Specificity of CD8-Targeted Fusosomes in Human PBMCs Using Single Cell RNA and T Cell Receptor Sequencing

Introduction:The ability to deliver genes to specific cell types in vivowould have a profound therapeutic impact for a diverse set of diseases. For example, targeting T cells for in vivodelivery of a chimeric antigen receptor (CAR) to treat B cell malignancies would improve access to CAR T therapies by overcoming the limitations of ex vivomanufacturing such as high costs, wait times and manufacturing failures. We have developed a novel paramyxovirus-based integrating vector (fusosomes) that specifically targets cell surface receptors for targeted gene delivery. Fusosomes, engineered to target CD8α, a cell surface protein expressed on CD8+ T cells, can bind and specifically deliver a genetic payload through membrane fusion. To evaluate the specificity of fusosome-mediated delivery to cells expressing CD8α in vitro, single cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (scTCR-seq) were performed on human PBMCs treated with CD8α-targeted fusosomes with a GFP payload. scRNA-seq is a tool that can be used to detect the transgene delivered by our fusosomes in specific cell populations by measuring mRNA expression of the receptor targeted by the fusosome (e.g., CD8α) and the genetic payload delivered by the fusosome in the same cell. Transcriptome information to understand potential pathway changes induced by delivery of the transgene is also captured.