Research Programme

To facilitate the goals of the project, STACCATO is divided into 3 scientific Work Packages (WP 1-3) with supporting WPs on Industry and
Academic Training (WP4), Dissemination and Exploitation (WP5) & Management and Organisation (WP6).

WP1 focuses on developing a new tool for cell
culture process development. ESRs will focus on adapting BD’s highly accurate molecular indexing technology and the nanowell-based Rhapsody platform. Through collaboration with those ESRs working on cell lines originating from non-model organisms they will develop and validate Rhapsody primer panels targeting pathways such as glycosylation. At the outset of the project will employ BD’s Precise single cell gene expression system. Precise utilises polyA capture probes and is capable of whole transcriptome analysis (WTA) on 96 cells sorted via flow cytometry. These initial low-resolution single cell exper-iments will confirm the expression of genes within pathways of interest to aid WP1 activities.

ESRs will develop a single cell mitochondrial DNA (mtDNA) sequencing method to identify mutations in the mitochondrial genomes of single cells. Secondments will enable the ESRs to apply the methodology to monitoring mtDNA mutations over the course of CHO cell HCDP culture and use mitochondrial genome engineering to improve the metabolic performance of CHO cells. ESRs will focus on developing mass spectrometry based single cell proteomics initially for CHO cells in collaboration with other ESRs and subsequently apply the method to insect cell lines and CAR T cells through secondments at iBET and PEI respectively. ESR’s will design bioinformatics and statistical methods for data integration. WP1 is lead by Dr. Siobhan Cashman of BD.

WP2 focuses on enhancing the upstream processes for therapeutic proteins, VLPs, oncolytic virus and gene therapies. ESR’s will conduct a series of screening experiments using the platforms developed in WP1 on libraries of producer cell lines for the production of recombinant proteins and oncolytic viruses to determine how heterogeneity affects production rates and product quality. The results of these analyses will enable the identification of cellular bottlenecks and subsequent utilisation of targeted genome engineering (e.g. CRISPR-Cas9) to optimise production. Through secondments ESRs will gain experience of insect cell culture systems and new cell engineering methods. ESRs will also learn new CRISPR-Cas9 approaches for cell engineering during their time in the project. ESRs will focus on using single cell data to determine the effect of cell culture media and HCDP bioprocess strategies on therapeutic protein producing CHO cells. These analyses will focus on cell health and how bioreactor conditions affect gene expression during the initiation of apoptosis and autophagy.

Through collaboration with ESRs they will correlate mtDNA mutations with media components, metabolic function and cellular growth rate. Another crucial layer of information from single cell proteomics will be gained by ESRs through secondments enabling the development of modelling approaches across multiple levels of the biological system. ESRs will utilise single cell data from insect cell lines to facilitate scale up, design feeding strategies and monitor continuous and hybrid perfusion cell culture for the production of influenza VLP based vaccines and rAAV vectors respectively. ESRs will build mathematical models based on the acquired data. ESRs will gain experience of gene therapy vector production. WP2 is lead by Prof. Paula Alves of iBET.

WP 3 focuses on enhancing manufacturing processes for emerging cellular therapies. ESRs will concentrate on utilising single cell analysis to understand the impact of manufacturing process on CAR T cell phenotypes such as their activation status, exhaustion level and functional activity. Through single cell analysis, ESRs will reveal the impact of the gene delivery vectors and investigate gene delivery  under non-stimulating conditions to enable the production of more  potent CAR-T cells. Both ESRs will spend secondment periods to develop novel Rhapsody gene panels targeting crucial aspects of CAR T cells. ESRs will utilise the technology to aid the development of a new 3D bioreactor system for the differentiation of stem cells to form
endocrine clusters in a 3D cell culture environment based on recombinant spider silk during secondments. Single cell data will be acquired to investigate the underlying biology of human embryonic stems cells (hESCs) and the cell to cell communication within the system in order to determine if the conditions achieved within the bioreactor can mimic the in-vivo environment. ESRs will also utilise single cell proteomics analysis and integrate those data with single cell RNASeq from matching stem cell populations. WP3 will be lead by Dr. Suvi Sorsa of TILT.