Knockdown of hypoxia-inducible factor-1 alpha by tumor targeted delivery of CRISPR/Cas9 system suppressed the metastasis of pancreatic cancer

The hypoxic tumor microenvironment of pancreatic cancer contributes to the progression and metastasis of tumor cells. Downregulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) with CRISPR/Cas9 is a promising approach to modulate tumor microenvironment and inhibit tumor metastasis. However, the in vivo delivery of CRISPR/Cas9 system remains a challenge. In the present manuscript, a tumor targeted lipid-based CRISPR/Cas9 delivery system was developed to suppress HIF-1 alpha. Plasmids encoding Cas9 and HIF-1 alpha-targeting sgRNA were successfully constructed and coencapsulated in R8-dGR peptide modified cationic liposome with PTX. R8-dGR-Lip exhibited enhanced BxPC-3 cell targeting and deep penetration into tumor spheroids. R8-dGR-Lip/PTX/pHIF-1 alpha successfully downregulated HIF-1 alpha and its downstream molecules VEGF and MMP-9, leading to enhanced antimetastatic effects. Besides, the blockade of HIF-1 alpha also promoted the cytotoxicity of PTX on BxPC-3 cell lines. Compared with pegylated liposomes, R8-dGR-Lip enhanced the distribution in tumor tissues. The targeted delivery of CRISPR/Cas9-HIF-1 alpha system and PTX significantly inhibited tumor growth. More importantly, inhibition of HIF-1 alpha suppressed the metastasis of pancreatic cancer and prolonged survival time. Since CRISPR/Cas9-HIF-1 alpha hardly affected HIF-1 alpha expression in normal hepatic cells, the designed R8-dGR-Lip/PTX/pHIF-1 alpha did not induce severe toxicity in vivo. This strategy broadened the in vivo application of CRISPR/Cas9 system. Downregulation of HIF-1 alpha may be a feasible approach for antimetastatic therapy.