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

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.

The emerging and dynamic field of Synthetic Biology has the potential to provide solutions to some of the key challenges faced by society, ranging across the healthcare, energy, food and environmental sectors. The UK government has recently a "Synthetic Biology Roadmap", which presents a vision and direction for Synthetic Biology in the UK. The report projects that the global Synthetic Biology market will grow from $1.6bn in 2011 to $10.8bn by 2016. It highlights that there is an urgent need for the UK to develop the interdisciplinary skills required to take advantage of the opportunities provided by Synthetic Biology.

The challenge to the academic and industrial research communities is to develop new translational approaches to ensure that these potential benefits are realised. These new approaches will range across the design and engineering of biologically based parts, devices and systems as well as the re-design of existing, natural biological systems across all scales from molecules to organisms. The techniques will encompass not only individual cells, but also self-assembled biomimetic systems, engineered microbial communities and multicellular organisms, combining multiple perspectives drawn from the engineering, life and physical sciences.

Realising these goals will require a new generation of skilled interdisciplinary scientists, and the training of these scientists is the primary goal of the SBCDT. Our programme will give the breadth of coverage to produce a "skilled, energized and well-funded UK-wide synthetic biology community", who will have "the opportunity to revolutionise major industries in bio-energy and bio-technology in the UK" (David Willetts, Minister for Universities and Science) in their future careers. This will be made possible through genuine inter-institutional collaboration in partnership with key industrial, academic and public facing institutions.

The potential impact of the SBCDT, and its potential national importance, are very therefore high, and the potential benefits to society are significant.