2019 – DEVELOPMENT OF CD90-TARGETED IN VIVO HEMATOPOIETIC STEM CELL GENE THERAPY

Hematopoietic stem cell (HSC) gene therapy is a promising treatment option for various genetic blood diseases/disorders. To enable efficient, precise, and safe modification of HSCs for gene therapies ex vivo and in vivo, robustly expressed and specific antigens are necessary to isolate and target HSCs. Here, we investigated the expression and cell-surface abundance of various commonly used HSC antigens such as CD34, CD90, CD117, and others using quantitative flow cytometry and bulk/single-cell RNAseq of human and NHP HSCs. We further studied the impact of mobilization on the transcription and cell surface presentation of antigens to inform the best route for the in vivo administration of HSC-targeted agents. Our analysis found the glycoprotein CD90 to be a robustly and stably expressed antigen on the surface of human and NHP HSCs regardless of donor or collection methodology, whereas expression of CD117 was downregulated on mobilized HSCs. As such, we developed second-generation targeted viral particles (VP) to the CD90 antigen, capable of highly specific transduction and editing of human HSCs in vivo using a murine xenograft model. CD90-targeted VPs targeted HSCs with over 100-fold more efficiency than any other hematopoietic subset in vivo. Additionally, modified HSCs were capable of unbiased repopulation of the hematopoietic hierarchy six weeks after VP administration and secondary transplantation. These results support previous studies identifying the HSC containing CD34+CD45+CD90+CD45RA- (CD34+CD90+HSCs) subset as responsible for long-term hematopoietic reconstitution. Thus, targeting CD90 on human HSCs provides a novel platform to modify quiescent HSCs for in vivo gene therapy without perturbing normal hematopoietic output.