Abdul-Razak, HH

Alonso-Ferrero, ME

Bartholomae, C

Blundell, MP

Bueren, JA

Gabriel, R

Gan, CHV

Garin, MI

Gregory, PD

Gueenechea, G

Holmes, MC

Howe, SJ

Kinnon, C

Molina-Estevez, FJ

Pantoglou, J

Prakash, V

Roberts, A

Rocca, C

Schmidt, M

Thrasher, AJ

von Kalle, C

Wang, J

Yanez-Munoz, RJ

UCL Discovery

Molecular Evidence of Ex Vivo Gene Editing in a Mouse Model of Immunodeficiency  Authors Hayder H. Abdul-Razak1, Céline Rocca1, Steve J. Howe2, Maria Eugenia Alonso-Ferrero2, Jianbin Wang3, Richard Gabriel4, Cynthia Bartholomae4, C. H. Victor Gan2, Marina I. Garín5, Alison Roberts6, Michael P. Blundell2, Versha Prakash1, Francisco Javier Molina-Estevez1, Julia Pantoglou1, Guillermo Güenechea5, Michael C. Holmes3, Philip D. Gregory3, Christine Kinnon2, Christof von Kalle4, Manfred Schmidt4, Juan A. Bueren5, Adrian J. Thrasher2, Rafael J. Yáñez-Muñoz1  1Advanced Gene and Cell Therapy Lab (AGCTlab.org), School of Biological Sciences, Royal Holloway, University of London, Egham, United Kingdom,2Institute of Child Health, University College London, London, United Kingdom,3Sangamo Biosciences, Richmond, CA,4Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany,5Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain,6King's College London, London, United Kingdom . Abstract Gene editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by the use of designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that gene editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. Here we show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in Prkdc scid fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, definite proof of the ability of these cells to mediate some level of reconstitution has been obtained by deep sequencing of tissues from some of the recipients, which carried the expected genomic signature of ZFN-driven gene correction (correction of the scid mutation associated to the introduction of a BsaWI diagnostic site). This signature has been observed in peripheral blood mononuclear cells (PBMC), thymus, spleen and purified spleen CD3 and CD8 T-cells. Levels are variable in the positive primary transplanted animals, higher with integration-proficient lentiviral vector (IPLV)-ZFN than integration-deficient lentiviral vector (IDLV)-ZFN, and were also observed in secondary recipients. In some primary and secondary transplant recipients we also detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. We consider the presence of the genome editing signature proof that reconstitution can be achieved, albeit inefficiently both in terms of the number of positive animals and the levels in different tissues. This Prkdc model is deficient in non-homologous end-joining, and this deficiency may be responsible for the frequency of a recurrent 44-bp deletion including the ZFN target site that we have observed in multiple samples in vitro and ex vivo. This highlights the importance of considering the possible effect of DNA damage repair defects on gene editing strategies. Acknowledgements: the authors received financial support from the 7th EU Framework Programme (PERSIST project, grant agreement no. 222878) and the Primary Immunodeficiency Association. H.H.A.-R. and C.R. contributed equally to this work 

Molecular Evidence of Ex Vivo Gene Editing in a Mouse Model of Immunodeficiency

http://discovery.ucl.ac.uk/1559133/1/Thrasher_Molecular%20Evidence%20of%20Ex%20Vivo%20Gene%20Editing%20in%20a%20Mouse%20Model%20of%20Immunodeficiency.pdf

Molecular Evidence of Ex Vivo Gene Editing in a Mouse Model of Immunodeficiency

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UCL Discovery