Development of highly sensitive methods for defining off target mutations to enable safe gene editing of haematopoietic cells for transplantation

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RNA guided endonucleases (RGENs), such as the CRISPR-Cas9 system have revolutionised our ability to edit the genome because they allow us to cut and edit the genome at the sequence defined by a RNA guide, which can be rapidly designed and manufactured. This has had a huge impact on molecular biology; allowing the genome of a wide variety of different cell types and organisms to be edited. The next challenge is to use this technology to edit the genome of human cells to treat disease. Haematopoiesis is one of the first areas in which this technology will be successfully implemented in the clinic because haematopoietic stem cell (HSC) transplantation has been performed for over 40 years and facilities for collecting, purifying and transplanting human HSCs are well established. One of the major barriers to using genome editing technology in patients is the potential risk of malignant transformation from unintended mutations during the editing process. In order to improve genome editing techniques to the point at which they can be safely used in human trials it is critical that assays are developed to define mutations in primary cells that have undergone genome editing. These off target effects need to be defined accurately in an unbiased genome wide fashion and the assays need to be highly sensitive so that rare off target mutations can be detected because malignancy arises from clonal transformation of single cells. To date, several methods have been described for determining off target effects but none of the techniques is able to define mutations sensitively and accurately in primary cells. This is a key stumbling block to the clinical application of these techniques because without rapid and cost effective methods to compare the accuracy of the huge variety of different techniques available for genome editing it will be difficult to develop safe methods of editing for use in human trials. I will develop a novel method that initially identifies all potential sites of in vitro RGEN in naked DNA (this has previously been shown to be a highly predictive of potential sites of off target activity). Subsequently these sites will be sequenced at great depth using biotinylated oligonucleotides designed to capture DNA at the target sites identified in vitro. Using this it should be relatively straightforward to make the assay is at least 100 times more sensitive than the best available methods. I will also use this approach to look for rare sites of unintended insertion of viral vectors and template sequences. The overall burden of mutations that occur during the editing process will also be investigated. This will be done by performing whole genome sequencing on haematopoietic stem and progenitor cells purified by flow cytometry both before and after the editing process. Methods will be used to minimise sequencing errors and software will be written to define the overall burden of mutations from the variability of the sequences derived from sequencing. These methods will be used to optimise two models of genome editing for the treatment of thalassaemia and sickle cell disease for potential clinical use. First a model has been developed in the host laboratory which aims to cure patients with transfusion dependent HbE beta thalassaemia, which accounts for around 50% of transfusion dependent thalassaemia, by deleting key transcription factor binding sites at the alpha globin gene. In addition an established method will be set up, which uses an RGEN to insert a DNA template to correct the beta globin gene in situ and simultaneously express a cell surface marker that allows purification of corrected cells. This will allow me to test the methods for quantification of off target effects on the two main strategies for using RGENs for editing cells.