Loncar, S. | Search bioRxiv

bioRxiv2023

Identification of genome safe harbor loci for human gene therapy based on evolutionary biology and comparative genomics

Gene transfer into CD34+ hematopoietic stem and progenitor cells (HSPCs) involving integrating viral vectors has unpredictable outcomes including potential adverse events like leukemogenesis, resulting from insertional mutagenesis. Therefore, identifying and characterizing genome safe harbor (GSH) sites where exogenous gene material can be safely integrated into adult progenitor and stem cells is critically important for therapeutic gene addition. Here, we present a novel approach to identify new GSH sites based on a proven system of stable transgene insertion: the evolutionarily conserved integration of parvovirus DNA into the germlines of host species. From a dataset of 199 unique endogenous parvovirus (EPV) integration events identified in host species genomes, 102 loci were mapped to the human genome with 17 being experimentally evaluated as GSHs in primary human CD34+ HSPCs. Nine promising GSHs resulted in cells edited using nucleofection alone or in combination with rAAV transduction. Of the nine GSH sites, six edited loci displayed sustained transgene expression in both erythroid and immune phenotypes while three clearly exhibited immune branch specific-regulation. Following this approach, additional GSH sites are likely to emerge from the remaining mapped loci for gene addition in hematopoietic stem and progenitor cells. Since it is unlikely that the GSH-lineage-restricted transgene expression is exclusive to hematopoietic stem cells, this approach extends the options for gene knock-ins while reducing the risks of insertional mutagenesis, unpredictable expression profiles, effects on differentiation, and increasing therapeutic effects.

genetics↗

bioRxiv2021

Comparative analysis reveals the long-term co-evolutionary history of parvoviruses and vertebrates.

Parvoviruses (family Parvoviridae) are small DNA viruses that cause numerous diseases of medical, veterinary, and agricultural significance and have important applications in gene and anticancer therapy. DNA sequences derived from ancient parvoviruses are common in animal genomes and analysis of these endogenous parvoviral elements (EPVs) has demonstrated that the family, which includes twelve vertebrate-specific genera, arose in the distant evolutionary past. So far, however, such paleovirological analysis has only provided glimpses into biology of parvoviruses and their long-term evolutionary interactions with hosts. Here, we comprehensively map EPV diversity in 752 published vertebrate genomes, revealing defining aspects of ecology and evolution within individual parvovirus genera. We identify 364 distinct EPV sequences and show these represent [~]200 unique germline incorporation events, involving at least five distinct parvovirus genera, that took place at points throughout the Cenozoic Era. We use the spatiotemporal and host range calibrations provided by these sequences to infer defining aspects of long-term evolution within individual parvovirus genera, including mammalian vicariance for genus Protoparvovirus, and inter-class transmission for genus Dependoparvovirus. Moreover, our findings support a model of virus evolution in which the long-term co-circulation of multiple parvovirus genera in vertebrates reflects the adaptation of each viral genus to fill a distinct ecological niche. Our discovery that parvovirus diversity can be understood in terms of genus-specific adaptations acquired over millions of years has important implications for their development as therapeutic tools - we show that these endeavours can now be approached from a rational foundation based on comparative evolutionary analysis. To support this, we published our data in the form of an open, extensible, and cross-platform database designed to facilitate the wider utilisation of evolution-related domain knowledge in parvovirus research.

evolutionary biology↗