Post cross-linked ROS-responsive poly(β-amino ester)-plasmid polyplex NPs for gene therapy of EBV-associated...

Area of Science:

• Biomedical Engineering
• Gene Therapy
• Nanotechnology

Background:

• Nasopharyngeal carcinoma (NPC) is prevalent in South China and Southeast Asia, often linked to Epstein-Barr virus (EBV) infection.
• Latent membrane protein 1 (LMP1), encoded by EBV, promotes NPC growth by upregulating NF-κB and PI3K pathways.
• Targeting LMP1 offers a potential therapeutic strategy for EBV-related NPC.

Purpose of the Study:

• To develop and evaluate a novel poly(β-amino ester) (PBAE) nanoparticle system for delivering CRISPR/Cas9 plasmids.
• To investigate the efficacy of this system in targeting and cleaving the Lmp1 oncogene in EBV-positive NPC cells both in vitro and in vivo.
• To assess the potential of this gene therapy approach for treating EBV-related NPC.

Main Methods:

• Development of post cross-linked ROS-responsive PBAE nanoparticles for CRISPR/Cas9 plasmid delivery.
• Optimization of nanoparticle composition, resulting in stable polyplex nanoparticles (NPs) with a diameter of ~230 nm and zeta potential of 22.3 mV.
• In vitro evaluation of NP transfection efficiency, gene editing ability against Mucin2 and Lmp1, and intracellular ROS levels in EBV-positive C666-1 cells.
• In vivo assessment of NP tumor penetration and tumor growth inhibition in a C666-1 xenograft mouse model.

Main Results:

• Optimized cross-linked NPs demonstrated enhanced cell uptake and higher transfection efficiency (53.5%) compared to non-cross-linked systems (40.6%) in C666-1 cells.
• The NPs achieved more efficient gene editing of the Mucin2 model gene (17.9%) and Lmp1 (8.5%) compared to controls.
• In vivo studies showed good tumor penetration and significant tumor growth inhibition in the xenograft model via Lmp1 cleavage, with reduced intracellular ROS levels.

Conclusions:

• The developed ROS-responsive PBAE nanoparticles effectively deliver CRISPR/Cas9 for targeted Lmp1 gene editing in EBV-related NPC.
• This targeted gene therapy approach demonstrates significant potential for inhibiting tumor growth and offers a promising strategy for treating EBV-associated nasopharyngeal carcinoma.