The STACCATO project was comprised of three scientific work packages:
The WP1 team, focussed on the development of experimental and computational methods for single cell RNA sequencing (scRNA-seq) using the BD Rhapsody platform, single cell mtDNA sequencing and the measurement of protein abundance in individual cells. The activities of WP1 enabled the first single cell studies of biopharmaceutical cell factories. The Rhapsody system was also successfully applied to chimeric antigen receptor (CAR)-T cell therapies and to human embryonic stem cell derived pancreatic aggregates. To study mtDNA mutations, a method was developed to detect mitochondrial heteroplasmy in individual CHO cells The consortium was also able to measure protein abundance for CHO cells and CAR-T cell therapies using BD’s AbSeq system.
WP2 centred on the analysis of cell factories used to produce recombinant proteins, gene therapies, vaccines, and oncolytic viruses. Over the course of the project the first-ever single cell study of CHO cells was accomplished. Additional experiments were carried out to investigate additional phenotypes and probe the correlation between gene expression and recombinant protein abundance in two mAb producing CHO cell lines. The 10X Genomics Chromium platform was also used to capture chromatin accessibility profiles in CHO cells for the first time. Further experiments in CHO cells focused on uncovering the relationship between the cell specific perfusion rate (CSPR) in high cell density perfusion culture (HCDP) and a high throughput platform for media screening has been designed. In addition the BD Rhapsody whole transcriptome analysis (WTA) was also used to capture scRNA-seq data for A549 producing oncolytic viruses. WP2 researchers working with insect cell systems developed customised cell culture strategies to increase titer for enhancing virus like particle (VLP) and recombinant adeno-associated virus (AAV). This work enabled the successful application of the Rhapsody system for the Sf9 and Hi5 insect cell lines, which allowed transcriptional heterogeneity to be captured at single cell resolution for the first time.
The third STACCATO work package focussed on the application of single cell technology for the analysis of exciting new therapies where living cells are utilised for treatment. For instance, STACCATO generated new knowledge that has the potential to improve the manufacture of CAR T cells by understanding heterogeneity introduced during the manufacturing process. STACCATO completed a range single cell analyses of CAR T-cells. STACCATO partners have also developed an efficient pancreatic differentiation protocol resulting in an increased yield of insulin-positive cells and functional glucose-stimulated insulin secretion.
Overcoming the inherent variability of cells grown in vitro to deliver effective, uniform and safe biological medicines is one of the most significant challenges faced by the industry today. The STACCATO research programme developed advanced experimental and computational methods to characterise manufacturing systems used in the biopharmaceutical industry with an unprecedented level of detail. During the project, the STACCATO ESRs contributed in total to 34 scientific publications comprising 11 published papers with a further 23 manuscripts in preparation for submission to peer-reviewed journals. STACCATO also contributed to 22 conferences. To further disseminate the knowledge gained throughout the project, we have created the STACCATO website, which includes a dedicated STACCATO Wiki, a searchable online platform hosted on the STACCATO website, that contains a collection of various terms related to the project. In addition, a series of podcasts were developed to communicate the research finding and experience to a broad audience.
