Editing the Genome of a Virus for Large DNA Construct delivery

We designed an easily customizable viral vector by knocking out the TK gene of the HSV viral genome, which controls the toxicity of the virus, and replaced it with the Tn7 site for ease of inserting large genes. In addition, we will replace LoxP sites with FRT sites for their use in Cre-LoxP recombination. A bacterial artificial chromosome (BAC) harboring the genome of HSV strain KOS-37 will be used in this design. This plan would allow us to create an HSV-based vector that can accept large gene payloads and is relatively safe since we will be working mainly with bacteria instead of viruses

Gene delivery techniques serve as important stepping stones for a wide range of research and clinical applications, including gene therapy. As the research in genetic engineering gets more sophisticated, the need for a delivery vector for a large gene construct grows. However, available viral vectors, such as AAV-based vector and lentivirus-based vector, could only harbor less than 10kb of a construct. One promising candidate for this task is a human simplex virus (HSV) which could carry more than 30 kb of transgene despite its natural cytotoxicity. Therefore, the goal of this project is to engineer a safe and customizable HSV-based viral vector as a tool for gene deliveries.

To edit the HSV viral genome to create a HSV viral vector with limited cytotoxicity, and a large carrying capacity of at least 30 kilobases. In addition, it needs to be easily customizable for a more efficient and affordable vector design for use in medical and research purposes.