPR/Cas system is a form of adaptive immunity in microorganisms that can be used by scientists to edit genes. A number of CRISPR/Cas systems have been identified but Dr. Koonin predicted that many more are undiscovered. Type II CRISPR/Cas9 is one of the most common Class 2 CRISPR/Cas gene editing systems used today in research studies, but it is not the simplest. The Type V-A CRISPR/Cpf1 effector, first described in 2015, is a single RNA-guided endonuclease that requires one RNA molecule to initiate DNA cutting. In contrast, Cas9 requires two RNA molecules. Another difference between Cpf1 and Cas9 is the manner in which each cuts DNA: Cpf1 cuts DNA in two different locations, leaving uneven strands, while Cas9 cuts DNA evenly at the same location, leaving blunt ends. This difference may allow easier, more efficient DNA insertion into genes during gene editing experiments.

Another alternative to the Type II CRISPR/Cas system is the Type VI C2c2 effector that targets and degrades invading RNA-based viral pathogens in bacteria. Dr. Koonin emphasized that the C2c2 effector is likely involved in cell dormancy and programmed cell death that provides the microorganism with time to develop adaptive immunity or to prevent the spread of infection, respectively. Some uses of C2c2-based RNA editing include cleavage of specific RNA sequences to reduce the amount of translated protein in a cell or insertion of fluorescent tags for RNA imaging studies in cells.

Sige Zou (ORIP)

ORIP-Funded Gene Editing (CRISPR/Cas9) Projects in Diverse Model Organisms

Dr. Zou highlighted the opportunities and challenges of CRISPR/Cas-based gene editing technologies. Overall, CRISPR/Cas9 and related techniques have given scientists unprecedented tools to create precision genetic modifications in diverse species. Researchers face challenges, however, in genome editing efficiency, accuracy and versatility. Large genes, genes with several genomic loci, and differences between species can affect the ability of CRISPR/Cas9 systems to produce genetic changes as designed. The ORIP is interested in the development of the CRISPR/Cas9 technology, particularly as it applies to the predictive value of precision animal models of human disease. To address this goal, 12 administrative supplements to currently funded projects were awarded involving Drosophila, Xenopus, fish, mice, rats, pigs and non-human primates. These projects span a number of human conditions, including inflammatory bowel disease, Fanconi anemia, cystic fibrosis, Parkinson’s disease, blood disorders and HIV-1 infection. Additionally, one funded project is testing Cas9 variants in non-human primates.

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Norbert Perrimon (Department of Genetics, Harvard Medical School) Impact of Genome Editing on LOF and GOF Screens in Drosophila Drosophila melanogaster is a highly useful, well-established genetic model for genome-wide functional studies. Dr. Perrimon noted that extensive collections of transgenic RNAi have been created to suppress gene expression in Drosophila. Available RNAi lines cover a majority of the animal’s 14,000 genes. Dr. Perrimon directs the highly successful Transgenic RNAi Project that has generated more than 10,000 gene silencing RNAi fly stocks for the research community. However, there are no equivalent sets of gene activation lines. With modifications to CRISPR-based gene editing, it is now possible to overexpress target genes using a crippled Cas9 nuclease (dCas9) fused to an activator domain (dCas9a; CRISPRa). Gene overexpression can reveal dramatic phenotypes and is useful for determining the function of redundant genes. It can also determine if one gene has an effect over another gene. Dr. Perrimon advocated the need for a genome-wide resource of gene overexpression in Drosophila.

Until very recently, endogenous gene activation using CRISPR was limited to cell culture; however, researcher’s in Dr. Perrimon’s group recently demonstrated that dCas9 in vivo can efficiently be used for gene over-expression in Drosophila. His group also investigated which activation domains worked best to induce gene over-expression in various cell types and species, including human. No one winner emerged, but SAM (synergistic activation mediator) and VPR (activation domains VP64, P65 and Rta) usually worked best. To generate over-expression of a protein, Dr. Perrimon described the requirement for guided RNAs to keep overexpression in appropriate physiological concentrations. Dr. Perrimon also noted the extraordinary versatility of CRISPR to not only direct gene over-expression but to also induce transcriptional repression. In this case, dCas9 is modified to include a repressor domain (dCas9i; CRISPRi) to cause knockdown phenotypes or in some experiments, lethality. It is not yet clear whether CRISPRi-mediated repression is stronger than RNAi in these conditions or if off target effects exist.

Marko Horb (Marine Biological Laboratory)

Expanding the Genetic Toolkit in Xenopus: Approaches and Opportunities for Human

Disease Modeling.

Dr. Horb introduced two species belonging to the amphibian genus Xenopus used extensively in biomedical research, Xenopus laevis and Xenopus tropicalis. Both species serve as models of development, regeneration and cell biology. Recently developed gene editing tools have created better opportunities for genetic studies in these animals. Dr. Horb directs the National Xenopus Resource (NXR) at the Marine Biological Laboratory (Woods Hole, MA) which serves as a stock center of inbred, transgenic and mutant Xenopus lines and creates transgenic and mutant Xenopus for the research community using CRISPR/Cas and TALEN-based gene editing tools. NXR scientists use oocyte-host transfer techniques to produce mutant lines more rapidly and efficiently than more traditional embryonic stem cell methodologies. Oocyte-host transfer also reduces the number of mosaic F0 animals and is better at transmitting mutations to the germ line. The NXR also provides advanced training in bioinformatics, imaging, genome editing and other specialized techniques. Perhaps uniquely, the NXR offers a research hotel service that provides scientists with housing during short-term visits to train, teach or use NXR resources for their research. Dr. Horb asked participants at the conference for suggestions for scientists facing reimbursement difficulties who wish to stay at the NXR research hotel to conduct research.

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Researchers in Dr. Horb’s laboratory recently discovered that DNA cutting with single guide RNAs (sgRNAs) and the commonly available S. pyogenes-derived Cas9 reagent was far less efficient in X. laevis than in X. tropicalis. Dr. Horb noted that X. laevis animals are maintained in water that is generally 3-80C colder than the temperature of water used to house X. tropicalis. His group determined that Cas9 is temperature dependent and has begun the search for Cas9 alternatives in psychrophilic (cold-adapted) bacteria. Other projects in the laboratory include the development of homozygous CRISPR/Cas mutants using euploid cell lines from neural stage embryos and somatic cell nuclear transfer. This approach involves transplantation of germ cell vegetal poles from later-stage (blastula) embryos injected with Cas9 and sgRNAs into wild-type embryos containing only soma (missing their own vegetal poles). Germ line graft-recipient embryos with CRISPR/Cas-induced mutations most often develop to adulthood with null mutations in place. Dr. Horb also mentioned recent work in which CRISPR/Cas is being used to create libraries of sgRNAs from genomic DNA or cDNA using newer CRISPR EATING (Everything Available Turned Into New Guides) techniques. The idea is to use multiple sgRNAs to target larger genomic regions that contain multi-exon genes. CRISPR EATING can be used to label these regions with fluorescent markers in order to visualize genomic regions during the cell cycle. Additionally, adaptations to the technique have the potential to generate large, regional mutations in genes that span hundreds of kilobases of DNA.

Steve Murray (The Jackson Laboratory)

High-Throughput Application of CRISPR: the Komp2 Experience Dr. Murray discussed the challenges faced in adapting CRISPR technology to high-throughput phenotyping screens. Dr. Murray described the current Knockout Mouse Phenotyping Program (KOMP2) at The Jackson Laboratory in Bar Harbor, MA, that is slated to produce 5500 knockout mouse lines by 2021. The goal is to build a comprehensive functional encyclopedia or catalogue of a mammalian genome through systematic phenotyping of knockout mice, standardized on a uniform C57BL/6N genetic background with defined, validated alleles. CRISPR-based mutagenesis is supplementing (and potentially replacing) embryonic stem cell (ES) line methodologies to produce knockout mice, resulting in improved consistency, speed and cost of the process. Dr. Murray also noted that the typical success rate of producing knockout mouse lines using CRISPR is greater than more traditional ES techniques. With respect to allele type in knockout mice for the KOMP2 program, the decision was made to use simple alleles to maximize genome coverage (at lower cost) and cryopreserve cells with complex, multipurpose alleles for future use by researchers. CRISPR-generated lines and phenotypic data are made immediately available to researchers as they come on line. Dr. Murray emphasized that CRISPR-based mutagenesis to produce knockout mouse lines has increased at a phenomenal rate with low failure rates. A CRISPR Knockout pipeline web based form has been created to assist in batch guide design based on the identity of Ensembl Exon IDs targeted for deletion. The project is currently refining methodologies to record data generated from batch guides and produce a guide summary using feedback from users.

One concern voiced by Dr. Murray is the potential for off target mutations using CRISPR technologies to produce knockout mice. However, he reported that no off-target mutations have been found among the 207 N1 mice generated to date. The project is now looking at optimizing complex allele generation with the goal to improve efficiency and lower the cost of production. The project is also exploring zygote electroporation as an alternative to direct microinjection of CRISPR mutagenesis reagents. In the future, emerging methodologies to induce mutagenesis (e.g., CRISPR/Cpf1 vs. CRISPR/Cas9) may be explored.

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Randall Prather (University of Missouri)

CRISPR Bacon: Knockouts Made Easy – A Sizzling Story! Dr. Prather began his talk by emphasizing the usefulness of pigs as biomedical models of human health and disease and described the functions and services offered by the National Swine Resource and Research Center at the University of Missouri. Among many projects, Dr. Prather noted that the Center produced the first pigs with modified genomes by CRISPR/Cas in early 2014 and currently offers 45 different genetic modifications of swine in support of research projects in cancer, cardiovascular disease, diabetes, regenerative medicine and other fields.

Dr. Prather also described recent experiments designed to determine if injection with CRISPR/Cas would affect in vitro development of swine zygotes. Three guide RNAs were designed to remove 58, 115 or 209 base pairs in the start codon of exon 2 in the TMPRSS2 gene. This gene encodes a protein that belongs to the serine protease family; and that is involved in prostate carcinogenesis. Results using combinations of the guide RNAs demonstrated that the CRISPR/Cas system did not affect the sex ratio of the blastocysts or the rate of their development at days 5, 6 or 7, when compared to non-injected controls. Genotyping of the TMPRSS2 gene in piglets produced from TMPRSS2-modified embryos demonstrated that the CRISPR/Cas system efficiently introduced multiple types of editing-induced errors ranging from 1 to 875 base pairs. There were far more deletions than insertions. As expected, different modifications appeared on each allele of the TMPRSS2 gene in CRISPR/Cas modified pigs and two animals carried to gestation were determined to be mosaics. Although Dr. Prather acknowledged that editing using the CRISPR/Cas system does not always produce desirable results, this technology allows us to modify DNA at lightning speed to answer basic and applied questions about human medicine and domestic animal biology. He conveyed confidence that any genetic modification imagined can most likely be produced at the National Swine Resource and Research Center.

Session 6: Poster Session There were 41 resource- and research-related posters presented and discussed during the session.

Session 7: Microbiota and Beyond: Opportunities and Concerns for Animal Resources Moderators: Manuel Moro (ORIP) and Maria Giovanni (NIAID)

Karen Nelson (J. Craig Venter Institute)

What are We Learning from the Microbiome in Health and Disease? Dr. Nelson and her colleagues published the first human metagenomics study in 2006 using whole genome shotgun sequencing to characterize human intestinal microbiota. Shotgun sequencing breaks up long strands of DNA into smaller, more manageable fragments. Once sequenced, computer programs reassemble fragments by identifying overlaps. Dr. Nelson and her colleagues analyzed unique DNA sequences and 16S ribosomal DNA sequences from fecal samples of healthy adults. Their results identified which microorganisms were present and the nature of metabolic pathways in use. Importantly, their study emphasized host plus microorganism contributions to metabolic functions in the gastrointestinal tract of healthy adults.

While whole genome shotgun sequencing eliminates the need to cultivate individual microorganisms and is highly effective at sequencing the genomes of many organisms, recent next

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generation sequencing (NGS), also known as high throughput, multiplex sequencing, allows simultaneous, automated sequencing in a fraction of the time and at significantly lower cost. The result is vast amounts of data that requires sophisticated bioinformatics approaches to ensure quality control and statistical analysis. Nonetheless, NGS is the basis for the NIH Roadmap (Common Fund) Human Microbiome Project to study whether changes in the microbiome are related to human health and disease. Dr. Nelson explained that the goals of the Human Microbiome Project were to:

Develop a reference set of up to 3000 bacterial genomes and a number of non-bacterial microorganisms (e.g., fungi, viruses, phage).

Conduct metagenomic analysis of microbial communities at individual body sites (e.g., skin, oral cavity, naso-pharyngeal tract, gastrointestinal tract, and female urogenital tracts) in healthy tissues.

Develop new tools for computational analysis, standard operating procedures, reagents and publically available data sets for further study.

Dr. Nelson emphasized that studies designed to provide deeper coverage of the microbiome in the distal gut microbiome are now possible. She referenced recent studies that found distinct microbial communities among body locations that are commonly shared among healthy adults, regardless of sex, age, weight, ethnicity or race. Further, differences in gut microbiomes found between BaAka hunter-gatherer-based and Bantu agricultural-based populations in Central Africa differ considerably from those of ‘Western’ populations. Future investigations that obtain periodic sampling from these and additional populations may reveal how environmental factors alter microbiomes over time. Similarly, longitudinal studies that characterize microbiome changes during life are likely to provide new insights about gastrointestinal health and the emergence and spread of multi-drug, antibiotic resistant bacteria in the elderly.

Craig L. Franklin (University of Missouri)

Variables that Influence Microbiota and Modulate Phenotypes

Dr. Franklin directs the Mutant Mouse Resource and Research Center (MMRRC) and is a Co-Investigator in the Rat Resource and Research Center (RRRC) at the University of Missouri. These Centers archive and distribute mutant mouse and rat strains and perform a host of services that support the biomedical community. Dr. Franklin began his talk by posing the important question, “How are models distributed [to the biomedical community] influenced by differing gut microbiota (GM)?” He explained that although the scientific literature is exploding with studies that link differing microbiota with health and disease, only a few studies have catalogued the extent of microbiota diversity in colonies of animal models. Researchers at the MMRRC and RRRC are closely examining factors that cause (or correlate with) variations in GM and how these variations affect phenotypes of animal models.

Previous research by the MMRRC/RRRC team determined that the source, age and strain affect the composition of GM in laboratory mice and rats. As resources, the MMRRC and RRRC have also examined variables that can be controlled such as diet, bedding, housing and other factors related to animal husbandry. One study examined the influence of 3 different diets (non-autoclaved, low fat/irradiated, high fat/irradiated), 2 types of cage bedding (paper chip, aspen), and two types of housing racks (static, ventilated) on fecal and cecal microbiota in groups of 12 mice. Although fecal microbiota of all animals appeared similar, the profiles of cecal microbiota differed greatly.

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Over the long term, differing diet showed negligible changes in microbiota. Housing in static microisolators on aspen bedding, however, created unique cecal microbiota profiles in mice. In another experiment, it was determined that the composition and diversity of GM changes with shipping of mice, leading to the question of whether the GM of mice distributed to different facilities would remain similar to or diverge from pre-shipment GM. It was found that GM populations diverged when mice were housed in different facilities within a single institution although all husbandry variables were constant. To address the question of whether variation in GM alters animal model phenotypes, Dr. Franklin described experiments in which an inbred strain of mice that develops inflammatory bowel disease was rederived by embryo transfer onto mice from 3 different vendors that harbored GM of differing composition and diversity. Results demonstrated that differences in GM were associated with differing lesion scores. In similarly designed rat studies, differences in naturally occurring GM were associated with differences in severity and incidence of experimental colorectal cancer.

Dr. Franklin then posed additional unanswered questions related to the ability of any mouse or rat resource to establish, maintain and rescue GM in mice and rats for research at the same or other facilities. He proposed that one or more workshops should be considered in the near future to determine how the research resource community should take into consideration variability in GM of contemporary rodent colonies. Topics could include reporting of microbiota in manuscripts, standardizing microbiota and development of novel tools to rescue or modulate microbiota. He expressed his own feelings that reporting microbiota profiles would be beneficial and may become commonplace as the costs of analysis continue to become more affordable. Following his talk, there was a brief discussion on how and when to determine if variation in gut microflora impacts results or if it is simply “noise.” Suggestions included looking at each individual animal’s tumor load, for example, and correlating that with their GM. If a microbe or microbial profile is suspected of influencing phenotype, findings can be interpreted in the context of more classic experiments (e.g., using antibiotics, gnotobiotics etc.) to ultimately understand the role of individual bacteria in the context of a complex microbiota. The panelists also emphasized the need to embrace complexity as another tool in understanding the role of the GM as it ultimately represents what we have in the human population.

Skip Virgin (PIDR, Washington University) Metagenomics as a Basis for Experimental Research Dr. Virgin and his colleagues are interested in trans-kingdom interactions among viruses, intestinal microbiota, and host immune systems. He defined the microbiome as the sum of all organisms that live in or on a host, including bacteria, viruses, fungi, meiofauna and other microorganisms. He defined the metagenome as the sum of all host and microbiome genes that may influence the health status of the host. The virome, defined as the collection of viruses in an individual’s body, is a major contributor to our metagenome. It has only recently been possible, however, to ‘see’ the virome using bioinformatics tools. There is clear need for, and use of, a viromics-dedicated bioinformatics/computational platform that takes into account RNA as well as DNA with the capability to analyze sequences by taxonomy.

The recognition that the gut microbiome affects the host immune system and influences enteric viral infection, replication and pathogenesis has impacted the direction of recent studies of intestinal disease. Viral infection or transcriptional activation of latent viral sequences triggers the production of anti-viral soluble factors (e.g., interferons, cytokines, antibodies) and invokes cell-

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based mechanisms that eliminate infected cells. Regulatory mechanisms of the immune system are also invoked to spare uninfected cells and tissues of the host; however, the cumulative effects of a potent antiviral response is often inflammation. Chronic inflammation is associated with a number of intestinal illnesses including inflammatory bowel disease and colorectal cancer. Dr. Virgin reviewed important new discoveries and emerging concepts that are shaping our understanding of microbiome and virome relationships within the contexts of immunity and disease. Three of the concepts highlighted by Dr. Virgin are:

Viruses in the systemic circulation can be mutualistic symbionts. Dr. Virgin presented data demonstrating that chronic infection with a type of herpesvirus in mice infected with lethal Listeria monocytogenes could be protective. Similarly, chronic herpesvirus infection provoked production of interferon-γ and reduced tumor burdens in animal models of transplanted tumors.

The virome can interact with or without the microbiome and host genetic variation to alter host phenotypes. Dr. Virgin explained that bacteria can control persistent enteric norovirus infection in a manner dependent on the interferon-lambda receptor. This indicated that transkingdom interactions (in this case between bacteria and a virus) can control the outcome of infection. .

Chronic and acute infections in laboratory mice changed gene expression profiles (compared to control, non-infected mice) and altered immune responses to vaccination against yellow fever. This insight may provide important clues as to why vaccinations against human are sometimes more effective in some regions of the world compared to others. Also, these experiments may help researchers create animal models that are better predictors of how vaccines will work in people.

Balfour Sartor (National Gnotobiotic Rodent Resource Center, UNC-Chapel Hill) Using Gnotobiotic Mice to Address Intestinal Bacterial Function Dr. Sartor is the Director of the National Gnotobiotic Rodent Resource Center. The Center provides germ-free and gnotobiotic mice to the research community. Germ-free mice are raised from the moment of birth in sterile environments and are free of all microorganisms. Gnotobiotic mice are raised in aseptic conditions and are colonized with known microorganisms. These animals, with or without selected genetic mutations, can be used to study host-microbiota relationships and have been used extensively in studies modeling inflammatory bowel disease and colorectal cancer.

Dr. Sartor’s research interests lie in the connections between commensal bacteria and diseases of the intestinal tract. He stated that while many studies have examined changes in bacterial profiles of the gut in health and disease, he is interested in the unique functions of species and strains of bacteria in the gut and how they affect the immune system. For example, Dr. Sartor explained that while germ-free, HLA-B27 rats do not develop inflammation in a model of colitis, colonization of the gut could produce mild to severe inflammation depending on the bacterial source introduced. In certain strains of mice, genetic deficiency of the anti-inflammatory cytokine Interleukin-10 (IL-10) leads to predictable gut inflammation. Colonization of IL-10 knockout mice with different, single strains of E. coli induced variable degrees of colitis. Alternatively, co-colonization of the gut with certain species of Lactobacillus can protect against E. coli-induced inflammation in this model. On the other hand, further research has demonstrated that the role of IL-10 in immune responses to commensal bacteria is not uniform: Certain bacteria in the class Clostridia can prevent and treat experimental colitis induced by human bacteria from feces or simplified human microbiota (SIHUMI) consortium sources.

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Dr. Sartor also described experiments that revealed the differential capability of Enterococcus faecalis and E. coli NC101 to induce colitis and tumorigenesis in animal models (both can induce colitis but only E. coli can induce colorectal cancer). E. coli produces a recently identified genotoxin, colibactin, encoded within the pks genomic island that can induce chronic DNA damage and modulate immune responses. Deletion of the pks island in E. coli reduces the tumorigenic potential of colibactin but does not affect the development of inflammation in murine models.

These and other studies have led researchers to appreciate that there are interactions between the host and commensal microorganisms and that each individual responds differently to the types and numbers of microbiota populations present. The search is ongoing for additional commensal protective bacterial species that may protect against causal agents of inflammatory bowel disease and colorectal cancer in animal models and humans.

Manuel Moro (ORIP) and Maria Giovanni (NIAID) Questions / Discussions for Session 7 Discussions following this session focused on the problem of reproducibility of similar or identical metagenome studies. For example, Dr. Nelson mentioned the very different results obtained from two separate metagenomic profiles performed on the same fecal sample by two independent laboratories. She noted that it is difficult to control how studies are performed and problems with contamination of microbes originating from reagents and laboratory personnel have encouraged efforts to standardize methodologies and quality control. Dr. Virgin mentioned that array-based science was often criticized until standardized platforms and procedures were adopted. He pointed out that virologists must recognize that they carry bacterial contaminants with them and efforts must be stringent to reduce compromised data. Other questions from the audience focused on the contribution of the gut microbiome to energy consumption (about 30%). The point was made that germ-free animals must eat considerably more than their routinely housed, laboratory counterparts or pet store mice, highlighting the contribution of the microbiome in energy autophagy regulation. Finally, the subject of the priobiome was brought up in the context of the potential presence of prions in brain or cerebral fluid; however, none of the speakers had experience in this field to comment.

Session 8: New NIH Policies and Good Resources Practices Moderators: Jack Harding (ORIP) and Matt Jorgensen (Wake Forest School of Medicine)

Jennifer Plank-Bazinet (ORWH)

New NIH Policies: Rigor and Transparency: Guidelines and Early Lessons Learned and

Studying Both Sexes in NIH-Funded Research The ability to understand the mechanisms responsible for health and disease depends on good study design, adequate data collection, and unbiased analysis of the results. Historically, science has been viewed as relatively self-correcting: new studies are published, interested scientists repeat them with or without small changes to test their own hypotheses, and the original results are either confirmed or refuted. Dr. Plank-Bazinet emphasized that while reproducibility is an unwavering tenet of the scientific method, a number of contemporary factors impede scientific self-correction:

Incomplete reporting of materials and resources Unexpected variability of materials or resources Poor training in study design and statistical analysis

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A reward system (e.g., promotion, grant application success) for high profile publications

Low recognition for publication of ‘negative’ results A hyper-competitive environment Historically low funding rates

Dr. Plank-Bazinet conveyed the goal of the NIH to demonstrate to its public stakeholders its commitment to support high quality, unbiased biomedical studies that provide the research community with thorough methodologies and readily identifiable resources. These updates are required for most research program grants and mentored career development awards to ensure that NIH-supported research meets the highest standards of rigor and transparency. Applicants will be asked to improve the manner in which they describe their proposed research, and reviewers will be asked to evaluate the quality of information provided to them in four key areas:

The scientific premise of the proposed research The scientific rigor of the experimental design, statistical analysis and transparency of

reporting positive and negative findings Consideration of biological variables, such as sex, gender, age, weight, and underlying

health conditions Authentication of key biological and / or chemical resources (e.g., cell lines,

antibodies, specialty chemicals, and other biologics)

Dr. Plank-Bazinet explained that the scientific premise should be presented in terms of the strengths and weaknesses of published studies used to support the application. The rigor of previous experimental designs, inclusion of appropriate statistical tests, incorporation of relevant biological variables, and authentication of key resources should be noted. She also noted that problems with previous resources that impeded reproducibility should be mentioned.

The proposed scientific design may be based on new observations or innovative, unproven ideas. Applicants should thoroughly explain how the proposed experimental design and methods could achieve robust and unbiased results. Appropriate statistical design, power analyses and randomization protocols should be included, including how outliers will be handled. The sources of reagents and materials should be provided to ensure that the research community will be able to reproduce and extend the proposed study, when appropriate. Grant applications should also describe how key biological and / or chemical resources will be regularly authenticated to ensure identity and validity.

Dr. Plank-Bazinet noted that the NIH recognizes that sex and gender may influence results in pre-clinical and clinical studies and that an imbalance has existed historically in the choice of sex in cell-, animal- and human-based studies. NIH policy changes require that sex be considered as a biological variable. The NIH policy changes do not require scientists to carry out “sex differences research.” Any research design proposing to study only one sex, however, must provide adequate justification. If both sexes will be studied, analysis of preclinical data by sex should be carried out when scientifically appropriate.

Dr. Plank-Bazinet also reminded conference participants that these updated requirements need to be incorporated into existing page limits. The scientific premise, scientific rigor, and consideration of biological variables are expected in the research design section of the application. Authentication of

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key biological and /or chemical resources will be requested in a separate form that will not be scored, but deficiencies will be discussed during peer review and addressed with program officials prior to award. The NIH Office of Extramural Research (grant.nih.gov/reproducibility/index.htm) and the NIH Office of Research on Women’s Health (orwh.od.nih.gov) includes this and additional information on their web sites.

Zoltan Varga (Zebrafish International Resource Center) Good Resource Practices: Disaster Planning, Emergency Management, Continuation of Operations, and Backing up of Resources at the Zebrafish International Resource Center The Zebrafish International Resource Center (ZIRC) is located at the University of Oregon (Eugene, OR). It serves the biomedical community as a central repository and distribution center for wild-type, transgenic, and mutant strains of zebrafish (Danio rerio), antibodies, gene probes and other markers for research. Other services include a veterinary fish health service and training in health, husbandry and cryopreservation of zebrafish sperm.

Dr. Varga described the aftermath of a fire that occurred at the ZIRC in May 2014. While the fire directly damaged research space only in one section of the facility, smoke, soot and ash contamination was extensive and affected the entire building. Operations were shut down immediately. Recovery and return to most normal operations took 9 months, however, resource imports were resumed only after 18 months. The University of Oregon lies in an area of the U.S. with high potential for seismic activity and is affiliated with the Disaster Resilient Universities Network, which fosters open communication, discussion, and resource sharing between emergency management practitioners at member universities. Dr. Varga noted that the ZIRC possessed a good relationship with emergency management officials at the University and a disaster/ emergency management plan had been created in 2009, 5 years before the fire and had been reviewed annually and improved.

At the time of the fire, Dr. Varga stated that the ZIRC was able to draw directly from their Emergency Management (EM) and Continuation of Operation (CoOp) plans to ensure the safety of personnel and animals, minimize and manage losses, rebuild damaged facilities, restore lost resources and when possible, resume normal operations. He emphasized the usefulness of the widely distributed Response Plan, which included a communication chain, a staff emergency response team, and succession of authority scheme. The success of the Response Plan was evident in the quick and efficient emergency response activities during and immediately after the fire was discovered. For example, personnel at the facility and emergency responders knew the locations of assembly points, emergency and first aid kits, and whom to contact for updated information in the event that one or more key personnel were missing or otherwise out of communication. The follow-up Continuity Plan was followed step by step in the aftermath of the fire, ensuring that critical functions and operations at the ZIRC were restored in priority order. For example, the Continuity Plan included instructions for the care and welfare of surviving animals (e.g., how quickly water flow needed to be restored to tanks, how often fish needed food, etc.) and provided coping, “what if” strategies and alternative sources of critical materials (e.g., if nitrogen supply for the cryogenic resource repository was lost, who was the vendor and was there another source?). The Continuity plan included copies of critical supplier standard operating procedures, and other protocols and was readily available for all personnel engaged in any function of the ZIRC.

Dr. Varga also discussed the Prevention Plan that includes action items generated by regular staff discussions, annual, routine Fire Safety Inspections, and Laboratory Safety Audits of the ZIRC

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http:orwh.od.nih.gov

  

building. He also emphasized that the success of the collective EM / CoOp plan lies in its availability to all personnel: it is available in digital form, as a mobile app, as well as traditional hard copy. Dr. Varga explained that out of state wireless and landline telephone contacts should be maintained and updated regularly, in the event that cell towers or landlines are out of operation during an emergency. He noted that fire departments and first responders need to be informed of the ZIRC’s chemical storage and inventory. Finally, Dr. Varga highlighted the importance of one or more offsite, back up repositories for the ZIRC (and similar resources). He noted that the National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, CO has preserved 2,335 samples (representing 1,292 alleles) of genetic material from the ZIRC and continues to receive regular shipments. While the NCGRP is considered to be one of the most secure locations in the U.S., Dr. Varga advocated during the discussion period - after his talk - that a “Plan C” may also be necessary to preserve valuable genetic resources, perhaps in a backup repository in another country. Mirror sites for data storage are critical for good backup plans, as is the “Cloud” and hard copy storage locations off site. He also strongly suggested that any plan, even a minimal one, will be helpful and should be in place before a disaster occurs. It can be revisited, expanded, and improved regularly or when possible to update information and refine procedures.

Melween Martinez (Caribbean Primate Research Center) Good Resource Practices: A Lesson Learned From Monkeys’ Escape In early summer 2015 a group of non-human primates left their housing through an undetected opening in a fence of the Caribbean Primate Research Center (CPRC), University of Puerto Rico. Dr. Martinez, Director of the CPRC, described the aftermath of the incident. Initially, a security officer noticed the breech and immediately called the Director of Security, per protocol. The local authorities were informed and response teams were mobilized. While a number of animals escaped, all but one animal was recovered (or returned on their own) within 24 hours. Unfortunately, news of the animals’ escape reached the local press and details of the situation were quickly exaggerated and sensationalized. One newspaper article warned the local population of the animals’ escape but provided photographs of a completely different species of non-human primate. Many newspaper articles warned of the imminent danger from the escaped animals, erroneously stating that the CPRC had injected them with dangerous infectious agents. Dr. Martinez quickly arranged a press conference with the media accompanied by a CPRC veterinarian. She was also joined by the Mayor to assure everyone that the escaped animals were not infectious and that many had returned on their own within hours of their initial escape. She reminded the press about the mission of the CPRC, its importance to the U.S. and international research community, and its economic impact in terms of local jobs. She and the Security Office reviewed the security measures in place and how CPRC and local officials handled the response to the animals’ escape. Fortunately, the CPRC had in place a response plan that was followed by all personnel, including a phone tree that spelled out whom to call following the discovery of an animal housing breech. The phone tree included CPRC officials, University officials, local law and emergency organizations, the IACUC chair/members, and internal and external press offices.

While dealing with the press, Dr. Martinez was forthright in how the response plan had worked and had not worked. She emphasized to the audience that personnel (at the animal resource facility) should never talk to the press on their own, but if contacted should pleasantly connect any one asking questions with the facility’s press officer. She emphasized it is important not be scared of the press, but to be sure that you have accurate, timely information that can be disseminated in clear language. Dr. Martinez also warned that any communications with the press must be truthful and

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transparent or trust with the local community could be lost. During discussions following her talk, Dr. Martinez agreed with participants that the establishment of a friendly, long-term relationship with the press before a problem occurs is critically important.

Stephanie Murphy (Director, DCM, ORIP) Concluding Remarks Dr. Murphy provided concluding remarks which included the following key themes, challenges, and opportunities that had emerged and had been discussed during the meeting:

Reproducibility of animal models Phenotype gaps Co-clinical modeling Complexity versus precision medicine Impact of genome editing Microbiota Protecting resources

Based on speaker presentations and attendee discussions at the Eleventh Comparative Medicine Resource Directors Meeting, the following next steps were suggested:

Explore further the concept of “microbiota” and its impact on animals models and research with animal models through a workshop or conference

Examine further what types of training will be needed relative to development and maintenance of resources as well as what type of training can be offered by resources

Consider what the future informatics needs will be for resources and how resources will handle the current deluge of data

Develop strategies that would allow resources to move towards having animal models more in parallel with patient management and treatment (co-clinical modeling)

Define what types of quality assurances (i.e. genetic, phenotypic, microbiotic, etc.) should be developed for resources

Consider other venues in Bethesda for future meetings that would facilitate more group interaction and discussion

Evaluation forms were provided to the participants, and feedback will be considered in planning the 2018 meeting. Participants were told that additional feedback could be communicated to Dr. Murphy, Director of DCM.

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