An automated platform for lentiviral transduction for HSC gene therapy promotes fitness of hematopoietic stem cells combined with higher transduction efficiency

Abstract

Abstract Although therapies for HSC gene therapy have been recently approved (Zynteglo™, Lyfgenia™ and Casgevy®), still certain challenges remain associated with high costs, scalability and safety related to leukemic events. These indicate a need for improved and standardized manufacturing steps allowing for large scale implementation of gene therapy interventions to a degree that addresses the population suffering from the long-term sequalae of these diseases. Automation is the key to achieve these goals and, to this end, the CliniMACS Prodigy as an automated, closed, GMP-compliant platform for HSC transduction, is an ideal candidate to streamline cell processing. We had previously shown that lentiviral (LV) transduction is facilitated on the CliniMACS Prodigy compared to manual steps when a VSVg pseudotyped LV expressing GFP was utilized. In the current study, we extended these observations by employing LVs pseudotyped with BaEV (Baboon Endogenous retrovirus) combined with a modification to our original transduction medium which now contains only the “basic” necessary cytokines: SCT, FLT3L and TPO. With the new modifications we observed comparable cell recovery and viability between comparator manual and automated conditions. Importantly, we demonstrate superior performance of the automated platform in terms of transduction efficiency. Moreover, BaEV pseudotyped vectors demonstrated superior transduction efficiency even with a very low MOI (0.75-5) in the presence of Vectofusin-1 transduction enhancer (histidine rich amphipathic peptide). In vivo experiments using NSG mice, also demonstrated better engraftment for the CD34+ cells processed on the CliniMACS Prodigy compared to the manually cultivated CD34+ cells and also compared to the “gold standard” i.e. uncultivated and unmanipulated CD34+ transplanted cells directly after enrichment (Day 0), suggesting that the automated platform promotes or preserves HSC fitness. HSC fitness was corroborated by the consistently higher number of CFUs generated by the cells processed on the CliniMACS Prodigy vs cells processed manually which showed a clear trend towards decreasing numbers of CFUs. To investigate the higher transduction efficiency and the sustained clonogenic capacity of the automated process, we conducted bulk transcriptome profiling via next generation RNA sequencing (bulk RNAseq) across all conditions. Principal component analysis (PCA) highlighted that samples cultivated on the CliniMACS Prodigy cluster closer to the control samples at Day 0, indicating that the automated cultivation process preserves of cellular features similar to day 0 HSPCs at experimental onset. Functional gene expression enrichment analysis revealed distinct expression patterns between the automated platform and the manual steps with the most enriched pathways downregulated in the automated platform relating to generic immune responses, including autoimmunity related pathways, and downregulation of genes involved in infectious disease and immune disease pathways. Remarkably, cells cultivated on the CliniMACS Prodigy also downregulated CD38 expression and Toll-like receptor 8 (TLR8), a gene which is known to recognize and degrade single-stranded RNA (ssRNA). Upregulated gene pathways included a) glycine, serine and threonine metabolism (a key metabolic hub associated with cell lifespan) b) cholesterol binding c) and sterol binding pathways. Notably, low-density lipoprotein receptor (LDL-R), the cellular receptor that serves for the recognition by the VSV glycoprotein, was accordingly upregulated in cells processed on the CliniMACS Prodigy. These results suggest that the CliniMACS Prodigy promotes higher stemness or better fitness of HSCs while simultaneously allowing for higher transduction efficiencies due to downregulation of immune related pathways and upregulation of cholesterol and sterol binding.