CRISPR-Cas9 Targeting of E7 Oncogene in HPV-positive Head and Neck Squamous Cell Carcinomas

Abstract

Background and Aims: Head and neck squamous cell carcinomas (HNSCCs) include cancers of the oral cavity, larynx, hypopharynx, and oropharynx. It is estimated that ~25% of HNSCCs are Human papillomavirus (HPV) positive. With the incidence on constant increase, HPV-positive HNSCCs have already overtaken the number of cervical cancer cases, another type of HPV-driven cancer. Current treatments include excision of tumour, chemotherapy and radiotherapy, while the 5-year survival rates are 45% with significant quality of life reduction and morbidity associated with first-line treatment regimens. Several prophylactic HPV vaccines exist and provide protection against infection from major high-risk HPV types, though without the ability to treat already established infections. These factors strongly indicate a pressing need for a new line of treatments to relieve the burgeoning pressure of HPV-positive HNSCCs. The discovery of clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease sequences (Cas) and its ability to induce site-specific modifications is set to revolutionise the field of human gene therapy. CRISPR/Cas9 system has the advantage over other genome-editing systems due to its low off-target effects and permanent editing capabilities. In case of HPV-positive tumours, this system lends itself in targeting major HPV oncogenes, particularly E6 and E7. Members of our lab recently demonstrated the absolute requirement of E7 for the survival of cervical cancer tumours using CRISPR/Cas9 technology. Therefore, we wanted to see whether this “oncogene addiction” occurs in HPV positive HNSCCs. This project explores the therapeutic potential of CRISPR/Cas9 editing technology for HPV-positive HNSCCs by targeting the HPV E7 oncogene in vitro. Methodology: HPV16-positive HNSCC cell lines were initially assessed for E7 expression levels via digital PCR and immunoblotting. To CRISPR-edit 16E7, cells were then transfected with plasmids bearing Cas9 gene and gRNAs targeting 16E7 by lipid-based transfection using a range of transfection methods, including chemical transfection and electroporation. To extenuate the problem associated with delivery of large plasmid DNAs, we then designed synthetic gRNA molecules and transfected HNSCC cells constitutively expressing Cas9 protein. Cell viability was then assessed by the MTT and RT-GLO assays at various times post-transfection. Results: We confirm that several HNSCC cell lines express the E7 protein and digital PCR revealed the absolute gene expression of E7 in these cells. We successfully deleted E7 using CRISPR, which was confirmed by immunoblotting. However, loss of E7 alone did not kill the cells, either using plasmid DNA or synthetic gRNA mediated CRISPR editing methods. To our surprise, loss of cell viability was only seen in cells that have had both E6 and E7 oncogenes CRISPR-edited. Conclusions: Here, we showed that unlike cervical HPV carcinomas, head and neck HPV-positive carcinomas require the loss of both E6 and E7 oncogenes to die. Future work aims to place focus on targeting both E6 and E7 oncogenes on a wider range of HNSCC cell lines both in vitro and in vivo.