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

红细胞（RBC）或红细胞是人体中最丰富的细胞类型。它们具有良好的生命周期特征和独特的生物物理特性，使其成为新疗法的有吸引力的平台。红细胞已经进化为氧气运输，作为天然的货物载体存在。它们的寿命约为120天，覆盖广泛的循环范围，可变形，并具有良好的表面积与体积比。自身标记物的存在使它们具有生物相容性，并且它们在体内安全降解。因此，感兴趣的是利用这些特性并工程化具有治疗益处的红细胞，包括延长寿命或将其开发为药物递送载体。红细胞生成是红细胞产生的过程。在最后阶段，定向祖细胞经历基因表达的巨大变化，以合成大量的红细胞特异性蛋白质，如带3和血红蛋白。这种对红细胞特异性基因表达的驱动导致通常存在于哺乳动物细胞中的许多基因网络的抑制。随着包括CRISPR-Cas9在内的强大基因工程技术的迅速发展，这个未使用的遗传物质库为我们提供了一个令人兴奋的机会来重新利用和功能化红细胞。这可以通过上调现有基因以及在早期成红细胞中插入新的遗传物质来实现。先前培养成红细胞的方法涉及从外周供体血液中分离干细胞，其在特定生长条件下可以扩增和分化以形成功能性未成熟红细胞（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。修改不容易保存，每次都必须从头开始。最近开发的永生化红细胞前体细胞系，布里斯托红细胞系成人（BEL-A），提供了一个模型细胞系统，其中遗传操作的前体细胞可以无限维持，储存和分化成功能性网织红细胞。CRISPR-Cas9已成功用于在BEL-A细胞中产生敲除，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性成红细胞细胞系的原理证明。我们的目标是在这一令人兴奋的创新基础上，扩展CRISPR-Cas9工具箱，以涵盖基因插入和激活。这将首次允许重新激活或新表达扩展RBC功能的遗传网络。在我的轮换期间，我们已经开发了包含spycatcher缀合带3蛋白的永生化成红细胞细胞系（未发表的工作）。带3是一种高丰度（每个细胞约120万拷贝）的红细胞膜蛋白，可促进阴离子交换并作为主要的锚定位点。这为我们提供了一个“即插即用”的中心点，将候选酶系在膜上并建立代谢途径。理想情况下，这将有助于优化途径组分的空间构型，并保护蛋白质在分化过程中不发生错误定位。使用从头设计的卷曲螺旋作为蛋白质-蛋白质相互作用位点也将在更复杂的途径的构建中进行探索。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加强红细胞对氧化应激的敏感性。这将潜在地改善成熟红细胞的长期储存，并且在理论上也有助于延长循环中的生命周期。非典型蛋白质的新用途还需要蛋白质工程以实现最佳表达、稳定性和功能。工程RBC将进行广泛的测试，以确保关键的天然细胞特性不受影响。该项目福尔斯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.