INFRAFRONTIER / IMPC Stakeholder Meeting 2017

Athens, 14 - 16 November 2017   -   Royal Olympic Hotel

"Advancing Personalised Medicine with Animal Models"

 

This was the first stakeholder meeting of INFRAFRONTIER, the European Research Infrastructure for phenotyping and archiving of model mammalian genomes. The meeting was jointly organized with the International Mouse Phenotyping Consortium (IMPC, www.mousephenotype.org) to which INFRAFRONTIER is contributing. Focus of this meeting was on ‘Advancing Personalised Medicine with Animal Models’.

The meeting was open to a wide range of INFRAFRONTIER stakeholders including Personalised Medicine initiatives, Rare Disease networks, funders, regulators and the INFRAFRONTIER user community to discuss advances in CRISPR/Cas9 technology to model human conditions. 

Programme in a nutshell

The Stakeholder Meeting was structured into 3 main themes:

1) Advancing Personalised Medicine with Animal Models (14 November)

  • CRISPR/Cas9 based approaches to model human conditions
  • Use cases for the utility of animal models for identifying targets for precision therapies

2) IMPC Open Annual Meeting (15 November)

  • IMPC CRISPR/Cas9 technology updates, and assessments of phenotyping pipeline, tests in development and new horizons
  • IMPC data analysis and outreach

3) Responsible Research (16 November)

  • Editing mammalian genomes: Ethical considerations
  • Reproducibility in experimental animal research

Meeting aims were to ...

  • ... raise awareness of INFRAFRONTIER / IMPC platforms among current Personalised Medicine initiatives, funders and policy makers
  • ... present use cases for the utility of animal models for identifying targets for precision therapies
  • ... share advances in CRISPR/Cas9 technology to model human conditions
  • ... strengthen interactions with Personalised Medicine initiatives and Rare Disease consortia

Advancing Personalised Medicine with Animal Models  

Personalised Medicine “refers to a medical model using

Characterisation of individuals’ phenotypes and genotypes

(e.g. molecular profiling, medical imaging, lifestyle data)

for tailoring the right therapeutic strategy for the right person at the right time,

and/or to determine the predisposition to disease

and/or to deliver timely and targeted prevention".

According to: Horizon 2020 and European Council Conclusions on personalised medicine for patients (2015/C 421/03) 

Despite recent successes in identifying causative mutations for human heritable diseases through the use of sequencing technologies, an associated gene has not been identified for approximately half of the reported diseases. Discovery of the genotype-phenotype relationships is a critical step towards understanding of the mechanism of these diseases and the development of new treatments. Kent Lloyd et al. highlighted four areas of animal-based research that are key for the succesful deployment of Personalised Medicine initiatives:

  • Gene Variant interpretation
  • Incorporating ‘–omic’ data
  • Environmental exposures
  • Integrative in vivo modelling

Lloyd et al., (2016) Animal-based studies will be essential for precision medicine. Science Translational Medicine, Vol 8 Issue 352, 17 August 2016

 

INFRAFRONTIER www.infrafrontier.eu and the International Mouse Phenotyping Consortium (IMPC) www.mousephenotype.org offer unique platforms for the functional validation of genetic variants identified in exome/whole-genome sequencing approaches and the development of mouse models with predictive utility for efficient translation. Generation of precision models is key to the development of new therapies for rare disease.

IMPC is creating a genome- and phenome-wide catalogue of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. Personalised Medicine initiatives will benefit greatly from this emerging data and biological resources which can be used to detect novel genotype-to-phenotype associations in diseases. Furthermore, new genome-editing technologies such as CRISPR/Cas9 now enable the efficient derivation of precision disease models incorporating patient-specific genetic variants as a means of recapitulating essential aspects of human disease in mouse and other model organisms.

INFRAFRONTIER facilitates access for the wider biomedical research community to the unique infrastructure and scientific expertise of the participating INFRAFRONTIER partners in open calls and EU Horizon 2020 supported Trans-national Access service calls. Access provision to the INFRAFRONTIER infrastructure supports the development of novel mouse and or rat models that will advance knowledge of human disease and will be of widespread use in biomedical science. Recent advances in genome editing technology are used to produce new mammalian models of human disease. INFRAFRONTIER provides open access to all newly developed disease models through the European Mouse Mutant Archive (EMMA). Access to this free-of-charge-service will be granted on the basis of the applicant's research plans and the potential impact of the proposed novel mouse line on the wider biomedical research community.

https://www.infrafrontier.eu/resources-and-services/infrafrontier-open-calls

 

The INFRAFRONTIER / IMPC Stakeholder Meeting presented an excellent opportunity to  better align INFRAFRONTIER / IMPC platforms with current Personalised Medicine initiatives to advance biomedical science and our knowledge of human disease.

Meeting Programme and abstracts

Day 1: All presentations

Martin Hrabĕ de Angelis, Helmholtz Center Munich (HMGU):

Introduction: Advancing Personalized Medicine with Animal Models

 

Maria Christoula, General Secretariat for Research and Technology (GSRT):

Research Infrastructures: Main Policy Aspects and Challenges

 

Martin Hrabĕ de Angelis, Helmholtz Center Munich (HMGU):

INFRAFRONTIER: Resources and Services to advance the understanding of human health and disease using mammalian models

 

George Kollias, BSRC Alexander Fleming:

Contribution of Disease Modelling to Precision Medicine Initiatives

 

Dimitris Kontoyannis, BSRC Alexander Fleming:

Phenotypos - The next Phase of the Greek Research Infrastructure for Phenotyping

 

Jason Heaney, Baylor College of Medicine (BCM):

Modeling human Disease Variants in Murine Orthologs with CRISPR/Cas9

 

Radislav Sedlacek,  Czech Center for Phenogenomics:

KLK5 and KLK7 Ablation fully rescues Lethality of Netherton Syndrome-like Phenotype

 

Tomoji Mashimo, Genome Editing R&D Center and Institute of Experimental Animal Sciences, Osaka University:

Efficient generation of conditional knockout mice and rats by CLICK

 

C.B Gurumurthy / University of Nebraska Medical Center, Omaha, USA

Easi-CRISPR for conditional and insertional alleles

 

Yann Herault, IGBMC, Translational Medicine and Neurogenetics Department:

In vivo chromosomal engineering in rodents to analyse structural variants through Crismere

 

Nadia Rosenthal, The Jackson Laboratory:

Of mice and CRISPR

 

Steve Brown, MRC Harwell / IMPC:

The IMPC and its role in Precision Medicine

 

Damian Smedley, Genomics England:

Use of model organism phenotype data to obtain novel insight into disease causes and mechanisms

 

Cat Lutz / JAX – Center for Precision Genetics, ALS Disease Modelling Unit:

Generation of an allelic series using CRISPR/Cas9 to study familial ALS

 

Jos Jonkers, Netherland Cancer Institute:

Dissection of breast cancer development and therapy resistance in mouse models

 

Enzo Medico, University of Torino

EurOPDX Consortium: PDX models as an emerging way to personalized medicine in translational cancer research

 

Andreas Roos, University of Newcastle

RD-Connect: Infrastructure for rare disease data and samples

 

Jerry Lanfear, ELIXIR:

CORBEL and contributions of the EU health related infrastructures to Personalised Medcine in Europe

 

Marisa Papaluca, European Medicines Agency (EMA):

Personalised Medicine Regulatory Issues

 

Colin Fletcher, NIH / IMPC:

KOMP 2 - Evolution of the Vision: Developing personalized gene therapy approaches

 

Monika Frenzel, Agence Nationale de la Recherche (ANR):

ICPerMed – The International Consortium for Personalised Medicine

 

Paul Lasko, Scientific Director, Institute of Genetics, Canadian Institutes of Health Research:

IRDiRC: looking toward the next ten years in rare diseases research

Summary - Advancing Personalised Medicine with Animal Models

Summary and conclusions of presentations and discussions at the INFRAFRONTIER / IMPC Stakeholder Meeting on Advancing Personalised Medicine with Animal Models

 

1 Mouse models and Individualized Medicine – A value proposition

David Valle @ ILAR Roundtable, Washington DC, Oct 3-4, 2017

Mouse models and Individualized Medicine:

  • Confirm causation
  • Understanding pathophysiology
  • Surrogates for treatment studies

http://nas-sites.org/ilar-roundtable/files/2017/10/VALLE-Precision-medicine_Valle_NAS_Oct2017.pdf

 

2 Animal-based studies are essential for precision medicine

  • Models must reflect -omic variation in patients in order to define downstream functional consequences and discriminate causal from correlative factors at relevant efficiency.
  • Effectively link omic data with environmental, behavioral, and lifestyle information to identify actionable findings.
  • Provide quick and accurate assessment of the scientific validity and clinical utility of gene-environment correlations.
  • Incorporate computational reasoning and semantic mapping efforts to enable cross-species phenotype comparisons, bridge the knowledge gap. and maximize the potential of patient data

Lloyd et al., (2016) Animal-based studies will be essential for precision medicine. Science Translational Medicine 

 

3 Idealized Precision Mouse Model

  • Patient variant introduced into the mouse genome

              – Construct validity, validates pathogenesis of variant

  • Mouse phenotype matches patient disease

              – Face validity, establishes similar pathophysiology

  • Phenotype is tested on multiple backgrounds

              – Modifier genetics identifies important ancillary pathways

  • Improved predictive validity of the model
  • Improved diagnostic/prognostic accuracy when combined with patients’ genomic information

http://nas-sites.org/ilar-roundtable/files/2017/10/BURGESS-ILAR-NAS.pdf

 

4 Advancing Personalised Medicine with Animal Models - Use Cases

Various use cases were presented with formal linkages between model organism communities and the clinical research community and Precision Medicine initiatives

 

Baylor College of Medicine

  • At Baylor BCM-KOMP2 and Centers for Mendelian Genomics (CMG) are formalising a partnership to advance human mendelian genomics
  • BCM-KOMP2 extensively modelled human disease variants and created mouse models of human disease relevant mutations using genome-editing technology
  • At Baylor model organism studies using drosophila and zebrafish within the Model Organism Screening Centres of the Undiagnosed Disease Network are critical for assessing pathogenicity

 

JAX Center for Precision Genetics

  • At the Center for Precision Genetics personalised gene therapy approaches are being developed based on a RNAi-mediated allele specific knockdown approach
  • In collaboration with ALS clinicians the Precision Genetics ALS Disease Modelling Unit developed a CRISPR/Cas9 based ALS model development and a tailored phenotyping pipeline
  • ALS-linked mutations are developed in stable genetic backgrounds but also explored in Collaborative Cross and Diversity Outbred mice. Harnessing diversity resources has the potential for discovery of new modifiers

 

GEMM Genome Editing Mice for Medicine

  • Genomics England is partnering with MRC Harwell in the GEMM program to support the development of precision mouse models
  • GEMM developed so far 42 precision models of human disease
  • GEMM demonstrates the importance of clinical and model organism phenotypes, as well as precision models, to achieve the vision of building the future of genomic medicine

 

Rare Diseases: Models and Mechanisms (RDMM) Network

  • RDMM catalyzes connections between people discovering new genes in patients with rare diseases, and basic scientists who can analyze equivalent genes and pathways in model organisms.
  • Catalyst Grants fund projects that will allow rapid confirmation of potentially disease-causing genes, and fuel pilot studies to improve understanding of how specific gene mutations cause disease.
  • Collaborations may expedite the understanding of disorders, enabling the design of new therapies to the ultimate benefit of those affected by rare diseases.

 

5 The International Mouse Phenotyping Consortium (IMPC) delivers Mouse Genetics for Precision Medicine

  • Comprehensive baseline (null allele) information on gene function and pleiotropy
  • Providing a comprehensive catalogue of mammalian gene function
  • Delivering insights into novel gene function and pleiotropy across diverse systems
  • Sophisticated pipelines to deliver and analyse human genetic variation in the mouse
  • An extensive new fund of disease models
  • Relevant pre-clinical models that will enable mechanistic and therapeutic insights
  • Increasing integration of IMPC and human/clinical genetics initiatives
  • Harnessing the infrastructure of mouse genetics centres worldwide to precision medicine initiatives

 

IMPC contributions - post KOMP

  • Complete a comprehensive, encyclopedic functional annotation of the genome
  • Precision Medicine

              – Tailored treatment to the individual

  • Allele, Genome, Environment
  • Harness Crispr-tunities

              – Model human disease alleles to understand mechanism, allelic series

  • Confirm Gene ID, define pathophysiology
  • “allelic heterogeneity”

               – Nulls on other genetic backgrounds (modifiers)

               – Non-coding DNA – common disease variants

 

6 INFRAFRONTIER Research Infrastructure

INFRAFRONTIER offers unique platforms for the functional validation of genetic variants identified in exome/whole-genome sequencing approaches and the development of mouse models with predictive utility for efficient translation. Generation of precision models is key to the development of new therapies for rare disease.

  • INFRAFRONTIER provides access to 6000 mouse models, including validated human disease models and widely used research tools, and thereby supports research to improving the understanding of gene function in human health and disease using the mouse model
  • INFRAFRONTIER facilitates access to state of the art genome-editing technologies such as CRISPR/Cas9 supporting the development of novel mouse and rat models precision models that will advance knowledge of human disease
  • INFRAFRONTIER facilitates access to sophisticated phenotyping pipelines to analyse human genetic variation in the mouse, and delivering insights into novel gene function and pleiotropy

 

7 Of Mice and CRISPR

Genome-editing is a key technology driving further the advancement of Personalised Medicine with animal models. CRISPR/Cas9 will expand available precision model organism resources and technology development for rapid assessment of variants

Scientific vistas CRISPR will open for Precision Medicine

  • Creation of complete allelic series of a genetic disease
  • Experimental validation of the phenotypic output of combinatorial gene variations
  • Humanization: the progressive reshaping of the whole mouse genome
  • Comprehensive understanding of genetic background and its effect on Mendelian disease mutation

Harness mouse diversity resources and CRISPR to functionalize the human genome

JAX Center for Precision Gnenetics: ALS-linked mutations were developed in stable genetic backgrounds and are also explored in Collaborative Cross and Diversity Outbred mice. Harnessing these diversity resources revealed the potential for discovery of new modifiers

Genome-editing technologies: Using Easi-CRISPR and CLICK are efficient technologies for the generation of conditional and insertional alleles, and can be used for the correction of disease associated phenotypes.

CRISMERE is a new technology for the efficient and rapid generation of large genomic variants in rats and mice supporting the modelling of copy number diseases with Intellectual Disability (ID) such as Down Syndrome (DS). These mouse models are invaluable tools for validating therapeutic approaches to rescue the cognitive defects in DS.

Advancing Personalised Medicine with Animal Models – Introduction

George Kollias, BSRC Alexander Fleming

Contribution of Disease Modelling to Precision Medicine Initiatives

In his introductory presentation George Kollias presented modeling rheumatoid arthritis as use case, and showed that a human TNF transgenic mouse model of spontaneous arthritis closely resembles the human pathology. Prof Kollias presented further disease models for Spondyloarthritis (TgA86 (tmTNFmu)) and spondyloarthropathies (TNF-ΔARE). The TNF-ΔARE mouse model was systemically phenotyped in the German Mouse Clinic (www.mouseclinic.de) which uncovered comorbidities.

 G. Kolyas

Reference:

Mesenchymal TNFR2 promotes the development of polyarthritis and comorbid heart valve stenosis. Maria Sakkou et al., published April 5, 2018
JCI Insight. 2018;3(7):e98864. doi:10.1172/jci.insight.98864.

Prof Kollias concluded his presentation stating that animal research is essential for modelling and understanding human disease, and summarised key points for disease modelling using animals and characteristics of animal models in the era of Precision Medicine.

 

OF ANIMALS and MEN

Species do differ!

  • Unity in Biology but also diversity and descent by modification
  • Size, metabolic rates, sensory systems, stress, cognitive functions
  • Life expectancy, reproductive rate, diets, microbiomes, pathogens

… but animal model research is essential

  • To reproduce the cause and biology underlying complex disease
  • To tweak the system with genetics and ensure safety for human
  • To experiment with new treatments before they are ready for the clinic
  • To understand essential biological mechanisms: the last 10 Nobel in Medicine and multiple landmark discoveries involved studies in animals.

For disease modelling using aimal models

  • Consider multi-layered pathways and disease heterogeneity

(select your model based on target biology)

  • Do not blame your animal model (blame your choice!)
  • Support conclusions by evidence (not statistical magic!)
  • Know your animal model (target biology, pathways, cellular mechanisms, comorbidities)
  • Establish disease-specific primary/dominant causalities
  • Design preclinical experiments more rigorously (avoid noise by genetic background, balance for gender and environment, and standardize induction and treatment protocols)
  • Standardize design, analyses and publication of research
  • Standardize statistics, interpretations and extrapolations

Characteristics of animal models in the era of Personalised Medicine

Towards next generation animal models that target personalized phenotypes through:

  • Mouse models for mechanistic understanding of complex diseases and biomarker development
  • “Pathogenesis Maps” aligning animal models to the different subsets of human disease
  • “Mouse Avatars” (e.g. PDX) and Humanized Mice for personalized drug efficacy studies
  • “Co-clinical trials” - real time integration of mouse and human data to guide therapeutic approaches in ongoing clinical trials
  • Prompt and efficient “human to mouse to human” discoveries
Advancing Personalised Medicine with Animal Models – use cases

Jason Heaney - Baylor College of Medicine (BCM):

Modeling human disease variants in murine orthologs with CRISPR/Cas9

Human genomic discovery programs are likely to encounter increasing numbers of apparently unique missense variants of unknown significance (VUS). These alleles in fact have great potential to inform novel biological and disease mechanisms as they reflect increasing context-dependent disease pathogenesis that integrates specific protein domain functions and interactions with differential threshold for dysfunction in specific tissues. Model organism studies will be critical for assessing pathogenicity and the BCM drosophila and zebrafish cores (Model Organism Screening Centres (MOSC) within the Undiagnosed Disease Network (UDN) will provide essential insight.

However, the mouse will offer the best preclinical model for comparative phenotypic analysis at a tissue level that can point to new tissue mechanisms, as well as, unexpected, undetected, and/or incompletely penetrant phenotypes in the undiagnosed patient. Moreover, the mouse model will be required for rapid testing of repurposed or new therapies. At Baylor Human Genomic discovery programs such as the Center for Mendelian Genomics (CMG) are formalising partnerships with BCM-KOMP2 in advancing human mendelian genomics.

Jason Heaney reported on the extensive modeling of human disease variants by BCM-KOMP2 to create and characterize mouse models of human disease relevant mutations (273 null alleles, 58 conditional knockout alleles, and 79 point mutation and other knock-in allele types using short and long ssODNs). Jason emphasized that traditional null alleles remain a valuable resource for precision disease modeling, and that variant knock-in alleles in mice present unique challenges that require close collaboration between genomic discovery and animal modeling teams.

Collaborations among clinicians, human geneticists and model organism researchers facilitate diagnosis and studies of undiagnosed conditions. Candidate causative genes and variants identified from a patient with an undiagnosed disease can be explored in a number of genetic model organisms. Using stateof-the-art genome engineering technologies in these model systems, one can assess whether the variants of interest lead to functional consequences in vivo, and obtain phenotypic information that may directly or indirectly relate to the patient’s condition. Integration of biological information from multiple species can be complementary or/and confirmatory.

Reference:

  • Wangler et al., (2017) Model organisms facilitate Rare Disease diagnosis and therapeutic research. Genetics
More use cases - JAX Center / Genomics England

Robert Burgess - JAX Center for Precision Genetics:

Developing personalized gene therapy approaches

Robert Burgess presented a use case on Charcot-Marie-Tooth Diseases (CMTs), inherited peripheral neuropathies, which are caused by mutations in tRNA synthetases. Human GARS mutations cause CMT-2D, and mouse models with good face and construct validity are available. Robert Burgess developed a therapeutic strategy based on eliminating the mutant Gars by a RNAi-mediated allele-specific knockdown approach. Allele specific RNAi was developed and tested in vitro. Subsequently, following scAAV9 delivery of Gars-targeting miRNA shuttles, mice were analyzed at 4 weeks of age to test efficacy and showed an ameliorated phenotype.

Translational potential

The translational potential of the approach is now being tested on a four year old female patient with severe motor neuropathy. Whole exome sequencing identified a de novo 12 base pair deletion in exon 8 of GARS. Assessment if the mutation is causative using mouse models and the development of a translational allele-specific knock down approach for the human disease allele is underway.

A Cure for Caroline: https://www.jax.org/news-and-insights/personal-stories/curing-caroline

 

Cat Lutz / JAX – Center for Precision Genetics, ALS Disease Modelling Unit:

Generation of an allelic series using CRISPR/Cas9 to study familial ALS

Cat Lutz presented a use case  on familial ALS, a complex and progressive neurological disease that affects the control of muscle movement caused by damage to motor neurons. The incidence of ALS is two per 100,000 people,  average age of onset is 55 years, and survival 3-5 years after diagnosis. Exome sequencing identified new variants that appear to cause ALS or contribute to susceptibility and/or severity of the clinical presentation. ALS-linked mutations were developed in stable genetic backgrounds and are also explored in Collaborative Cross (CC) & Diversity Outbred (DO) mice. Harnessing these diversity resources revealed the potential for discovery of new modifiers.

Working with ALS clinicians and researchers the Precision Genetics ALS Disease Modelling Unit developed a new CRISPR/Cas9 based ALS model development pipeline with 28 mutations engineered in 9 new ALS genes to date. A tailored phenotyping pipeline has been established with bi-weekly body weight and neurological scoring, and at 1 year of age testing of grip strength, gait analysis, adhesive removal test, and erasmus ladder.

 

Damian Smedley, Genomics England:

Use of model organism phenotype data to obtain novel insight into disease causes and mechanisms

Damian Smedley provided an overview of the Genomics England project which aims to sequence 100000 genomes with a scientific focus on cancer and rare genetic disease.

Damian highlighted then the role of clinical and model organism phenotyping and presented the Genomics England process for clinical data collection and panel assignment and protocols for automated variant prioritisation. First families are now diagnosed e.g. a de novo deletion in SLC2A1 wasidentified as the cause of Glut 1 deficiency syndrome. For the expected numerous rare disease cases without a clear diagnosis from the standard pipeline, CRISPR/Cas9 precision animal models are required for functional validation and ultimately improved treatment.

To facilitate the development of precision mouse models Genomics England is partnering with the MRC Harwell Institute in the GEMM Genome Editing Mice for Medicine program.

GEMM published open calls in 2016/2017 and supported the production of 42 precision mouse model development projects leading to disease models for X-linked intellectual disability, Non-ketotic hyperglycinemia, Thyroid Cancer, Epilepsy, infantile-onset dyskinesia and others.

Damian Smedley concluded by stating that clinical and model organism phenotypes, as well as precision animal models, are key to achieving the vision of building the future of genomic medicine.

The IMPC and its role in Precision Medicine

Steve Brown, MRC Harwell / IMPC:

The IMPC and its role in Precision Medicine 

Steve Brown presented the goals and opportunities of the global International Mouse Phenotyping Consortium

IMPC delivers Mouse Genetics for Precision Medicine:

  • Comprehensive baseline (null allele) information on gene function and pleiotropy
  • Providing a comprehensive catalogue of mammalian gene function
  • Delivering insights into novel gene function and pleiotropy across diverse systems
  • Sophisticated pipelines to deliver and analyse human genetic variation in the mouse
  • An extensive new fund of disease models
  • Relevant pre-clinical models that will enable mechanistic and therapeutic insights
  • Increasing integration of IMPC and human/clinical genetics initiatives
  • Harnessing the infrastructure of mouse genetics centres worldwide to precision medicine initiatives

IMPC is transforming the mammalian genome landscape

  • IMPC has delivered nearly 7,000 gold-standard mouse lines
  • Robust and reproducible phenotyping platforms delivered phenotype data from over 5,000 lines
  • Substantive step towards a comprehensive catalogue of mammalian gene function
  • Transforming the opportunities for rare disease and precision medicine initiatives
  • Establishing the context and data for a new era of cross-species analysis via mouse and human multidimensional genetic and phenotype datasets

 

References:

  • Meehan et al., (2017) Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium. Nature Genetics
  • Lloyd et al., (2015) Precision Medicine: Look to the mice. Science
NIH / IMPC program integration

Colin Fletcher, NIH / IMPC:

Developing personalized gene therapy approaches

In his presentation Colin Fletcher emphasized the importance of the unbiased approach of the IMPC by aiming to provide a comprehensive catalogue of mammalian gene functiion and promoting access to unannotated genes.

The Nature Commentary describes how most research continues to focus on genes known before the human genome was sequenced (75% of publications are focused on the same 10% of genes). IMPC is able to provide the insights and preliminary data that can drive research into the “dark” genes. Colin Fletcher then highlighted possible contributions IMPC can make beyond the current KOMP funding

IMPC - post KOMP

Precision Medicine

–      Tailored treatment to the individual

  • Allele, Genome, Environment

Harness CRISPR-tunities

–      Model human disease alleles to understand mechanism, allelic series

  • Confirm Gene ID, define pathophysiology
  • “allelic heterogeneity”

–      Nulls on other genetic backgrounds (modifiers)

–     Non-coding DNA – common disease variants

Using genome editing approaches to model human conditions

A key area where model organisms can make significant contributions to Personalised Medicine initiatives is the interpretation of gene variants. New genome editing technologies such as CRISPR/Cas9 enable now the efficient derivation of precision mouse models incorporating patient-specific genetic variants to recapitulate essential aspects of human disease. The versatility of genome editing technology further allows in vivo chromosal engineering to analyse structural variants. A dedicated session on the disruptive genome editing technology was held with excellent contributions from leading experts providing background information on the technology and state of the art approaches to generate conditional and insertional alleles.

 

 

Soren Warming / Genentech:

In-depth analysis of CRISPR off-targets in genetically engineered mice and rats

Most researchers are aware of the potential for off-target effects with CRISPR, but there is no consensus on what to look for, what to worry about, and how to deal with concerns about animals created using CRISPR. Genentech routinely analyzes all G0 founders for a list of potential off-targets using deep sequencing. From the analysis of in-house projects there is evidence that about 25% of the model development projects have off-target cutting by Cas9. While some correlation exists between predicted scores and off target profiles, it is also evident that existing algorithms are still lacking in their predictive power. Based on the comprehensive data-set Genentech has generated, a list of recommendations for CRISPR project design and subsequent analysis of the resulting animals was provided.

Recommendations

  • Ideally: two independent mouse/rat lines generated using different sgRNAs
  • When possible, use sgRNAs with specificity scores >70. Algorithms then likely to predict most or all off-targets. Follow up with ampli-seq
  • For KOs: more freedom to design optimal sgRNAs in introns
  • For low specificity sgRNAs, use un-biased methods like GUIDE-seq, Digenome-seq, or CIRCLE-seq to identify most likely off-targets. Follow up with ampli-seq
  • Consider specificity-optimized Cas9
  • Benefits of screening G0 founders by deep sequencing:
    • identifies founders with highest on-target contribution
    • identifies founders w/o off-target (or identifies off-targets to screen for in G1s)

 

 

Tomoji Mashimo, Genome Editing R&D Center and Institute of Experimental Animal Sciences, Osaka University:

Efficient generation of conditional knockout mice and rats by CLICK

References:

ssODN-mediated knock-in with CRISPR/Cas for large genomic regions in zygotes

     K Yoshimi et al. Nat Commun 2016 Jan 20;7:10431 

     http://resou.osaka-u.ac.jp/ja

Allele-specific genome editing and correction of disease-associated phenotypes in rats using CRISPR/Cas platform,

      K Yoshimi et al. Nat Commun 2014 Jun 26;5:4240

      http://www.kyoto-u.ac.jp/ja/research/research_results/2014/140626_1.html

 

 

C.B Gurumurthy / University of Nebraska Medical Center, Omaha, USA

Easi-CRISPR for conditional and insertional alleles

References:

Easi-CRISPR protocol

     http://www.biorxiv.org/content/early/2017/05/23/141424

GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system, a novel microinjection-independent genome editing method

     https://www.nature.com/articles/srep11406

Easi-CRISPR + GONAD = a total solution

     http://www.biorxiv.org/content/early/2017/08/04/172718

 

 

Yann Herault, IGBMC, Translational Medicine and Neurogenetics Department:

In vivo chromosomal engineering in rodents to analyse structural variants through Crismere

Yann Herault presented a new CRISPR/Cas9 based technology termed ‘Crismere’ which significantly improves chromosomal engineering. The timescale of projects could be reduced from 3-5 years to 6 months, no additional minigenes and loxP sites are needed, no more ES cells work, and Crismere can be applied in rats! Using Crismere the easy production of deletions, inversions and duplications from a few bp to 24,4 Mb are possible. Yann emphasized that the generation of structural variants is now straightforward, but the genotyping determination must be done carefully.

As a use case Yann Herault presented the modelling of copy number diseases with Intellectual Disability (ID). These are rare disorders, with Down Syndrome (DS) being the most common cause of ID and chromosomal aneuploidy. Such Down Syndrome mouse models are invaluable tools for validating therapeutic approaches to rescue the cognitive defects in DS.

References

 

 

Nadia Rosenthal, The Jackson Laboratory:

Of mice and CRISPR

What scientific vistas will CRISPR open for precision medicine?

  • Creation of complete allelic series of a genetic disease
  • Experimental validation of the phenotypic output of combinatorial gene variations
  • Humanization: the progressive reshaping of the whole mouse genome
  • Comprehensive understanding of genetic background and its effect on Mendelian disease mutation

How to model complex traits with CRISPR?

  • Multiplex mutations in a single mouse
  • Stack variations by crossing multiple mutant mice for combinatorial power
  • Reengineer functional portions of the mouse genome (eg. immunoglobulins)
  • Expand the genetic diversity of mouse backgrounds on which to test mutations

Harness mouse diversity resources and CRISPR to functionalize the human genome

Goal 1: “reading” the human genome

Decoding human variation with mouse diversity pipelines:

  • Genetic mapping of variable disease phenotypes
  • Validation of candidate modifiers with mutation
  • Mapping gene networks onto human genome data

Validation of candidate modifiers with mutation

Crossing CRISPR mutants into CC inbred panels

Goal 2: “writing” the human genome

Curing complex diseases in mouse diversity panels:

  • Testing therapeutics targeted to complex disease traits
  • “Repairing” these traits with CRISPR editing

Reference

  • Liu et al., Of mice and CRISPR: The post-CRISPR future of the mouse as a model system for the human condition. EMBO Rep. 2017 Feb;18(2):187-193
Contribution of European RIs and other large EU initiatives to Personalised Medicine

EUROPEAN RESEARCH INFRASTRUCTURES

Research Infrastructures (RIs) are described as facilities, resources and related services used by the scientific community to conduct top-level research in their respective fields.  European RIs are built to correspond to the long-term needs of European research communities covering all research areas. They play an important role in expediting knowledge and technology and bringing together a wide diversity of stakeholders to look for solutions to current problems the society is facing. As stated by the European Commission, RIs are a center of the knowledge triangle of research, education and innovation, producing knowledge through research, diffusing it through education, and applying it through innovation.

The EU Member States and Associated Countries operate a diverse system of RIs ranging from sectors of Physical Sciences to Health, and in their location of single sited to those built on distributed capacity and specialty. In 2016, European Strategy Forum on Research Infrastructures (ESFRI) published a list of current RIs in the European Roadmap categorized into Landmarks, which refer to successfully implemented ESFRI projects delivering science services or effectively constructing them. Health related RIs of the ESFRI roadmap such as BBMRI, EATRIS, ECRIN, ELIXIR, EU-OPENSCREEN, INFRAFRONTIER and ISBE, and new emerging RIs implemented as Starting Communities can contribute to the agenda of Personalised Medicine for Europe.

 

 

Enzo Medico, University of Torino

EurOPDX Consortium: PDX models as an emerging way to personalized medicine in translational cancer research

http://europdx.eu/

"The EurOPDX EDIReX project: Towards a European research infrastructure on patient-derived cancer models"aims toestablish a cutting-edge European Research Infrastructure offering Trans-national Access to PDX resourcesfor academic and industrial cancer researchers, including the distribution of cryopreserved samples to third parties, the structured biobanking of user-developed models, and the performance of drug efficacy studies. This will imply in particular theset-up of the EurOPDX public repository of models, andfurther work on establishing standards in the field.

Reference:

Byrne AT et al., Interrogating open issues in cancer medicine with patient-derived xenografts. Nat Rev Cancer. 2017 Sep 15;17(10):632

 

 

Andreas Roos, University of Newcastle

RD-Connect: Infrastructure for rare disease data and samples

https://rd-connect.eu/

RD-Connect is an integrated platform connecting databases, registries, biobanks and clinical bioinformatics for rare disease research.

To help researchers study rare diseases, RD-Connect links different data types - omics (e.g. genomics), clinical information, patient registries and biobanks - into a common resource. RD-Connect enables scientists and clinicians around the world to analyse genomics data and share them with other researchers. By making data accessible beyond the usual institutional and national boundaries, RD-Connect speeds up research, diagnosis and therapy development to improve the lives of patients with rare diseases

Thompson et al., RD-Connect: An integrated platform connecting databases, registries, biobanks and clinical bioinformatics for rare disease research. J Gen Intern Med. 2014 Aug;29 Suppl 3:S780-7.

 

 

Jerry Lanfear, ELIXIR:

CORBEL and contributions of the EU health related infrastructures to Personalised Medcine in Europe

https://www.elixir-europe.org/

ELIXIR unites Europe’s leading life science organisations in managing and safeguarding the increasing volume of data being generated by publicly funded research.

ELIXIR coordinates, integrates and sustains bioinformatics resources across its member states and enables users in academia and industry to access vital data, tools, standards, compute and training services for their research.

ELIXIR provides data infrastructure for Europe’s 500,000 life-science researchers

ELIXIR is organised in five technical platforms: Data, Interoperability, Tools, Compute and Training.

The four use cases of ELIXIR connect the technical activities to the real needs of user communities in the life sciences: Marine metagenomics, Crop and forest plants, Human data, Rare diseases.

 

 

CORBEL – Shared services for life sciences:

Contribution from the Health Related Infrastructures - BBMRI, ECRIN, EATRIS, ELIXIR, EU-OPENSCREEN, INFRAFRONTIER, ISBE and MIRRI - to a Personal Medicine Roadmap for Europe

http://www.corbel-project.eu/home.html

Rapid developments in '-omics' technologies such as genomics, proteomics or metabolomics, together with advances in a number of other areas (e.g. liquid biopsies and biological therapies) are creating the possibility for scientists to develop tools to stratify medicine and move towards personalised diagnosis and treatment strategies. Health related RIs collaborating in the H2020 project CORBEL(Coordinated Research Infrastructures Building Enduring Life-Science Services) can contribute to the agenda of Personalised Medicine (PM) for Europe. These RIs play a fundamental role in accelerating the PM implementations, particularly addressing the five most high impact challenges identified by the Coordination and Support Action PerMed consortium in the Strategic Research and Innovation Agenda (SRIA). These regard patients & citizens, data and information technologies, translational research, innovations, and health systems.

Challenges in Personalised Medicine

While Personalised Medicine presents an opportunity for the European citizens to increase their chances of a successful outcome of any disease, the implementation of PM is a challenge in Europe and beyond due to fragmented activities, lack of generic solutions and insufficient cross-sectional communication between different stakeholders involved in PM (healthcare specialists, regulators, researchers, industry representatives, and patient organizations). The Coordination and Support Action PerMed was initiated to accelerate the coordination efforts between European key stakeholders, to avoid duplication in competition and to ensure maximum transparency and openness while preparing Europe for leading the global way in Personalised Medicine.

Following its mission, PerMed generated a Strategic Research and Innovation Agenda (SRIA) on Personalised Medicine based on analysis of recent strategic reports, interviews and consultations of all relevant operational sectors important to the implementation of PM. SRIA identifies five most high impact challenge areas of PM today:

1)     Citizens and Patients: public engagement and involvement of citizens/patients in science,

2)     Data and Information and Communications Technologies (ICT): integrating Big Data and ICT solutions to generate the required knowledge,

3)     Research Efforts: translation of discoveries into clinical research,

4)     Market Access: bringing new PM innovations to the market, and

5)     Health Systems: shaping up the healthcare into a knowledgeable system that is able to adapt fast to new approaches.

SRIA also presents several prioritized recommendations that have the highest potential impact and outcome when implementing the Personalised Medicine for the benefit of citizens and society as a whole. These highlight not only the importance of patients, regulatory frameworks, ICT, and the dialogue between different stakeholders, but also the relevance of European Research Infrastructures whose outcomes and achievements can be harnessed, developed further, and applied for the needs of Personalised Medicine.

A particular strength of RIs is their dynamic nature and responsiveness to the multidisciplinary needs of PM challenges that are difficult to tackle in sectorial way.

Contribution of  INFRAFRONTIER to the Personalised Medicine challenges in Europe

The PerMed Strategic Research Agenda identifies five most high impact challenge areas of PM today:

1)     Citizens and Patients: public engagement and involvement of citizens/patients in science,

2)     Data and Information and Communications Technologies (ICT): integrating Big Data and ICT solutions to generate the required knowledge,

3)     Research Efforts: translation of discoveries into clinical research,

4)     Market Access: bringing new PM innovations to the market, and

5)     Health Systems: shaping up the healthcare into a knowledgeable system that is able to adapt fast to new approaches.

 

Challenge 3. Research Efforts: Translating Basic to Clinical Research and beyond

Personalised Medicine (PM) must be supported by robust knowledge of the disease and patients through excellent basic research conducted across Europe. In order for PM to reach its promise and anticipated impact, translation of discoveries and communication across the continuum of research are required.

 

INFRAFRONTIER is comprehensively contributing to address the PM challenge 3.The INFRAFRONTIER mouse clinics are founding partners and key contributors to the effort of the International Mouse Phenotyping Consortium (IMPC) to build the first truly comprehensive functional catalogue of a mammalian genome. The IMPC dataset is of increasing interest to groups working with large biobank, phenotype and genetic datasets in human. The comparative analysis of cross-species datasets will potentially be transformed by a complete catalogue of mouse mutant phenotypes. As a consequence, we expect IMPC to make pivotal contributions to data-driven discovery in medical research. The IMPC provides a key pillar to diverse priorities in biomedical sciences and actively aligns its activities with rare disease programs, genome projects and the National Institutes of Health (NIH) Precision Medicine Initiative.

INFRAFRONTIER and IMPC offer unique platforms for the functional validation of genetic variants identified in exome/whole-genome sequencing approaches and the development of mouse models with predictive utility for efficient translation. Generation of precision models is key to the development of new therapies for rare disease.

INFRAFRONTIER facilitates access for the wider biomedical research community to the unique infrastructure and scientific expertise of the participating INFRAFRONTIER partners in open calls and EU Horizon 2020 supported Trans-national Access service calls. Access provision to the INFRAFRONTIER infrastructure supports the development of novel mouse and or rat models that will advance knowledge of human disease and will be of widespread use in biomedical science. Recent advances in genome editing technology are used to produce new mammalian models of human disease. INFRAFRONTIER provides open access to all newly developed disease models through the European Mouse Mutant Archive (EMMA). Access to this free-of-charge-service will be granted on the basis of the applicant's research plans and the potential impact of the proposed novel mouse line on the wider biomedical research community.

https://www.infrafrontier.eu/resources-and-services/infrafrontier-open-calls

 

Rare disease programmes: The models generated by the IMPC and accessible by mouse repositories like INFRAFRONTIER/European Mouse Mutant Archive (EMMA) have important utility in examining the function of rare disease genes, as well as potentially providing pre-clinical models for therapeutic assessment. Close links have been forged with rare disease communities both across INFRAFRONTIER member countries and internationally through the International Rare Disease Research Consortium (IRDiRC).

 

100,000 genome project and the Precision Medicine Initiative: Both in the UK and the US there is increasing emphasis on the matching of extensive genome information with health records and other sources of human phenotype data. In the UK, the 100 000 genomes project will focus initially on rare disease phenotypes and prostate cancer. However, it is expected that the project will expand to incorporate other sources of phenotype information. In the US, the Precision Medicine Initiative is likely to take a similar approach. But in either case, the catalogue of gene function being developed in the IMPC will be an important validation and extension of the gene-phenotype associations that emerge from these human genetics projects, contributing to target discovery and validation.

INFRAFRONTIER and the IMPC are very well geared to support these projects, and continual informatics developments are feeding into the IMPC program to ensure that we maximize the opportunity to provide and learn from relevant mouse models. For example, the development of sophisticated tools such as the Phenodigm algorithm provides the community with tools to analyse mouse phenotype data to identify new pre-clinical models for human disease.

Meehan et al., (2017) Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium. Nature Genetics

ICPerMed – The International Consortium for Personalised Medicine

Monika Frenzel, Agence Nationale de la Recherche (ANR):

ICPerMed – The International Consortium for Personalised Medicine

Monika Frenzel provided a comprehensive overview of the International Consortium for Personalised Medicine (ICPerMed) which brings together over 30 European and international partners representing ministries, funding agencies and the European Commission (EC). Together, they work on coordinating and fostering research to develop and evaluate personalised medicine approaches.

Despite all efforts, only a limited number of Personalised Medicine approaches have so far managed the long road from basic biomedical research to clinical application. A lot of investment is made in personalised medicine related research. However, the research efforts in this highly innovative and rapidly changing field are fragmented. Therefore research funders assembled under the umbrella of ICPerMed address this fragmentation challenge on the European as well as on the international level.

ICPerMed is implementing the Strategic Research Agenda (SRIA) of PerMed which identified key challenges across the whole healthcare value chain and beyond:

1. Citizens and Patients

2. Data & ICT

3. Research Efforts

4. Market Access

5. Health Systems

The ICPerMed Action Plan describes actionable research and support activities identified by the ICPerMed Consortium to address key PM challenges

https://www.icpermed.eu/media/content/ICPerMed_Actionplan_2017_web.pdf

Implementation of the Action Plan is supported by the ERA-NET ERA PerMed which launched its first and co-funded Joint Call for Proposals to support transnational research projects in Personalised Medicine and to encourage and enable interdisciplinarity, in combining pre-clinical and/or clinical research with bio-informatics components.  

http://www.erapermed.eu/

IRDiRC – International Rare Diseases Research Consortium

Paul Lasko, Scientific Director, Institute of Genetics, Canadian Institutes of Health Research:

IRDiRC: looking toward the next ten years in rare diseases research

Paul Lasko, former IRDiRC chair, provided a short overview of IRDiRC which now includes over 40 members from all over the world who collectively have committed over $2 billion US to rare disease research. Paul Lasko reported on the significant progress of IRDiRC:

Diagnostics (goal: most rare diseases by 2020)

– Nearly 4,100 rare diseases for which there is a genetic test available, as compared with 2,200 in 2010.

Therapies (goal: 200 new therapies by 2020)

– Achieved! 222 as of end 2016.

 

Paul Lasko also highlighted technical achievements and contributions of genomics technologies to RD diagnosis, but also emphasized remaining challenges.

RD diagnosis today - still work to do

  • Genomics technology exists to diagnose many rare diseases that affect most rare disease patients.
  • This technology is being transferred from research programs to healthcare but implementation in healthcare is spotty
  • Many genetic diseases still elude diagnosis by genomics—substantial discovery research is still needed to improve diagnostic yield.

 

Further challenges of RD research related to the complexity of understanding disease mechanism, for which animal models can make the needed contributions.

 

Future of rare diseases research 2017-2027: An IRDiRC perspective

Paul further briefly summarized the IRDiRC goals for 2017 which are summarized in  

Austin C et al.,  Future of Rare Diseases Research 2017-2027: An IRDiRC Perspective.

Clin Transl Sci. 2018 Jan;11(1):21-27. doi: 10.1111/cts.12500. Epub 2017 Oct 23.

 

Goal 1, IRDiRC goals 2017:

All patients coming to medical attention with a suspected rare disease will be diagnosed within one year if their disorder is known in the medical literature; all currently undiagnosable individuals will enter a globally coordinated diagnostic and research pipeline.

The publication identifies Rare Disease mechanism discovery as an important element to target goal 1 and states that the number of unsolved patients following whole exome sequencing argues that more disease genes and variants await discovery, thus the discovery effort must be expedited. So far, most of the known disease variants fall in coding regions of the genome, but much less is known, for example, about the role of non-coding region variants and structural variants in disease. Functional analyses at scale will need to be developed to facilitate variant interpretation in conjunction with data sharing.

This is an area where animal models and in particular precision mouse models engineered with state of the art genome editing technology such as CRISPR/Cas9 can make invaluable contributions.

 

Finally, Paul Lasko suggested specific actions how the infrastructure and expertise of INFRAFRONTIER and IMPC can be aligned with RD research efforts:

  • Prioritizing RD-relevant mouse models, animal models are specifically mentioned in European Joint Program in Rare Diseases. 
  • Proactive seeding of collaborations with clinical researchers, building on the example of the Rare Diseases Models and Mechanisms initiative.
  • Possibly interacting with IRDiRC to develop a priority list of rare diseases to be de-risked through public efforts to enable therapeutic development.