Oncogenic Viruses: A Potential CRISPR Treatment Neha Matai ∗ February 26, 2021 Abstract Oncogenic viruses promote carcinoma development by establishing long-term latent infections, obstructing tumor suppressor pathways, and transforming host cells into unchecked, proliferating malignancies. Epstein- Barr Virus, the first oncogenic virus to be discovered, can promote lym- phomagenesis in T cells, NK cells, and most commonly, resting memory B cells through the expression of oncoprotein LMP-1 and other proteins which may aid in B cell transformation. Human Papillomavirus is a com- mon infectious agent found in epithelial cells and has been shown to pro- mote malignant transformation in host cells through the overexpression of oncoproteins E6 and E7. Hepatitis C Virus, whose life cycle is not fully known yet, can also lead to carcinoma development in hepatocytes, and HCV oncoproteins NS3, NS4A, and NS5B have been associated with onco- genic roles. A novel genetic approach involving a CRISPR/Cas treatment designed to dysregulate these viral oncogenes can be used to combat these infections and their resulting carcinomas. While the long-term effects of CRISPR treatments are still being researched, this gene therapy offers a robust selection of potential treatments regarding long term diseases such as oncogenic viral infections. 1 Introduction The human population is prone to cancer through a wide variety of variables; in fact, 20% of which are caused by infectious agents such as bacteria, viruses, and other pathogens.1 Oncogenic viruses alone are responsible for 12% of all cancers diagnosed worldwide. More specifically, 80% of viral cancer cases occur in the developing world today.2 The first oncovirus was discovered in 1964 when researchers located the Epstein-Barr Virus in Burkitt’s Lymphoma cells using electron microscopy.3 Since this turning point in viral oncology, six more viruses have been confirmed to cause oncogenesis. ∗Advised by: Everardo Hegewisch Solloa 1 [Hau09] 2 [Hau09] 3 [MHT17] 1 While infections from oncoviruses are common, they rarely lead to malig- nancies and take a long period of time to transform host cells into an oncogenic state. For cancers to arise, these viral infections must be accompanied by chronic inflammation, environmental mutagens, or immunosuppression.4 Additionally, oncogenic viruses do not follow a uniform path to oncogenesis since some of the viruses are considered to be direct carcinogens, while others are indirect carcinogens. Direct carcinogens cause cancer cell transformation by expressing oncogenes, while indirect carcinogens cause chronic infection or inflammation, which leads to carcinogenesis.5 The seven known oncogenic viruses have been characterized by the class of genetic material they possess (e.g., DNA or RNA). The five known DNA oncoviruses include Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Human Herpesvirus-8 (HHV-8), Human Papillomavirus (HPV), and Merkel Cell Polyomavirus (MCPyV). On the other hand, there are two oncogenic RNA viruses, which include Hepatitis C Virus (HCV) and Human T-Cell Lymphotropic Virus-1 (HTLV-1).6 Oncogenic viruses can be transmitted via the exchange of bodily fluids and direct skin to skin contact.7 Viral cancers only arise 15 to 40 years after primary infection since chronic infection is a critical component of oncogenic transfor- mation. Oncoviruses can achieve chronic infection by switching from a lytic to a latent stage after initially infecting host cells.8 Viruses in a lytic phase actively induce the host cell to biosynthesize a wide variety of functional and structural proteins to produce more viral progeny. Contrarily, viral latency does not in- volve infectious virus production since, in a latent stage, viruses integrate a copy of their genome into the host cell’s genome during viral replication and rely on host cell replication for survival.9 The latent stage ensures that the virus exists in host cells without detection from the immune system. It is often latency that promotes oncogenesis since viruses in the lytic phase create a higher risk of DNA damage detection that results in programmed cell death, inhibiting the virus and host cell from replicating further. It is noted that cancer cells have little to no evidence of viruses in the lytic stage.10 The most common treatments for viral cancers are currently chemother- apy, such as lytic-inducing chemotherapies used for virus-related lymphomas, and resection, which is the surgical removal of tumorous tissue.11 Additionally, preventative treatments include vaccinations, which exist for some oncoviruses. Other oncogenic viruses are treated using radiotherapy, immunotherapy, or an- tiviral drugs prescribed on a case to case basis.12 However, these treatments cannot guarantee complete eradication of infection and many cases result in 4 [MC10] 5 [MC10] 6 [MHT17] 7 [MSFP14] 8 [MC10] 9 [CCY15] 10 [MC10] 11 [MHT17]and [BDA+13] 12 [CKQ19] 2 reactivation of the virus.13 Recent advancements in molecular biology research have allowed researchers to explore gene-editing tools as potential therapeutics for oncogenic viruses. Clustered regularly interspaced short palindromic repeats (CRISPR) technologies have now been considered for use in therapies to treat cancers, chronic viral infections, or genetic diseases.14 In situ, the CRISPR/Cas system is a bacterial and archaeal ”adaptive immune system” that recognizes bacteriophage genomes complementary to a guide RNA (gRNA), which is bound to a Cas protein. Once CRISPR/Cas has associated with its complementary target sequence in the phage genome, it induces a double-stranded break in the complementary DNA strand, inhibiting the phage’s ability to replicate.15 CRISPR/Cas was discovered as a potential gene-editing tool in 2013 for its abil- ity to induce double-stranded breaks in a sequence-specific manner, resulting in either nonhomologous end-joining (NHEJ) or homology-directed recombination (HDR).16 The ability to induce site-specific gene edits allows scientists to knock out a gene or introduce a gene with higher efficiency. Not only has CRISPR/Cas allowed scientists to further understand gene functions, but it also has the po- tential of serving as a therapy via either the deletion or correction of a mutated gene. CRISPR diagram.png Figure 1: Double-stranded breaks are induced by the Cas-gRNA complex 13 [GP16] 14 [LdS19] 15 [LdS19] 16 [LdS19]and [WHK15] 3 Currently, novel virus-targeted therapies are being researched for oncogenic viruses, which include new immunotherapies, cellular therapies, and antibody therapies.17 Gene therapies are treatments which consist of the application of zinc-finger nucleases (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR to treat a disease. The concept of gene editing for repairing mu- tated genes constitutes a new field of research that is being applied to various diseases and disorders. Here I will propose a novel CRISPR/Cas based therapy for treating Epstein-Barr Virus, Human Papillomavirus, and Hepatitis C Virus. 2 Epstein-Barr Virus (EBV) The Epstein-Barr Virus is also known as human herpesvirus-4 and is one of the eight human viruses in the herpesviridae family 2. Today, EBV has infected 90% of the world’s adults.18 However, the majority of EBV carriers maintain lifelong, asymptomatic infections.19 EBV is a double-stranded DNA virus that is transmitted primarily through saliva. After decades of research, it is confirmed that EBV is able to infect a wide variety of cells that encompasses B cells, T cells, NK cells, glandular and squamous epithelial cells, and smooth muscle cells. However, the virus is most commonly known to have oncogenic potential in B lymphocytes since it is able to create undetected DNA damage during a latent stage by inserting its genetic material into the host genome and then transforming B lymphocytes into proliferating lymphoblastoid cells through the large amounts of oncogenic protein made during a long span of latency.20 2.1 Lifecycle and Genome Properties of EBV In its primary stage of infection, EBV is in a lytic phase and most commonly replicates in squamous epithelial cells and local lymphocytes.21 Cases of in- fectious mononucleosis can sometimes occur as a result of an abnormal EBV- specific immune response.22 After EBV has established a local infection in a lymph node, the secondary phase of infection begins when EBV spreads to B lymphocytes, transforming them into virus-producing ’factories.’ Once in B cells, EBV downregulates the expression of its growth-transforming gene so that it can remain undetected in the host cell under a latent stage. Resting memory B cells infected with latent EBV then continue to circulate as part of the B cell pool. In a healthy host, EBV typically remains in a latent stage or is eradicated by the immune system, yet in cases when EBV re-enters the lytic stage of in- fection at a mucosal surface, new viral particles are released and can go on to infect more cells.23 17 [MHT17] 18 [MHT17] 19 [PGS+15] 20 [CCY15] 21 [CCY15] 22 [PGS+15] 23 [CCY15] 4 The genome of Epstein-Barr Virus is a 172kb double-stranded DNA genome that encodes for lytic gene products and latent gene products. EBV infection is initiated when structural proteins gp350 and gp220 bind to CD21 receptors of B cells. EBV structural protein gp42 then completes viral fusion into B cells by binding to the human leukocyte antigen class II receptor.24 Lytic gene products are then expressed during the early stages of EBV infection and con- sist of the early antigen (EA) complex, a complex of non-structural proteins, and the viral capsid antigen (VCA), which is composed of distinct structural antigen complexes. Both the expression of EA and VCA are significantly re- duced or not present after EBV transitions into a latent stage.25 EBV then expresses latent proteins which fall under two main categories: nuclear antigens (EBNA-1,2,3A,3B,3C,LP), and latent membrane proteins (LMP-1,2A,2B).