Drug delivery systems, Department of Pharmacy
Delivery of CRISPR-Cas9 vectors lung cancer therapeutics
KRAS is the most frequently mutated gene in human cancers and despite its direct involvement in malignancy and intensive effort, no effective pharmacological inhibitor of KRAS has been developed. Due to its important role in cell signalling, picomolar affinity between KRAS and GTP, and the relatively smooth protein surface, KRAS has proved to be an impossible target for novel small molecule drugs making it an exciting candidate for nucleic acid-based therapies.
In comparison to systemic chemotherapeutic anti-cancer drugs, the direct localized administration of nucleic acids via the pulmonary route allows higher retention in lung tissues and reduces systemic toxicity for better treatments of lung cancer. Using either siRNA, miRNA or the efficient CRISPR/Cas9 system, mutant KRAS alleles can be specifically targeted resulting in sudden cessation of signalling and ultimate cell death. Inhibition of an initiating oncogene often leads to extensive tumour cell death, a phenomenon known as oncogene addiction.
In order to deliver nucleic acids specifically to lung cancer cells, we will use cell surface proteins signatures that were unique to cancer cells. In order to deliver the payload, we use affibodies against cell a surface protein, ERBB3, a member of the EGFR Receptor Tyrosine Kinase family. We found high levels of ERBB3 on the surface of lung cancer cell lines and Kras mutant tumour models. The affibody will be purified from a bacterial expression system, coupled to fluorescent dyes and their uptake, cellular localization and anti tumour effect alone will be evaluated.
Next, lung cancer cell lines known to carry Kras mutations (A549, A427 etc) will be specifically transfected with the Cas9-sgRNA specific to the mutant allele, thereby deleting it. The frequency of deletion and allele specificity will be measured by digital droplet PCR and T7EI PCR. Subsequently, hallmarks of cancer cells, such as cell survival, proliferation, migration and anchorage independent growth will be evaluated.