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

开发高度灵敏的方法来定义脱靶突变，以实现对移植用造血细胞的安全基因编辑

• 批准号: MR/R008108/1
• 负责人: James Davies
• 金额: $ 132.64万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FR008108%2F1
• 关键词: Development; highly; sensitive; methods; defining

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.

RNA引导的核酸内切酶（RGEN），如CRISPR-Cas9系统，已经彻底改变了我们编辑基因组的能力，因为它们允许我们在由RNA引导物定义的序列处切割和编辑基因组，这可以快速设计和制造。这对分子生物学产生了巨大的影响;允许编辑各种不同细胞类型和生物体的基因组。下一个挑战是利用这项技术编辑人类细胞的基因组来治疗疾病。造血是该技术将在临床上成功实施的第一个领域之一，因为造血干细胞（HSC）移植已经进行了40多年，并且用于收集，纯化和移植人类HSC的设施已经建立。 在患者中使用基因组编辑技术的主要障碍之一是编辑过程中意外突变的恶性转化的潜在风险。为了将基因组编辑技术改进到可以安全地用于人体试验的程度，关键是要开发测定法来定义已经经历基因组编辑的原代细胞中的突变。这些脱靶效应需要以无偏倚的全基因组方式准确定义，并且测定需要高度灵敏，以便可以检测到罕见的脱靶突变，因为恶性肿瘤源于单细胞的克隆转化。迄今为止，已经描述了几种用于确定脱靶效应的方法，但是没有一种技术能够灵敏且准确地定义原代细胞中的突变。这是这些技术的临床应用的关键绊脚石，因为如果没有快速且具有成本效益的方法来比较可用于基因组编辑的各种不同技术的准确性，将难以开发用于人体试验的安全编辑方法。我将开发一种新的方法，该方法最初识别裸DNA中体外RGEN的所有潜在位点（这在以前已被证明是脱靶活性的潜在位点的高度预测）。随后，将使用生物素化的寡核苷酸对这些位点进行深度测序，所述生物素化的寡核苷酸被设计用于捕获体外鉴定的靶位点处的DNA。使用这一点，它应该是相对简单的，使测定是至少100倍以上的灵敏度比最好的可用方法。我还将使用这种方法来寻找病毒载体和模板序列意外插入的罕见位点。还将研究编辑过程中发生的突变的总体负担。这将通过在编辑过程之前和之后对通过流式细胞术纯化的造血干细胞和祖细胞进行全基因组测序来完成。将使用方法来最小化测序错误，并编写软件来定义来自测序序列变异性的总体突变负担。这些方法将用于优化两种基因组编辑模型，用于治疗地中海贫血和镰状细胞病，以供潜在的临床应用。首先，已经在宿主实验室中开发了一种模型，其旨在通过删除α珠蛋白基因处的关键转录因子结合位点来治愈患有输血依赖性HbE β地中海贫血的患者，所述输血依赖性HbE β地中海贫血占输血依赖性地中海贫血的约50%。此外，将建立一种已建立的方法，该方法使用RGEN插入DNA模板以原位校正β珠蛋白基因，同时表达允许纯化校正细胞的细胞表面标记物。这将使我能够测试对使用RGEN编辑细胞的两种主要策略的脱靶效应进行量化的方法。

项目成果

BET inhibition disrupts transcription but retains enhancer-promoter contact.

• DOI: 10.1038/s41467-020-20400-z
• 发表时间: 2021-01-11
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Crump NT;Ballabio E;Godfrey L;Thorne R;Repapi E;Kerry J;Tapia M;Hua P;Lagerholm C;Filippakopoulos P;Davies JOJ;Milne TA
• 通讯作者: Milne TA

Scientific Business Abstracts of the 113th Annual Meeting of the Association of Physicians of Great Britain and Ireland.

大不列颠及爱尔兰医师协会第 113 届年会科学商业摘要。

• DOI: 10.1093/qjmed/hcz175
• 发表时间: 2019
• 期刊: monthly journal of the Association of Physicians
• 作者: Cacciottolo TM

MLL-AF4 cooperates with PAF1 and FACT to drive high-density enhancer interactions in leukemia.

• DOI: 10.1038/s41467-023-40981-9
• 发表时间: 2023-08-25
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Crump, Nicholas T.;Smith, Alastair L.;Godfrey, Laura;Dopico-Fernandez, Ana M.;Denny, Nicholas;Harman, Joe R.;Hamley, Joseph C.;Jackson, Nicole E.;Chahrour, Catherine;Riva, Simone;Rice, Siobhan;Kim, Jaehoon;Basrur, Venkatesha;Fermin, Damian;Elenitoba-Johnson, Kojo;Roeder, Robert G.;Allis, C. David;Roberts, Irene;Roy, Anindita;Geng, Huimin;Davies, James O. J.;Milne, Thomas A.
• 通讯作者: Milne, Thomas A.

Targeted high-resolution chromosome conformation capture at genome-wide scale

• DOI: 10.1101/2020.03.02.953745
• 发表时间: 2020-03
• 期刊: bioRxiv
• 作者: D. Downes;M. Gosden;Jelena M. Telenius;Stephanie J. Carpenter;L. Nussbaum;Sara de Ornellas;M. Sergeant;Chris Eijsbouts;R. Schwessinger;J. Kerry;N. Roberts;Arun Shivalingam;A. El-Sagheer;A. M. Oudelaar;T. Brown;Veronica J. Buckle;James O J Davies;J. Hughes

Analysis of sub-kilobase chromatin topology reveals nano-scale regulatory interactions with variable dependence on cohesin and CTCF.

• DOI: 10.1038/s41467-022-29696-5
• 发表时间: 2022-04-19
• 期刊: Nature communications
• 影响因子: 16.6