Gene Editing Immortalised Erythroblasts to Generate Engineered Red Cells with Novel Functionality

红细胞(RBC)是人体内含量最丰富的细胞类型。它们具有独特的生命周期和独特的生物物理性质，使其成为新疗法的诱人平台。在进化为氧气运输后，红细胞以天然货物载体的形式存在。它们的寿命约为120天，覆盖广泛的循环范围，可变形，具有良好的表面积与体积比。自我标记物的存在使它们具有生物相容性，它们在体内可以安全地降解。因此，开发这些特性并设计具有治疗作用的红细胞是很有意义的，包括延长寿命或将其开发为药物输送载体。红细胞生成是红细胞产生的过程。在最后阶段，承诺的祖细胞经历基因表达的戏剧性变化，以合成大量的红系特有蛋白，如带3和血红蛋白。这种对红系特异性基因表达的驱动，导致了许多通常存在于哺乳动物细胞中的基因网络的抑制。随着包括CRISPR-CAS9在内的强大基因工程技术的快速发展，这个未使用的遗传材料库为我们提供了一个重新调整红细胞用途和使其功能化的令人兴奋的机会。这可以通过上调现有基因以及在早期红细胞细胞中插入新的遗传物质来实现。以前培养红细胞的方法包括从外周供者血液中分离干细胞，在特定的生长条件下，干细胞可以扩大和分化为功能不成熟的红细胞(网织红细胞)。虽然这一系统已被证明是必不可少的，但这一过程既耗时又费力。更改不容易保存，每次都必须从头开始该方法。永生化红细胞前体细胞系布里斯托尔红系成体(BEL-A)的最新发展提供了一种模型细胞系统，在该系统中，基因操纵的前体细胞可以无限地维持、储存和分化为有功能的网织红细胞。CRISPR-Cas9已成功用于在BEL-A细胞中产生基因敲除，以去除特定的血型。这提供了多个编辑可以组合并导致第一个报道的多相容红细胞细胞系的原理证据。我们的目标是在这一令人兴奋的创新的基础上，扩展CRISPR-Cas9工具箱，以涵盖基因插入和激活。这将第一次允许重新激活或以新颖的方式表达扩大红细胞功能的遗传网络。在我的轮值期间，我们开发了一种永生化的红细胞细胞系，其中包含SpyCatcher共轭带3蛋白(未发表的工作)。带3是一种高度丰富的红细胞膜蛋白(约120万拷贝/细胞)，促进阴离子交换，是主要的锚定部位。这为我们提供了一个“即插即用”的中心点，将候选酶连接到膜上，并建立代谢途径。理想情况下，这将有助于优化途径组件的空间配置，并保护蛋白质在分化过程中不受错位定位。在构建更复杂的通路时，还将探索使用从头设计的螺旋线圈作为蛋白质-蛋白质相互作用位点。最初的努力将集中在应用这些新的方法，通过增加抗氧化酶或途径来增强红细胞对氧化应激的敏感性。这可能会改善成熟红细胞的长期储存，并在理论上有助于延长流通中的生命周期。非典型蛋白质的新用途还需要蛋白质工程来实现最佳的表达、稳定性和功能。工程红细胞将接受广泛的测试，以确保关键的本地细胞特性不会受到影响。该项目属于EPSRC合成生物学领域。

Red blood cells (RBCs), or erythrocytes, are the most abundant cell type in the human body. They possess a well characterised life cycle and unique biophysical properties that make them an attractive platform for novel therapeutics. Having evolved for oxygen transportation, RBCs present as natural cargo carriers. They exhibit a life-span of about 120 days, cover an extensive circulatory range, are deformable and boast a favourable surface to volume ratio. The presence of self-markers makes them biocompatible and they are safely degraded in the body. It is therefore of interest to exploit these properties and engineer erythrocytes with therapeutic benefits including, extending life-span or developing them as drug delivery vehicles. Erythropoiesis is the process of RBC production. During the final stages, committed progenitor cells undergo dramatic changes in gene expression to synthesise vast amounts of erythroid specific proteins, such as band 3 and haemoglobin. This drive towards erythroid specific gene expression, results in the suppression of many gene networks normally present in mammalian cells. With powerful genetic engineering techniques, including CRISPR-Cas9, rapidly evolving, this library of unused genetic material presents us with an exciting opportunity to repurpose and functionalise erythrocytes. This could be achieved through the upregulation of existing genes as well as the insertion of new genetic material in early erythroblast cells. Previous approaches to culturing erythroblasts have involved stem cell isolation from peripheral donor blood which, under specific growth conditions, can expand and differentiate to form functional immature erythrocytes (reticulocytes). Whilst this system has proved indispensable, the process is time consuming and laborious. Alterations cannot be easily saved and the method must be started from scratch each time. The recent development of immortalised erythrocyte precursor cell lines, Bristol Erythroid Line Adult (BEL-A), provides a model cellular system where genetically manipulated precursor cells can be infinitely maintained, stored and differentiated into functioning reticulocytes. CRISPR-Cas9 has successfully been used to generate knockouts in BEL-A cells to remove specific blood groups. This provides proof of principle that multiple edits can be combined and led to the first reported multi-compatible erythroblast cell line. We aim to build on this exciting innovation and extend the CRISPR-Cas9 toolbox to encompass gene insertion and activation. This would, for the first time, allow for reactivation or novel expression of genetic networks that expand RBC functionality. During my rotation, we have developed an immortalised erythroblast cell line comprising spycatcher conjugated band 3 protein (unpublished work). Band 3 is a highly abundant (~1.2million copies per cell) RBC membrane protein that facilitates anion exchange and serves as a major anchorage site. This provides us with a 'plug-and-play' centre point to tether candidate enzymes to the membrane and build metabolic pathways. Ideally, this will help optimise spatial configuration of pathway components and also protect proteins from mislocalisation during differentiation. The use of de novo designed coiled-coils as protein-protein interaction sites will also be explored in the construction of more complex pathways. Initial efforts will focus on applying these novel approaches to strengthen erythrocyte sensitivity to oxidative stress by increasing antioxidant enzymes or pathways. This will potentially improve long-term storage of mature RBCs and also theoretically facilitate a prolonged life cycle when in circulation. The novel use of atypical proteins will also require protein engineering to achieve optimal expression, stability and function. Engineered RBCs will undergo extensive testing to ensure key native cellular properties are not compromised. This project falls within the EPSRC synthetic biology area.