Abstract
The unique biophysical properties of RBCs render them attractive candidates for the development of novel therapeutic vehicles. Recent advancements in immortalised cell line technologies has provided the field with great potential to produce genetically engineered RBC products at scale. However, our ability to successfully harness these cells for novel diagnostic and therapeutic purposes is both dependent on improving their terminal differentiation capacity, in addition to developing efficient and precise genetic engineering to enable the introduction of novel therapeutic characteristics. Much of the work in this thesis has therefore focussed on addressing these matters in the Bristol erythroid - Adult cell line (BEL-A).Interrogation of whole genome sequencing (WGS) reads, by aligning that of a BEL-A subclone (c79) to the available human reference genome sequence (GRCh38), provided key datasets detailing predicted BEL-A germline and/or somatic mutations. Multiple putative disruptive polymorphisms were identified in several erythroid-specific genes, which are hypothesised to contribute to inherent BEL-A terminal differentiation defects. CRISPR-Cas9 genome editing was applied to demonstrate proof of principle genome repair of ZFPM1, showcasing the value of WGS callsets to direct potentially beneficial genome edits.
Multiple antioxidant enzymes were successfully overexpressed using cDNA lentivirus methods, with their corresponding expression profiles determined throughout terminal differentiation. This work demonstrated a capacity of BEL-A cells for additional antioxidant enzyme expression but also highlighted how the functional benefits of overexpressed proteins can become limited by the bioavailability of ancillary proteins and molecules that exist naturally in an erythroid context.
Finally, considerable effort was made to establish a robust BEL-A line containing CRISPR activation machinery, testing this on multiple genes and benchmarking its output against that of standard lentiviral cDNA overexpression. These studies have helped identify several suitable applications of CRISPRa in erythroid cells, and, in turn, has expanded our CRISPR-Cas9 toolkit and future potential to generate precisely modified cRBCs.
Although not yet successful in overcoming the current BEL-A cell line terminal differentiation defects, the work in this thesis has substantially contributed to ongoing research efforts that aim to harness immortalised erythroid cells as therapeutic products.
| Date of Award | 21 Mar 2023 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Ash M Toye (Supervisor) & Jan Frayne (Supervisor) |
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