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Generation, quality control, and analysis of the first genomically humanised knock-in mice for the ALS/FTD genes SOD1, TARDBP (TDP-43), and FUS

Anny Devoy, Georgia Price, Francesca De Giorgio, Rosie Bunton-Stasyshyn, David Thompson, Samanta Gasco, Alasdair Allan, Gemma F. Codner, Remya R. Nair, Charlotte Tibbit, Ross McLeod, Zeinab Ali, Judith Noda, Alessandro Marrero-Gagliardi, José M Brito-Armas, Michelle Simon, Edward O’Neill, Jackie Harrison, Gemma Atkins, Silvia Corrochano, Michelle Stewart, Lydia Teboul, View ORCID ProfileAbraham Acevedo-Arozena, View ORCID ProfileElizabeth M.C Fisher, View ORCID ProfileThomas J. Cunningham
doi: https://doi.org/10.1101/2021.07.05.451113
Anny Devoy
1Department of Neuromuscular Diseases, UCL Institute of Neurology, Queen Square, London, WC1N 3BG
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Georgia Price
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Francesca De Giorgio
1Department of Neuromuscular Diseases, UCL Institute of Neurology, Queen Square, London, WC1N 3BG
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Rosie Bunton-Stasyshyn
1Department of Neuromuscular Diseases, UCL Institute of Neurology, Queen Square, London, WC1N 3BG
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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David Thompson
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Samanta Gasco
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Alasdair Allan
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Gemma F. Codner
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Remya R. Nair
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Charlotte Tibbit
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Ross McLeod
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Zeinab Ali
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Judith Noda
3Research Unit, Hospital Universitario de Canarias; ITB-ULL and CIBERNED, La Laguna, 38320, Spain
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Alessandro Marrero-Gagliardi
3Research Unit, Hospital Universitario de Canarias; ITB-ULL and CIBERNED, La Laguna, 38320, Spain
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José M Brito-Armas
3Research Unit, Hospital Universitario de Canarias; ITB-ULL and CIBERNED, La Laguna, 38320, Spain
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Michelle Simon
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Edward O’Neill
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Jackie Harrison
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Gemma Atkins
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Silvia Corrochano
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Michelle Stewart
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Lydia Teboul
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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Abraham Acevedo-Arozena
3Research Unit, Hospital Universitario de Canarias; ITB-ULL and CIBERNED, La Laguna, 38320, Spain
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  • ORCID record for Abraham Acevedo-Arozena
  • For correspondence: elizabeth.fisher@ucl.ac.uk aacevedo@ull.edu.es t.cunningham@har.mrc.ac.uk
Elizabeth M.C Fisher
1Department of Neuromuscular Diseases, UCL Institute of Neurology, Queen Square, London, WC1N 3BG
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  • ORCID record for Elizabeth M.C Fisher
  • For correspondence: elizabeth.fisher@ucl.ac.uk aacevedo@ull.edu.es t.cunningham@har.mrc.ac.uk
Thomas J. Cunningham
2UK MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK
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  • ORCID record for Thomas J. Cunningham
  • For correspondence: elizabeth.fisher@ucl.ac.uk aacevedo@ull.edu.es t.cunningham@har.mrc.ac.uk
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SUMMARY

Amyotrophic lateral sclerosis - frontotemporal dementia spectrum disorder (ALS/FTD) is a complex neurodegenerative disease; up to 10% of cases are familial, usually arising from single dominant mutations in >30 causative genes. Transgenic mouse models that overexpress human ALS/FTD causative genes have been the preferred organism for in vivo modelling. However, while conferring human protein biochemistry, these overexpression models are not ideal for dosage-sensitive proteins such as TDP-43 or FUS.

We have created three next-generation genomically humanised knock-in mouse models for ALS/FTD research, by replacing the entire mouse coding region of Sod1, Tardbp (TDP-43) and Fus, with their human orthologues to preserve human protein biochemistry, with exons and introns intact to enable future modelling of coding or non-coding mutations and variants and to preserve human splice variants. In generating these mice, we have established a new-standard of quality control: we demonstrate the utility of indirect capture for enrichment of a region of interest followed by Oxford Nanopore sequencing for robustly characterising large knock-in alleles. This approach confirmed that targeting occurred at the correct locus and to map homologous recombination events. Furthermore, extensive expression data from the three lines shows that homozygous humanised animals only express human protein, at endogenous levels. Characterisation of humanised FUS animals showed that they are phenotypically normal compared to wildtype littermates throughout their lifespan.

These humanised mouse strains are critically needed for preclinical assessment of interventions, such as antisense oligonucleotides (ASOs), to modulate expression levels in patients, and will serve as templates for the addition of human ALS/FTD mutations to dissect disease pathomechanisms.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ^ now at Laboratorio de InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón; Spanish HIV BioBank, Instituto de Investigación Sanitaria Gregorio Marañón (IISGM) C/Dr. Esquerdo 46, 28007 Madrid, Spain

  • ↵◆ now at United Kingdom Dementia Research Institute Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Maurice Wohl Clinical Neuroscience Institute, London, UK, SE5 9RT

  • ↵# now at Grupo Enfermedades Neurológicas, Hospital Clínico San Carlos, IdISSC, Madrid, 28040, Spain

  • ↵* Co-senior authors

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 05, 2021.
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Generation, quality control, and analysis of the first genomically humanised knock-in mice for the ALS/FTD genes SOD1, TARDBP (TDP-43), and FUS
Anny Devoy, Georgia Price, Francesca De Giorgio, Rosie Bunton-Stasyshyn, David Thompson, Samanta Gasco, Alasdair Allan, Gemma F. Codner, Remya R. Nair, Charlotte Tibbit, Ross McLeod, Zeinab Ali, Judith Noda, Alessandro Marrero-Gagliardi, José M Brito-Armas, Michelle Simon, Edward O’Neill, Jackie Harrison, Gemma Atkins, Silvia Corrochano, Michelle Stewart, Lydia Teboul, Abraham Acevedo-Arozena, Elizabeth M.C Fisher, Thomas J. Cunningham
bioRxiv 2021.07.05.451113; doi: https://doi.org/10.1101/2021.07.05.451113
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Generation, quality control, and analysis of the first genomically humanised knock-in mice for the ALS/FTD genes SOD1, TARDBP (TDP-43), and FUS
Anny Devoy, Georgia Price, Francesca De Giorgio, Rosie Bunton-Stasyshyn, David Thompson, Samanta Gasco, Alasdair Allan, Gemma F. Codner, Remya R. Nair, Charlotte Tibbit, Ross McLeod, Zeinab Ali, Judith Noda, Alessandro Marrero-Gagliardi, José M Brito-Armas, Michelle Simon, Edward O’Neill, Jackie Harrison, Gemma Atkins, Silvia Corrochano, Michelle Stewart, Lydia Teboul, Abraham Acevedo-Arozena, Elizabeth M.C Fisher, Thomas J. Cunningham
bioRxiv 2021.07.05.451113; doi: https://doi.org/10.1101/2021.07.05.451113

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