Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs

Area of Science:

• Biotechnology
• Gene Editing
• Molecular Biology

Background:

• CRISPR/Cas gene editing holds therapeutic promise but requires safe and efficient delivery vectors.
• Clinical translation is limited by the lack of vectors for short-term expression of CRISPR/Cas components.
• Virus-like particles (VLPs) are emerging as potential delivery systems for CRISPR/Cas technology.

Purpose of the Study:

• To characterize and compare three retrovirus-based VLPs (gRVLPs, egRVLPs, LVLPs) for CRISPR/Cas delivery.
• To evaluate the efficiency and specificity of these VLPs in gene editing applications.
• To establish robust methods for VLP characterization and validation.

Main Methods:

• Optimized production and unified assays for RVLP characterization (NTA, IFC, Cas9-ELISA).
• Comparative testing of gRVLPs, egRVLPs, and LVLPs for gene editing in 293T cells.
• Assessment of on- and off-target editing in human-induced pluripotent stem cells (hiPSCs) targeting CCR5.

Main Results:

• Enhanced gRVLPs (egRVLPs) demonstrated the most efficient gene editing.
• Superior performance of egRVLPs attributed to enhanced Cas9 cargo transfer and/or release.
• egRVLPs achieved nearly 100% CCR5 knockout in hiPSCs with minimal off-target activity.

Conclusions:

• egRVLPs represent the most efficient retrovirus-like particles for CRISPR/Cas delivery among those tested.
• Established characterization methods facilitate VLP validation for predictable and safer applications.
• This study advances the development of VLPs for clinical gene editing strategies.