Reprogrammable bacterial nanosyringes to deliver RNA and gene editors

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• The engineered Photorhabdus virulence cassette (PVC) system is effective for protein delivery but lacks RNA packaging and injection capabilities.
• Expanding PVC applications to RNA delivery is crucial for advancing gene therapy and molecular tools.

Purpose of the Study:

• To engineer a novel platform, DART (PVC Docker-based All-purpose RNA Injection Delivery Tool), for intraluminal RNA loading and delivery using the PVC system.
• To demonstrate DART's capability to protect and deliver diverse RNA types and facilitate gene editing applications.

Main Methods:

• Engineered the PVC system by incorporating the U1A RNA-binding domain for intraluminal RNA anchoring.
• Developed DART for protective loading of various RNAs (Pepper RNA, guide RNA, siRNA, miRNA, mRNA).
• Co-delivered Cas9 with DART for in vitro gene editing of EGFP, KRAS, and PD-L1 targets.

Main Results:

• Successfully achieved intraluminal RNA loading and protective packaging of diverse RNA molecules within the PVC.
• Demonstrated effective in vitro gene editing of reporter (EGFP), oncogenic (KRAS), and immune-related (PD-L1) targets.
• Showcased significant antitumor effects in a mouse model via DART-mediated KRAS knockout.

Conclusions:

• DART represents a novel intraluminal nanosyringe platform that expands PVC capabilities from protein to RNA delivery.
• DART facilitates versatile RNA delivery and gene editing, showing therapeutic potential, particularly in cancer treatment.
• This internal engineering approach complements external strategies like SPEAR, broadening the utility of PVC-based systems.