Metal-organic frameworks as non-viral vectors for CRISPR/Cas9 system to targeted knockdown of telomerase gene in cancer

Globally cancer is the leading cause of death. Just in 2022, Australia recorded approximately 162,163 new cases and 49,996 deaths. Current cancer therapies are often toxic to normal cells and cause drug resistance. Telomerase, a ribonucleoprotein coded by the hTERT gene, plays an important role in cellular immortalization and carcinogenesis. Telomerase activity is upregulated in most cancer cells; however, it is absent in non-cancerous somatic cells. This unique feature may provide a window of opportunity for targeting telomerase as a strategy for cancer therapeutics. Research over a decade has developed many therapeutics to target the telomerase including immunotherapy, small molecule inhibitors, antisense oligonucleotides, and gene therapy. Currently, although therapies in preclinical and clinical trials show effective telomerase inhibitory effects till date none of these therapies have been approved by FDA. Therefore, future avenues for the development of promising anti-telomerase therapies are open. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, due to its simplicity, versatility and high efficiency has appeared as a powerful gene-editing tool for cancer. CRISPR/Cas9 system offers permeant hindrances of expression protein by specific disruption of the target gene. However, the cellular delivery of intact CRISPR tools into cells efficiently and safely is still an ongoing challenge. The currently used viral delivery vehicles to carry the CRISPR tool limits their use because of immunogenicity and low tolerance. Metal-Organic Frameworks (MOFs) are a class of coordination polymers that emerged as promising materials for therapeutic agent/biomolecule delivery systems. Among the different MOF carriers, Zeolitic Imidazole Frameworks (ZIFs), are composed of transition metal ions connected by imidazolate linker (e.g., 2-methyilimidazole), and have emerged as biomolecule delivery vehicles with interesting properties. Despite the vast studies available on ZIFs, its efficiency in delivering CRISPR plasmid tool that targets the specific functional gene in cancer cells is yet to be explored. This thesis aims to develop a biocompatible Zn-based MOF for effective in vitro cancer treatment. The body of the work constitutes a significant understanding of a gene (CrhTERT) encapsulation by non-porous carbonate-rich MOF called ZIF-C (CrhTERT@ZIF-C), cellular uptake and its efficiency in chromosomal gene editing at telomerase gene. The anticancer properties of the gene-edited cells were thoroughly assessed. Finally, the efficiency of ZIF-C in delivering the CRISPR tool in a tumour mimic environment was investigated. The results of this project suggest that the ZIF-C shows a successful loading of CrhTERT with a high loading capacity of ~85%. The loaded plasmid in ZIF-C is highly protected against enzymatic degradation. CrhTERT@ZIF-C is efficiently endocytosed by cancer cells and the subcellular release of CrhTERT leads to telomerase knockdown. The resultant inhibition of hTERT expression decreases cellular proliferation and causes cancer cell death. Furthermore, hTERT knockdown shows a significant reduction in tumour metastasis and alters protein expression. Collectively the thesis work shows the high potential of ZIF-C-based biocomposites as a promising general tool for gene therapy of different types of cancers.