Mechanisms of Infection and Transduction by A New HSV Gene Therapy Vector - Project Summary Recombinant viral vectors have been used to deliver genes to cells for immunotherapy, immunization, and gene therapy, the latter including three adenovirus-associated virus (AAV) vectors and one herpes simplex virus 1 (HSV-1) recombinant virus. Despite these successes, AAV has a limited pay load capacity of 4 kbp, so larger genes cannot be accommodated. Also, the previous HSV vectors express ICP0, which causes cell toxicity, so transgene expression is transient. Thus, there is a continuing medical need for viral gene therapy vectors that have a larger capacity and that are not cytotoxic, so expression is durable. HSV has unique potential as a gene therapy vector because of the large genome capacity for transgenes and for its natural persistence in neurons with durable expression of the latency-associated transcript (LAT). Initial HSV gene vectors showed short-term expression due to cytotoxicity by HSV ICP0, but recent gene therapy vectors have 1) all viral immediate-early (IE) genes deleted to reduce cytotoxicity, 2) internal inverted repeat (IR) sequences deleted to increase payload capacity, and 3) the transgene bounded by insulator sequences to allow long-term expression of the transgene. We have found that re-introducing the viral genomic IR sequences in- creases replication of HSV gene therapy vectors in complementing cells and, surprisingly, increases expression of a transgene in normal cells under transduction conditions. We have further found that the IR sequences in- crease late gene transcripts during lytic infection and increase the accumulation or maintenance of transduced viral genomes, and we hypothesized that the internal inverted repeats promote the folding or intranuclear asso- ciations of the viral genome that promote lytic late and transduced gene transcription. The proposed studies are highly significant and will examine the mechanisms by which the presence of IR sequences affects lytic infection and transduction by the vector as well as open new areas of study of the basic mechanisms of lytic and latent infection by wildtype HSV-1. We will investigate the new recombinant viruses and the effect of the IR sequences on lytic replication and transduction capabilities through the following specific aims: 1. Determine the role of genome structure and epi- genetics in replication of a new HSV gene therapy vector by defining the effect of the internal inverted repeat sequences on late viral transcription in lytic infection of complementing cells. 2. Determine the effects of the IR sequences on gene transduction in normal human cells. Identification of a new HSV gene therapy vector and definition of its mechanisms of action are highly signifi- cant because of the medical need for vectors with higher capacity and long-term expression with no cytotoxicity. Furthermore, the studies provide a model system for defining the role of viral genome structure in late gene transcription and maintenance and expression of genes in the LAT locus in latent infection. This will open many opportunities for future studies of the HSV-1 wildtype genome during lytic and latent infection.
