Systemic Delivery of CRISPR/Cas9 Targeting HPV Oncogenes Is Effective at Eliminating Established Tumors

Abstract

The recent advancements in CRISPR/Cas9 engineering have resulted in the development of more targeted and potentially safer gene therapies. The challenge in the cancer setting is knowing the driver oncogenes responsible, and the translation of these therapies is hindered by effective and safe delivery methods to target organs with minimal systemic toxicities, on-target specificity of gene editing, and demonstrated lack of long-term adverse events. Using a model system based on cervical cancer, which is driven by the ongoing expression of the human papillomavirus E6 and E7 proteins, we show that CRISPR/Cas9 delivered systemically in vivo using PEGylated liposomes results in tumor elimination and complete survival in treated animals. We compared treatment and editing efficiency of two Cas9 variants, wild-type (WT) Cas9 and the highly specific FokI-dCas9, and showed that the latter was not effective. We also explored high-fidelity repair but found that repair was inefficient, occurring in 6%-8% of cells, whereas non-homologous end joining (NHEJ) was highly efficient, occurring in $80% of the cells. Finally, we explored the post gene-editing events in tumors and showed that cell death is induced by apoptosis. Overall, our work demonstrates that in vivo CRISPR/Cas editing treatment of preexisting tumors is completely effective despite the large payloads.

CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 and E7 induces senescence in cervical cancer cells

“…Integration of E6 and E7 regions within the host cell in combination with non-viral risk factors facilitate progression of HPV infections to invasive cancer. Recent work has shown that inactivation of high-risk HPV E6 and E7 genes by CRISPR/Cas9 systems restored the p53 and pRb networks resulting in induction of cell cycle arrest and apoptosis as final outcome (15–17). Studies from Jubair et al, indicated that systemic administration of Cas9/gRNAs against HPV16 E7 Caski cell and HeLa cell tumors in mice were cleared by apoptosis (17).…”

“…Recent work has shown that inactivation of high-risk HPV E6 and E7 genes by CRISPR/Cas9 systems restored the p53 and pRb networks resulting in induction of cell cycle arrest and apoptosis as final outcome (15–17). Studies from Jubair et al, indicated that systemic administration of Cas9/gRNAs against HPV16 E7 Caski cell and HeLa cell tumors in mice were cleared by apoptosis (17). Similarly, lentiviral transduction of Cas9/gRNA against HPV 18 E6 and E7 genes for a 10-day period induced cell death in HeLa cells (15).…”

“…CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 is a dsDNA editing method where the Cas9 nuclease from Streptococcus pyogenes , guided by small RNAs, can be used to edit target DNA. Inactivation of E6 and E7 genes using CRISPR/Cas9 methodology has been reported to induce arrested cell growth and apoptosis but not senescence (15–17). However, application of CRISPR/Cas9 technology for treatment of cervical cancer needs to be addressed in more detail for a successful clinical application.…”

Applications of CRISPR‐Cas9 in gynecological cancer research

“…The pathogenic mechanism of HPV is mainly through the viral oncoproteins E6 and E7, where E6 protein binds to the host pro‐apoptotic tumor suppressor, p53, and E7 binds to the retinoblastoma tumor suppressor protein, retinoblastoma protein (pRb), eventually causing cell proliferation, deformation, and canceration . As CRISPR‐Cas9 can efficiently cut and knock out pathogenic genes, it brings new hope for cervical cancer treatment, and it has in recent years made great progress in cervical cancer treatment (summarized in Table ).…”

“…The knockout of HPV‐infected E6/E7 genes through CRISPR‐Cas9 can induce cervical cancer cell apoptosis, cause cell cycle arrest, and inhibit cervical cancer metastasis. Apoptosis of cervical cancer cells is more pronounced when CRISPR‐Cas9 is combined with the chemotherapy drug, cisplatin . Using CRISPR‐Cas9 to knock out the MSTN gene in the cervical cancer cell line HeLa caused an increase in the expression of intracellular fatty acid oxidase, and triggered depolarization of the mitochondrial membrane, which affected the structure and function of mitochondria, leading to cytochrome c (Cyt‐c) release and caspase‐3 and caspase‐7 activation, eventually inducing apoptosis and inhibiting tumor growth.…”