Generation and validation of recombinant herpes simplex type 1 viruses (HSV-1) using CRISPR/Cas9 genetic disruption

Herpes simplex virus type 1 (HSV-1) is a large DNA virus that has been popular for oncolytic virus development in pre-clinical research and clinical trials. An oncolytic HSV-1 encoding granulocyte-macrophage colony stimulating factor (GM-CSF), designated talimogene laherparepvec (T-VEC) was approved for the treatment of patients with advanced melanoma in 2015. There are numerous advantages of HSV-1 for oncolytic development, including the ease of recombinant engineering, presence of non-essential genes allowing attenuation of pathogenicity and space for foreign transgene expression. In addition, most recombinants retain sensitivity to acyclovir providing an additional safety feature. In this chapter, we will focus on the key methods for the development of oncolytic HSV-1 vectors and some of the commonly utilized laboratory protocols used to characterize and assess the structure and oncolytic activity of recombinant HSV-1 viruses.