Self-Repairing Herpesvirus Saimiri Deletion Variants

Abstract

Herpesvirus saimiri (HVS) is discussed as a possible vector in gene therapy. In order to create a self-repairing HVS vector, the F plasmid vector moiety of the bacterial artificial chromosome (BAC) was transposed via Red recombination into the virus genes ORF22 or ORF29b, both important for virus replication. Repetitive sequences were additionally inserted, allowing the removal of the F-derived sequences from the viral DNA genome upon reconstitution in permissive epithelial cells. Moreover, these self-repair-enabled BACs were used to generate deletion variants of the transforming strain C488 in order to minimalize the virus genome. Using the en passant mutagenesis with two subsequent homologous recombination steps, the BAC was seamlessly manipulated. To ensure the replication capacity in permissive monkey cells, replication kinetics for all generated virus variants were documented. HVS variants with increased insert capacity reached the self-repair within two to three passages in permissive epithelial cells. The seamless deletion of ORFs 3/21, 12–14, 16 or 71 did not abolish replication competence. Apoptosis induction did not seem to be altered in human T cells transformed with deletion variants lacking ORF16 or ORF71. These virus variants form an important step towards creating a potential minimal virus vector for gene therapy, for example, in human T cells.

Bacterial-Artificial-Chromosome-Based Genome Editing Methods and the Applications in Herpesvirus Research

“…In the second round of recombination, the I-SceI site is cleaved by the expression of the I-sceI enzyme, where the sequence duplications mediate the recombination, thus achieving scarless editing. The technology referred to as “ en passant mutagenesis ” has been shown to be effective in inducing large-scale changes in viral genomes without scarring [ 76 ] and for removing F-derivative sequences from the viral genome during reconstruction in cells [ 77 ]. However, the accumulation of point mutations or deletions within the I-SceI recognition site during the second round of homologous recombination [ 78 , 79 ], as well as the self-ligation of the I-SceI enzyme cleavage site, can lead to high background noise [ 79 , 80 ], negatively impacting screening efficiency (after the second round of recombination, the screening efficiency drops from 95% to 1–15%) [ 81 ].…”