Inactivation of the Human Papillomavirus E6 or E7 Gene in Cervical Carcinoma Cells by Using a Bacterial CRISPR/Cas RNA-Guided Endonuclease

Abstract

High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16-and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers. IMPORTANCEHuman papillomaviruses (HPVs) are the causative agents of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome, where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest, leading to cancer cell death. We propose that viral vectors designed to deliver E6-and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers. C ervical carcinomas arise as a result of infection by human papillomavirus (HPV), most commonly high-risk HPV serotype HPV-16 or HPV-18 (1-4). Tumorigenesis results primarily from the deregulated expression of two viral oncoproteins, termed E6 and E7, which induce the degradation of the host cell tumor suppressors p53 and retinoblastoma (Rb) protein, respectively (2-7). This suggests that inactivation of either E6 or E7 expression in cervical carcinoma cells should result in the induction of p53 or Rb, respectively, as these proteins normally remain fully intact in cervical carcinoma cells. In fact, repression of E6 expression not only induces p53 but also upregulates the downstream effectors of p53 function, including proapoptotic proteins and the cyclin-dependent kinase inhibitor...

Expression of CRISPR/Cas single guide RNAs using small tRNA promoters

“…However, as discussed previously by ourselves and others (Cox et al 2015;Kennedy et al 2014), effective delivery of both the Cas9 gene and cognate sgRNAs into tissues in vivo will likely require the development of adenoassociated virus (AAV)-based vectors, as only AAV can be readily produced at titers sufficient to transduce enough cells in vivo to exert the desired phenotypic effect (Kotterman and Schaffer 2014). However, AAV vectors have a DNA packaging limit of ∼4.7 kb of which ∼290 bp must be dedicated to the two invariant AAV inverted terminal repeats (ITRs), leaving only ∼4.4 kb as the payload capacity.…”

Long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele-specific MYC expression in HeLa cells

“…Loss of HPV viral proteins E6 and E7 in cervical cancer cells could induce p53 or Rb expression, leading to cell cycle arrest and cell death 29. Knock down of E6 and E7 protein by RNA interference could induce senescence in HeLa Cells 30.…”

Crispr-Cas System: A Feasible Solution for Getting Rid of Persistent Viral Infection?

“…Study showed CRISPR-Cas system can restrict HBV infection under simulative in vivo infection scenario (Lin et al, 2014a). Studies also showed that CRISPR-Cas system works out in intervening Human Papilloma Virus (HPV) infection (Kennedy et al, 2014;Hu et al, 2014b;Zhen et al, 2014).…”