title: Next-Generation Cancer Therapies: The Therapeutic Potential of RNA-Directed Gene-Editing
Creators: name: Jubair, Luqman Khaleel
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Cancer therapies
subject: Gene-editing
subject: CRISPR
subject: Oncogene-addicted cancers
subject: Papillomavirus

description: The discovery of the bacterial Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated Cas9 protein, and its ability to edit genes in mammalian cells is poised to revolutionise our ability to treat genetic diseases, particularly in the cancer setting where the driver genes are known. Indeed, CRISPR/Cas9 was shown to effectively edit any gene of interest with high efficiency and at low cost. However, a major challenge to treating cancer is the heterogeneity of the genetic alterations and the ongoing accumulation of new mutations that enable cancers to survive and drug resistance to emerge. With the increasing knowledge of cancer biology, thanks to the advances in DNA sequencing technologies, the low cost, and the ability to edit genes with CRISPR/Cas9 with high efficiency, it is now possible to develop more targeted anticancer therapeutics with promising outcomes. Despite the exceptional CRISPR/Cas9 gene-editing features, the early studies found several limitations to its potential use as a human gene therapy, such as the targeting specificity of CRISPR/Cas9, the in vivo delivery of the treatment with minimal systemic toxicity, the in vivo efficacy under immunocompetent conditions, the immunogenicity of CRISPR/Cas9 and the delivery vehicle when delivered systemically, and whether the introduced edits would induce an immune response. Particularly for cancer treatment, challenges can be broadly categorized into: the in vivo efficacy of CRISPR/Cas9 therapeutics, the in vivo delivery of the treatment to target tissue with high transfection efficiency, and the heterogeneity of genetic mutations in cancer, and thus targeting a known gene may not be enough to cure cancer. To enable the utilisation of CRISPR/Cas9 as an anticancer therapy, the above-mentioned challenges need to be addressed. We utilised the well-established Human Papillomavirus (HPV)-driven cervical cancer models due to their addiction on the expression of HPV oncogenes, namely E6 and E7, for their survival and progression, and thus enabled us to assess the efficacy and the delivery of CRISPR/Cas9 therapies, independent of cancer heterogeneity. To improve the on-target specificity, we assessed the feasibility of using the highly specific variant of Cas9, the catalytically inactive Cas9 fused to the dimerization-dependent cutting domain, FokI, or FokI-dCas9, and compared its editing efficiency on target genes and the effect on downstream protein expression compared to the wild type (WT) Cas9. Our results proved that the FokI-dCas9 is ineffective as a cancer therapeutic, particularly when the target genes are short. We further explored the repair mechanism of the CRISPR/Cas9-mediated double-stranded breaks (DSB) and found that the high-fidelity homology-directed repair (HDR) was modest, accounting for ≈ 8%, compared to the random non-homology end joining (NHEJ) repair, which accounted for ≈ 80% of the edited cells, with a significant inhibition of cancer cell proliferation. We also showed that the cell death was apoptotic, mediated by the reactivation of the tumour suppressor p53 protein when E6 gene was targeted, or the restoration of retinoblastoma protein (Rb) when E7 gene was targeted. To test the feasibility of the intravenous delivery of CRISPR/Cas9, we optimised a protocol to package the treatment in PEGylated liposomes by using the Hydration-of-Freeze-Dried-Matrix (HFDM) method and showed that these liposomes effectively protected the payload against serum nucleases. Furthermore, the intravenously administered CRISPR/Cas9 against HPV 16E7 and HPV 18E7 oncogenes, coated in PEGylated liposomes, effectively cleared established cervical cancer xenografts in immunocompromised mouse models. Next, we aimed to explore if the in vivo efficacy would be affected by the immunogenicity of the treatment under immunocompetent conditions. We showed for the first time that CRISPR/Cas9 therapies eliminated HPV16E7-driven tumour xenografts entirely in syngeneic mice, with no significant inflammation or hepatic toxicity. In addition, an ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. Therefore, we explored the immunogenicity of cell death and showed for the first time that CRISPR/Cas9-mediated cell death was not immunogenic. Overall, this research demonstrates that CRISPR/Cas9 therapeutics are very effective for the treatment of oncogene-addicted cancers. We showed that the PEGylated liposomes can be an ideal delivery vehicle for CRISPR/Cas9 therapies despite the large payloads, with no significant immune response or toxicity, and provided new insights into harnessing CRISPR/Cas9 technology as an anticancer therapeutic.