Human Papillomavirus Oncogene Manipulation Using Clustered Regularly Interspersed Short Palindromic Repeats/Cas9 Delivered by pH-Sensitive Cationic Liposomes

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology enables targeted gene editing, but cancer gene therapy with this approach requires improvements to enable safe and efficient delivery of CRISPR/Cas9 to tumors. We developed and evaluated a self-assembled liposome to selectively deliver CRISPR/Cas9 to cancer tissues. Our CRISPR/Cas9 system effectively inhibited proliferation of human papillomavirus (HPV) 16-positive cervical cancer cells and induced apoptosis by inactivating the HR-HPV16E6/E7 oncogene. Based on this system, we prepared a long-circulating pH-sensitive cationic nano-liposome complex with a high cell targeting and gene knockout rate. Intratumoral injection of cationic liposomes targeted to splicing HPV16 E6/E7 in nude mice significantly inhibited tumor growth without significant toxicity in vivo. Liposomes that targeted HPV16 E6/E7 splicing were established as a basis for treatment of HPV16-positive cervical cancer drug candidates. Our study demonstrates that this liposome offers an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine-based cancer therapeutics.

Recent Advances in Nanosystems and Strategies for Vaginal Delivery of Antimicrobials

“…In vivo experiments in mice showed that these nanoliposomes significantly inhibited tumor growth without toxicity. Therefore, this system can be considered a valuable delivery carrier of nonviral gene editing elements, offering a promising route to precision medicine-based cancer therapeutics [69].…”

Delivery Approaches for Therapeutic Genome Editing and Challenges

“…The introduction of a tunable tissue-specific method for delivering LNPs opens potential therapeutic applications for lipid-mediated delivery of Cas9. In a separate study, an alternative strategy for tissue-targeted delivery of Cas9 was demonstrated using pH-sensitive liposomes [ 200 ]. The authors demonstrated that pH-sensitive liposomes disassembled in the presence of the tumor microenvironment, releasing the Cas9 protein and allowing for gene editing.…”

Nanovesicle-Mediated Delivery Systems for CRISPR/Cas Genome Editing

“…Delivery systems can be broadly categorized into lipids, polymers, peptide/protein nanovesicles, and extracellular vesicles ( Figure 1, Table 1). [30] hydrophilic head groups. These amphipathic lipids self-assemble in a physiological aqueous environment, forming vesicles with a bilayer structure.…”

“…Another study utilized DOTAP-based cationic liposome for delivery of pCas9 and viral protein-specific sgRNA to treat cervical cancer [30]. In this study, liposomes composed of DOTAP, DOPE, cholesterol, and DSPE-PEG-2000 were complexed with human papillomavirus 16 E6/E7 (HPV 16 E6/E7)-specific sgRNA and pCas9.…”