CRISPR-Cas9 lipid nanoparticles for targeting cyclin-dependent kinases in the tumor microenvironment

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Research

Background:

• Cyclin-dependent kinases 4 and 6 (CDK4/6) play crucial roles in cancer progression.
• Targeting CDK4/6 is a significant strategy for cancer therapy.

Purpose of the Study:

• To develop a novel cancer treatment using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) plasmid DNA (pDNA) encapsulated in lipid nanoparticles (LNPs).
• To investigate the efficacy of a gene knockout strategy targeting CDK4/6 for cancer treatment.

Main Methods:

• pDNA-LNP formulation was prepared and characterized using a microfluidic system.
• Cytotoxicity and apoptosis induction were assessed using the Annexin-V-FITC-PI method.
• Gene expression levels of CDK4 and CDK6 were quantified using real-time quantitative PCR (qPCR).
• In vivo anti-tumor activity was evaluated in a preclinical cancer model.

Main Results:

• pDNA-LNP exhibited a hydrodynamic diameter of 90.0 ± 0.1 nm with a PDI of 0.1 and negative zeta-potential.
• Significant apoptosis induction and reduction in CDK4/6 expression were observed in treated cells.
• In vivo studies showed decreased tumor size and increased median survival time (MST) from 31 to 51 days with 0.5 μg pDNA-LNP.
• A dose of 1 μg pDNA-LNP indicated potential toxicity, necessitating further dose optimization.

Conclusions:

• CRISPR-Cas9 pDNA encapsulated in LNPs is a promising strategy for cancer treatment.
• This gene knockout approach effectively suppresses tumor growth and enhances survival.
• Further studies are required to optimize the dosing for safe and effective therapeutic application.