Inhalable nanoparticles for in vivo genome editing mediated by crispr-cas9 delivery for undruggable KRAS driven lung cancers

Introduction

KRAS is the most frequently mutated oncogene. Due to its important role in cell signalling, picomolar affinity to GTP, and smooth protein surface, KRAS has proven to be an impossible target for small-molecule drugs and KRAS-tumours are considered untreatable.

In contrast to small molecule inhibitors, CRISPR/Cas9 based genome-editing can be exploited to correct KRAS mutations. While direct pulmonary delivery of nucleic acids has been successful for treating infections, efficient intracellular delivery at low toxicity remains difficult. For targeted delivery of DNA to lung cancer cells, we propose using affibodies against ERBB3, an EGFR Receptor Tyrosine Kinase family member. Affibodies are small, single domain proteins with high affinities to their targets.

This project aims to address two major obstacles: 1. The `undruggable` nature of KRAS driven lung cancers; 2. Efficient and safe pulmonary delivery of nucleic acids to target cells.

Materials and Methods

KRAS mutant cell lines, A549, A427, hop62, H358 were used for this study. Bacterially overexpressed trivalent ERBB3 affibodies were purified by Ni-affinity chromatography. Size and zeta potential of affibody-DNA conjugates were measured by dynamic light scattering. Plasmid delivery and functionality were examined using flow cytometry, western blot and PCR. Cell viability was evaluated by MTT and cell migration via Boyden-chamber assays.

Results and discussion

Mutation specific sgRNAs for KRAS mutants, G12S, G12D and G12C were cloned into the pSpCas9-GFP plasmid. After transfection to KRAS mutant lung cancer cell lines, high on-target genome editing efficiency was demonstrated by the T7-EndonucleaseI assay and digital droplet PCR. Edited cells showed decreased cell viability and cell migration, confirming this approach for KRAS tumours.

All lung cancer cell lines tested (A431, A549, A427) were found to have high levels of ERBB3 at the membrane. A trivalent ERBB3-affibody was purified by affinity chromatography. The affibody-pDNA conjugate had favorable sizes <200nm, suitable for in vivo delivery, and efficiently delivered pDNA which edited the KRAS mutant allele in lung cancer cell lines. Importantly, affibodies against ERBB3 could independently inhibit PI3K/Akt signaling, making them doubly beneficial. The therapeutic benefit of the affibody-pDNA conjugates was confirmed in xenograft mouse models.

Conclusions

Our data demonstrate the efficient lung cancer-targeted delivery of CRISPR/Cas9 machinery to induce mutant specific genome editing, without causing any severe additional inflammation.

We are the first to study targeted, non-viral pDNA delivery specifically to cancer cells using affibody-DNA conjugates and aim to develop dry powder formulations in order to obtain a clinically relevant dosage form.