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

Autor: Christina Chaivorapol, Chamith Fonseka, Terry J. Fry, Shirisha Amatya, Zach Frye, Saurabh Paliwal, Jagesh V Shah, Samantha Davis, Patricia Cruite, Christopher Bandoro, Hina Iftikhar, Nikolas Balanis, Michael Laska, Lauren Pepper
Rok vydání: 2021
Předmět:
Zdroj: Blood. 138:3983-3983
ISSN: 1528-0020
0006-4971
DOI: 10.1182/blood-2021-149990
Popis: Introduction: The ability to deliver genes to specific cell types in vivo would have a profound therapeutic impact for a diverse set of diseases. For example, targeting T cells for in vivo delivery of a chimeric antigen receptor (CAR) to treat B cell malignancies would improve access to CAR T therapies by overcoming the limitations of ex vivo manufacturing 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. Methods: scRNA-seq was performed using the 10X Genomics system on human PBMCs in vitro. Activated and resting PBMCs from a single donor were transduced with CD8α-targeted fusosomes. Cells were then harvested 3 days post-transduction for scRNA-seq and scTCR-seq. Following library preparation and Illumina sequencing, read processing and bioinformatics analyses were performed using 10X Genomics Cell Ranger and the Seurat R package. Results: In the PBMCs transduced with fusosomes, > 9,000 cells were barcoded with > 1,900 median genes detected per cell. scRNA-seq identified multiple cell types in PBMCs with approximately 25% of cells expressing CD8α transcripts. Fusosome-associated transcripts were seen in about 54% of the cells expressing CD8α and in particular, T cells classified as CD8+ using known markers and classification algorithms based on reference data sets. Subsequently, scTCR-seq data were used to confirm the identity of T cells. Comparison of the results showed an overlap of > 87% of cells classified as T cells by the two independent methods. Visualization by UMAP and inference based on a reference dataset showed that naïve (Tn), central memory (Tcm), effector memory (Tem) and mucosal-associated invariant T cells were transduced by the CD8α-targeted fusosomes. In addition, fusosome-associated transcripts were detected in about 19% of NK cells where approximately 62% of these NK cells also expressed CD8α. Overall, our CD8α-targeted fusosomes have a specificity of > 93% in resting PBMCs based on CD8α expression. A subset of cells may have detectable GFP transcripts at the time of analysis, but not CD8α transcripts due to limited sequencing depth per cell. Summary: Our in vitro scRNA-seq and scTCR-seq data demonstrate that our CD8α-targeted fusosomes are highly specific for cells expressing CD8α transcripts in resting PBMCs. These data highlight the potential for utilizing single cell sequencing technologies to comprehensively characterize the specificity of our fusosomes, and to identify key biological pathways that may play a role in specificity, transduction efficiency and clinical efficacy. As next steps, we will use similar approaches to characterize in vivo transduction in animal models. Disclosures Iftikhar: Sana Biotechnology: Current Employment. Balanis: Sana Biotechnology: Ended employment in the past 24 months. Fonseka: Sana Biotechnology: Current Employment. Bandoro: Sana Biotechnology: Ended employment in the past 24 months. Cruite: Sana Biotechnology: Current Employment. Davis: Sana Biotechnology: Current Employment. Amatya: Sana Biotechnology: Current Employment. Frye: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Laska: Sana Biotechnology: Current Employment. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Shah: Sana Biotechnology: Current Employment. Paliwal: Sana Biotechnology: Current Employment. Chaivorapol: Sana Biotechnology: Current Employment.
Databáze: OpenAIRE
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