Engineering of complex alleles
Lead Research Organisation:
University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics
Abstract
Mutations associated with human disease can be engineered into the equivalent genes in mice, and the resulting genetically modified mice provide important insights into disease mechanism, revealing new targets for future medicines. Furthermore, these mouse models of human disease have considerable utility for preclinical testing of new medicines and can help the development of new tools to help disease diagnosis.
To date, most disease mutations that have been explored using mouse models are restricted to mutations which affect the DNA encoding proteins. Investigations into genetic variation in humans over the last decade, however, have revealed that mutations and variation in non-protein-coding DNA sequences and the overall structure of human genes play an important role in defining disease and disease-risk. For example, the three dimensional architecture of a gene and its location within a larger domain (spanning 10's to 100's of thousands of DNA sequence bases) impacts gene regulation enormously. This can be perturbed by large variations in the structure of this domain or smaller mutations that interfere with the domain's boundaries. We need to develop tools that enable manipulation and assessment of DNA sequences at much larger scale then conventionally performed to allow an accurate investigation and modelling of human disease in mouse.
Significant differences also exist between mouse and human in these non-coding sequences and overall gene structure. Consequently, more sophisticated modelling of human disease mutations in the mouse is required. This is important both with respect to accurately modelling the human disease in mouse but also for testing new medicines that act on human gene products or new generation therapies that even on human gene sequences.
Methods have been reported by a few research teams around the world, but there is no clear best practice established for engineering the mouse genome in this way. UK researchers are able to access technologies for achieving simple modifications of the genome, through core facilities within their universities or via access to national and international programmes. There is, however, currently no capacity within the UK for the more sophisticated large-scale engineering in the mouse that our understanding of disease biology now demands.
Our cluster will address this unmet need. We will explore and optimize the different experimental parameters and compare different methodologies. We aim to establish robust pipelines for engineering complete human genes and chromosomal segments into the mouse. We will establish proof-of-concept models which encompass key applications of this technology, achieving better disease models and helping our understanding of the biology of disease in areas of cancer, haematology and neurodegeneration. The technology and the resulting models will assist in our understanding of disease and the development of new therapies.
To date, most disease mutations that have been explored using mouse models are restricted to mutations which affect the DNA encoding proteins. Investigations into genetic variation in humans over the last decade, however, have revealed that mutations and variation in non-protein-coding DNA sequences and the overall structure of human genes play an important role in defining disease and disease-risk. For example, the three dimensional architecture of a gene and its location within a larger domain (spanning 10's to 100's of thousands of DNA sequence bases) impacts gene regulation enormously. This can be perturbed by large variations in the structure of this domain or smaller mutations that interfere with the domain's boundaries. We need to develop tools that enable manipulation and assessment of DNA sequences at much larger scale then conventionally performed to allow an accurate investigation and modelling of human disease in mouse.
Significant differences also exist between mouse and human in these non-coding sequences and overall gene structure. Consequently, more sophisticated modelling of human disease mutations in the mouse is required. This is important both with respect to accurately modelling the human disease in mouse but also for testing new medicines that act on human gene products or new generation therapies that even on human gene sequences.
Methods have been reported by a few research teams around the world, but there is no clear best practice established for engineering the mouse genome in this way. UK researchers are able to access technologies for achieving simple modifications of the genome, through core facilities within their universities or via access to national and international programmes. There is, however, currently no capacity within the UK for the more sophisticated large-scale engineering in the mouse that our understanding of disease biology now demands.
Our cluster will address this unmet need. We will explore and optimize the different experimental parameters and compare different methodologies. We aim to establish robust pipelines for engineering complete human genes and chromosomal segments into the mouse. We will establish proof-of-concept models which encompass key applications of this technology, achieving better disease models and helping our understanding of the biology of disease in areas of cancer, haematology and neurodegeneration. The technology and the resulting models will assist in our understanding of disease and the development of new therapies.
Technical Summary
Our cluster addresses the technological challenge of generating complex alleles in the mouse. Whereas simple and conditional gene knock-outs/ins are easily generated at scale, engineering more complex alleles, e.g. humanization of entire mouse genes, loci, chromosomes or disease-associated structural variants, remains a major undertaking with methodology choice unclear. High-throughput core facilities cannot address such alleles resulting in significant community need. Working in ES cells, our cluster aims to develop new methodologies, optimizing, testing and combining CRISPR/Cas9-driven gene targeting, recombinase/integrase cassette exchange and synthetic BAC approaches to develop a robust platform for complex allele generation in mouse.
We aim to profile the utility of such technology in three key areas by generating proof-of-concept models. Firstly, the technology will enable more accurate models of human disease, and two examples in the field of neurodegeneration are envisaged, including a simple repeat expansion disorder, assisting our understanding of this class of disease mutation. Secondly, the technology will serve as a tool for dissecting the regulatory landscape of a gene cluster, providing insights into how perturbations in gene regulation can both contribute to disease and provide future targets for therapies. Lastly, we aim to combine the technology with conditional mutagenesis to allow conditional expression of a regulatory mutation in non-coding DNA in the field of cancer genetics.
The developed technology platform would enable all pathogenic changes in human genomic DNA, not just coding mutations, to be modelled in mouse. Preclinical assessment of advanced therapeutics, such as CRISPR- or antisense-oligonucleotide-based treatments, requiring human target sequences to be present within the model, are made feasible. Securing the generation of complex alleles in the mouse, can extend the application of this model organism to all human genetic disease
We aim to profile the utility of such technology in three key areas by generating proof-of-concept models. Firstly, the technology will enable more accurate models of human disease, and two examples in the field of neurodegeneration are envisaged, including a simple repeat expansion disorder, assisting our understanding of this class of disease mutation. Secondly, the technology will serve as a tool for dissecting the regulatory landscape of a gene cluster, providing insights into how perturbations in gene regulation can both contribute to disease and provide future targets for therapies. Lastly, we aim to combine the technology with conditional mutagenesis to allow conditional expression of a regulatory mutation in non-coding DNA in the field of cancer genetics.
The developed technology platform would enable all pathogenic changes in human genomic DNA, not just coding mutations, to be modelled in mouse. Preclinical assessment of advanced therapeutics, such as CRISPR- or antisense-oligonucleotide-based treatments, requiring human target sequences to be present within the model, are made feasible. Securing the generation of complex alleles in the mouse, can extend the application of this model organism to all human genetic disease
