Oncogene editing in human papillomavirus (HPV) by CRISPR Cas9

By Daniel Lo

Proto-oncogenes are genes that control cell growth. Mutation of a proto-oncogene can be induced by point mutations (insertions and deletions), gene amplification or chromosomal translation. These mutations result in excessive copies of proto-oncogenes, resulting in proto-oncogenes become oncogenes (Chial H, 2008). Oncogenes can be permanently activated leading to uncontrolled cell growth which can lead to cancer.

In most of the studies and investigations conducted on oncogenic editing, the human papillomavirus (HPV) oncogenes – E6 and E7, are often involved. The appearance and progression of HPV-induced cervical cancer are entirely dependent on E6 and E7 (Yim E. , 2005). Thus, experimental outcomes can be easily interpreted, whether editing activity was shown or if the edits are deemed successful by observing the change in tumour since the growth and elimination of the tumour is entirely dependent on E6 and E7. E6 and E7 induce the degradation of the endogenous tumor suppressors p53 and retinoblastoma (Rb) protein, respectively (Pal A. ,2020). The tumour suppressor proteins’ activity prevents the formation of tumours as they control cell division and replication. When a tumor suppressor gene is mutated, its function will either be lost or suffer from a significant reduction in efficiency, accounting for the abnormal growth and division of cells, resulting in cancer. This suggests that silencing E6 and E7 in cervical cancer cells should bring about the normal functioning of p53 and Rb, as these proteins usually remain entirely intact in them. Both expressions of p53 and Rb result in cell cycle arrest, senescence, and apoptosis, allowing them to efficiently prevent the formation of tumours and eliminate tumours upon their formation (Richards, J. E. ,2011).

Viral oncogene suppression, targeting E6 and E7 has previously shown potential for the treatment of HPV-positive tumours (Morrison, M. et al. ,2011). Thus, with the increasingly widespread use of genome editing technologies, scientists have adopted a rather innovative approach to fight cancer by oncogenes editing. Such approaches involve silencing them so that they become dysfunctional and lose their oncogenic properties.

E6 and E7 are especially fit targets for CRISPR/Cas9 editing since they both contain multiple possible NGG-PAM sequences for sgRNA to recognise. More importantly, as E6 and E7 are only found in the HPV genome, none of the possible sgRNA sequences would have a corresponding on-target locus in normal human cells, reducing the risk of off-target effects. Therefore, silencing E6 and/or E7 using CRISPR-Cas9 has been proven to be able to interfere with the growth and division of cervical cancer cells, which has been translated recently into a phase I clinical trial.

CRISPR/Cas9 applied in oncogenes editing are seen successful A study on CRISPR/Cas9 applied in oncogene editing provided a proof of principle for the idea that a vector-delivered Cas9/sgRNA editing system holds enormous potential as an effective treatment for HPV-induced cancers

(Kennedy, E. et al. , 2014). The ideal treatment of HPV-induced cancers would be to permanently inactivate the viral E6 and E7 oncogene by targeted mutagenesis, through which the death of HPV-transformed cells is likely to result. This hypothesis has now been put into practice by the use of RNA-guided DNA endonucleases (RGNs) derived from the type II bacterial CRISPR/Cas system.

E6 and E7 in HeLa cells are used as targets for double stranded-DNA cleavage. The scientists designed sgRNAs complementary to nucleotides of the E6 open reading frame (ORF) and that of the E7 ORF (Cong, L. et al. ,2013). By inducing DSBs in the E6 and E7, they were inactivated. Then the inactivated endogenous E6 and E7 genes were integrated into the HeLa cell genome. The Surveyor assay was used to detect RGN-induced indels at the predicted Cas9 cleavage site. The presence of indels was observed in both the E6 and E7 genes at the predicted Cas9 target site.

A previous study has shown that the loss of either E6 or E7 oncogenic function in HeLa cells, and other HPV-transformed cells, could give rise to not only cell cycle arrest but also apoptosis (DeFilippis R. et al. ,2003) To prove whether the silencing of E6 and E7 by RGNs would result in the expected HeLa cell death, the expression of Cas9/sgRNA combinations specific for E6 or E7 is tested out by a transduction experiment to determine the percentage of viable cells. It was observed that both the E6- and E7-specific sgRNAs, under the presence of S. pyogenes Cas9, induced nearly full elimination of the transduced HeLa cell culture. This result demonstrates that targeted disruption of HPV E6 or E7 has the potential to induce the specific elimination of HPV-transformed cells.

Since oncogenes editing is carried out in vivo, the editing efficiency and off-target effects of CRISPR in vivo is also an underlying concern for the development of such a treatment approach. The CRISPR system was first discovered in prokaryotes before it was adopted for genome editing in eukaryotic cells. Hence, it is expected that the editing efficiency of CRISPR might be lower for some in vivo target sites in human cells. Such variation in editing efficiency might be due to the structural difference of how DNA is present in prokaryotic and eukaryotic cells. Unlike in prokaryotic cells, DNA in eukaryotic cells is coiled up around histones and further packed to form some large, complex chromatin structures, which may hinder binding of Cas9 to its targets. Moreover, both in vitro and in vivo experiments have shown that nucleosomes, the basic unit of chromatin, have negative impact over Cas9, inhibiting its binding and cleavage to the target sites (AC. Komor, A. et al. ,1970). The low editing efficiency of CRISPR in vivo in human cells, is mainly a problem when it comes to application in cancer treatment. Due to the limited editing efficiency, full elimination of tumour cells may not be achieved and tumour cells left may continue to grow and divide uncontrollably, contributing to the progression of cancer. Moreover, in terms of off-target effects, they can cause unwanted gene mutations or chromosomal rearrangements where DNA double stranded breaks at unintended sites in healthy cells can cause secondary cancers (Ghosh, D. et al. ,2019). Since oncogene editing is carried out in vivo, such off-target effects are likely to have a direct or maybe worse, permanent impact on patients.

In conclusion, despite possible side effects or hindrance to such a treatment approach, it is still a relatively new one with plenty room for improvement and has already made much progress with the application of CRISPR. In time, tackling the evolving problem for cancer treatments, heterogeneity of tumours cells, and raising the editing efficiency, also reducing off-target effects of in vivo CRISPR editing, this treatment approach can very likely become a widely adopted one for clinical practice.

References:

Chial, H. (2008). Proto-oncogenes to Oncogenes to Cancer.

Yim, E. and Park, J., 2005. The Role of HPV E6 and E7 Oncoproteins in HPV-associated Cervical Carcinogenesis. Cancer Research and Treatment, 37(6), p.319.Pal, A., & Kundu, R. (2020, January 21). Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy.

Pal, A. and Kundu, R., 2020. Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy. Frontiers in Microbiology, 10.

Richards, J. E. (2011). Tumor Suppressor Protein.

Morrison, M., Morreale, R., Akunuru, S., Kofron, M., Zheng, Y., & Wells, S. (2011, October 15). Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility.

Kennedy, E., Kornepati, A., Goldstein, M., Bogerd, H., Poling, B., Whisnant, A. et al. (2014, October). Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease.

Cong, L., Ran, F., Cox, D., Lin, S., Barretto, R., Habib, N et al. (2013, February 15). Multiplex genome engineering using CRISPR/Cas systems.

DeFilippis, R., Goodwin, E., Wu, L., & DiMaio, D. (2003, January). Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells.

AC. Komor, A., L. Bortesi, C., W. Li, F., KT. Jensen, L., EI. Campos, D., C. Kuscu, S. et al. (1970, January 01). Modulating chromatin accessibility by transactivation and targeting proximal dsgRNAs enhances Cas9 editing efficiency in vivo.

Ghosh, D., Venkataramani, P., Nandi, S., & Bhattacharjee, S. (2019, January 8). CRISPR-Cas9 a boon or bane: The bumpy road ahead to cancer therapeutics.