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引导的内核酸酶（RGENS）（例如CRISPR-CAS9系统）彻底改变了我们编辑基因组的能力，因为它们允许我们按照RNA指南定义的序列切割和编辑基因组，该指南可以快速设计和制造。这对分子生物学产生了巨大影响。允许编辑各种不同细胞类型和生物的基因组。下一个挑战是使用该技术编辑人类细胞的基因组来治疗疾病。造血症是该技术将在诊所成功实施的第一个领域之一，因为造血细胞（HSC）移植已经进行了40多年，并且已经建立了收集，纯化和移植人类HSC的设施。 在患者中使用基因组编辑技术的主要障碍之一是在编辑过程中意外突变发生恶性转变的潜在风险。为了改善基因组编辑技术，可以在人类试验中安全地使用它们，这一点至关重要的是，必须开发测定以定义经过基因组编辑的原代细胞中的突变。这些关闭目标效应需要以公正的基因组范围的方式准确地定义，并且测定必须高度敏感，以便可以检测到罕见的关闭靶突变，因为恶性肿瘤是由单个细胞的克隆转化引起的。迄今为止，已经描述了确定目标效应的几种方法，但是这些技术都无法在原代细胞中敏感和准确地定义突变。这是这些技术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，那么很难开发安全的编辑方法以用于人类试验。我将开发一种新的方法，该方法最初识别裸体DNA中体外RGEN的所有潜在位点（以前已证明这是对靶向活性的潜在位点的高度预测）。随后，这些位点将使用旨在在体外鉴定的目标位点捕获DNA的生物素化寡核苷酸在深度进行测序。使用此方法应该相对简单地使测定至少比最佳可用方法高100倍。我还将使用这种方法来寻找病毒载体和模板序列的意外插入的罕见位点。还将研究在编辑过程中发生的突变的总负担。这将通过在编辑过程之前和之后通过流式细胞仪纯化的造血干细胞和祖细胞上进行整个基因组测序来完成。方法将用于最大程度地减少测序误差，并写入软件，以定义突变的整体负担，从测序得出的序列的变异性。这些方法将用于优化两种基因组编辑模型，用于治疗thalassyal和镰状细胞疾病，以实现潜在的临床用途。首先，在宿主实验室中开发了一个模型，该模型旨在治愈依赖于输血的HBEβ地中海贫血的患者，该患者通过删除α球蛋白基因的关键转录因子结合位点来占依赖于输血的50％。另外，将建立建立的方法，该方法使用RGEN插入DNA模板以校正原位β球蛋白基因并同时表达允许纯化校正细胞的细胞表面标记。这将使我能够测试量化目标对使用RGEN进行编辑单元格的两个主要策略的关闭目标影响的方法。

项目成果

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

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

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

• DOI: 10.1101/2021.08.10.455796
• 发表时间: 2021-01-01
• 期刊: Preprint at bioRxiv
• 作者: Aljahani, A.

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