Nuclear-targeted CRISPR/Cas delivery by using aptamer-modified polymer lipid nanoparticles.

Area of Science:

• Biotechnology
• Nanomedicine
• Gene Therapy

Background:

• Efficient nuclear delivery of CRISPR/Cas ribonucleoproteins (RNP) is crucial for non-viral gene editing systems.
• Current non-viral methods face challenges in achieving precise and effective nucleus-directed delivery.

Purpose of the Study:

• To develop and evaluate a novel polymer-lipid hybrid nanoparticle system for enhanced nuclear delivery of CRISPR/Cas9.
• To functionalize nanoparticles with the AS1411 aptamer for targeting nucleolin and facilitating nucleus-directed transport.

Main Methods:

• Fabrication of AS1411 aptamer-functionalized polymer-lipid hybrid nanoparticles.
• Confocal microscopy to confirm nanoparticle accumulation within cell nuclei.
• AI-assisted deep convolutional neural networks (CNN) for precise quantification of nanoparticle and DAPI colocalization.
• In vitro assessment of CRISPR/Cas9 gene knockout efficiency and breast cancer cell proliferation inhibition.

Main Results:

• Aptamer-modified nanoparticles demonstrated successful accumulation within cell nuclei.
• AI-based analysis precisely quantified nanoparticle delivery and colocalization.
• Aptamer-functionalized nanoparticles reduced GFP-positive cells to 30.0% (vs. 40.8% for untargeted nanoparticles).
• Targeting the Lcn2 gene showed enhanced knockout efficacy and potent inhibition of breast cancer cell proliferation.

Conclusions:

• Aptamer-mediated nuclear targeting significantly enhances CRISPR/Cas9 editing efficacy.
• This nucleus-targeted nanoparticle system holds potential for advancing non-viral gene therapies.
• The developed system offers a promising strategy for improving the efficiency and precision of gene editing applications.