title: 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. Importance: Human 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Kennedy
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Anand V R
authors.initials: AV
authors.lastname: Kornepati
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Goldstein
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Hal P
authors.initials: HP
authors.lastname: Bogerd
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Brigid C
authors.initials: BC
authors.lastname: Poling
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Whisnant
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Kastan
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA bryan.cullen@duke.edu.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Cullen
mesh_terms: D052685:Alphapapillomavirus / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004720:Endonucleases / Q000378:metabolism*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000821:virology
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D004720:Endonucleases
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