Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy.

Gene transfer into CD34(+) hematopoietic stem and progenitor cells (HSPCs) involving integrating viral vectors has unpredictable outcomes including potential severe events like leukemogenesis, resulting from insertional mutagenesis. Therefore, identifying and characterizing genome safe harbor (GSH) sites where exogenous genetic material can be safely integrated is critically important for therapeutic gene addition. Here, we present an approach to identify GSH candidates based on a proven system of stable transgene insertion: the evolutionarily conserved integration of parvoviral DNA into the germlines of host species. By analyzing the preservation of endogenous parvoviral elements (EPVs) from phylogenetically diverse vertebrate genomes, 102 EPV loci were mapped to the human genome, and 17 underwent experimental evaluation as GSHs in CD34(+) HSPCs. This resulted in at least six loci tolerant to transgene insertion, low transcriptome disturbances, and three loci showing myeloid immune branch-specific regulation. Thus, our approach produced a catalog of candidate GSHs suitable for therapeutic transgenesis in human cells.