Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

Improvement of HSV-1 based amplicon vectors for a safe and long-lasting gene therapy in non-replicating cells.

A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.

Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

Targeted oncolytic herpes simplex virus type 1 eradicates experimental pancreatic tumors.

As many other cancers, pancreatic ductal adenocarcinoma (PDAC) progression is associated with a series of hallmark changes for cancer cells to secure their own growth success. Yet, these very changes render cancer cells highly sensitive to viral infection. A promising strategy may rely on and exploit viral replication for tumor destruction, whereby infection of tumor cells by a replication-conditional virus may lead to cell destruction and simultaneous release of progeny particles that can spread and infect adjacent tumor cells, while sparing healthy tissues. In the present study, we used Myb34.5, a second-generation replication-conditional herpes simplex virus type 1 (HSV-1) mutant in which ICP6 gene expression is defective and expression of the HSV-1 γ134.5 gene is regulated by the cellular B-myb promoter. We found that B-myb is present in experimental PDAC and tumors, and is overexpressed in patients' tumors, as compared with normal adjacent pancreas. Myb34.5 replicates to high level in human PDAC cell lines and is associated with cell death by apoptosis. In experimental models of PDAC, mice receiving intratumoral Myb34.5 injections appeared healthy and tumor progression was inhibited, with evidence of tumor necrosis, hemorrhage, viral replication, and cancer cell death by apoptosis. Combining standard-of-care chemotherapy with Myb34.5 successfully led to a very impressive antitumoral effect that is rarely achieved in this experimental model, and resulted in a greater reduction in tumor growth than chemotherapy alone. These promising results warrant further evaluation in early phase clinical trial for patients diagnosed with PDAC for whom no effective treatment is available.