Advancing the UCOE-based gene expression platform fo r improved bioma nufacturing and gene therapy applications.

Background.
Ubiquitous chromatin opening elements (UCOEs) consist of at least one promoter region with associated methylation-free CpG island from housekeeping genes and confer stable transgene expression. UCOE-based vectors have seen commercial success in biomanufacturing and provide unprecedented stability of expression from lentiviral vectors (LVs) for gene therapy applications. However, advances in understanding of transcriptional and post-transcriptional control mechanisms imply design of UCOE-based vectors can be improved. This project aims to improve UCOE-based vectors for (i) biomanufacturing and (ii) gene therapy. The workplan will use the prototypical UCOE from the human HNRPA2BI-CBX3 (A2UCOE) and the murine Rps3 loci.

Importance of project.
The need for reproducible and stable ubiquitous or tissue specific function is at the core of basic research investigating gene and protein function, effective gene therapy and industrial protein biomanufacturing processes. Therefore given the generic nature of the UCOE-based vector developments planned in this project, the impact from this work will be far reaching in both academic and industrial sectors. The development of robust and versatile next generation UCOE-based vectors to be developed will greatly expedite the work of researchers within academia, biomanufacturing. and gene therapy settings by providing access to state-of-the-art technology in the gene expression field.

Research methods.
The candidate will acquire a broad knowledge of basic molecular biology and gain training in a wide range of mammalian cell tissue culture and molecular biology (e.g. recombinant DNA. cell transfection, lentiviral vectors, DNA/RNA analysis, flow cytometry) research techniques. Importantly, the project will provide first-hand experience of taking a basic science investigation through to consideration for commercial scale use. The skills acquired will be used to address the following project aims:

Aim 1. Improving the efficiency of plasmid vectors in biomanufacturing.
Pre-mRNA splicing, 3'end formation and transcription termination are coupled, rate limiting steps in gene expression. Thus intronic, polyadenylation & transcription termination elements will be added to existing EMD Millipore A2 and Rps3 UCOE plasmid vectors to effectively provide all of these post-transcriptional events.
NOTE: promising designs to be tested at 5L bioreactor scale-up during industrial placement.

Aim 2. Generation of UCOE-based L Vs with enhanced safety features.
Adapt existing LVs in academic lead 's laboratory to generate generic minimised A2UCOE cassettes for the insertion of any gene of interest. LV designs aimed at minimising A2UCOE genotoxicity, which can occur via aberrant splicing and read through transcripts from the A2UCOE, will be addressed by inclusion of appropriately located miRNA target sites.

Aim 3. Generation ofUCOE-based Sleeping Beauty (SB) transposon vectors.
Although increasingly attractive for gene therapy applications, UCOEs have yet to be tested within SB vectors. Thus A2UCOE based cassettes as those in LVs (Aim 2) will be tested in SB vectors.

Aim 4 Explore UCOE mechanisms of action for future advances.
Conduct a bioinfofmatics investigation to highlight occupied transcription factor (TF) binding sites common to UCOEs that may underlie their function. Functionally test activity of identified TF binding sites by mutational analysis and cell expression studies.