Cyclization-enhanced poly(β-amino ester)s vectors for efficient CRISPR gene editing therapy.

Area of Science:

• Biomaterials Science
• Gene Therapy
• Polymer Chemistry

Background:

• Poly(β-amino ester)s (PAEs) are versatile non-viral gene delivery vectors with evolving structures.
• Highly branched PAEs (HPAEs) have demonstrated enhanced gene delivery efficacy.
• A need exists for novel PAE architectures with improved transfection capabilities.

Purpose of the Study:

• To introduce and characterize a new class of cyclic PAEs (CPAEs) for gene delivery.
• To evaluate the gene transfection performance of CPAEs compared to HPAEs.
• To assess the potential of optimized CPAEs for CRISPR-Cas9 gene editing in patient-derived cells.

Main Methods:

• Synthesis of cyclic PAEs (CPAEs) using an A2 + B4 + C2 cyclization strategy.
• Characterization of CPAE structures using 2D NMR and photoluminescence.
• In vitro evaluation of DNA delivery, intracellular uptake, gene expression, and biocompatibility.
• Application of MCPAEs for delivering CRISPR-Cas9 plasmid for gene editing in RDEB keratinocytes.

Main Results:

• Two sets of CPAEs with distinct ring sizes and topologies were synthesized and confirmed.
• Macrocyclic CPAEs (MCPAEs) exhibited significantly enhanced DNA intracellular uptake and gene expression compared to HPAEs.
• MCPAEs demonstrated excellent biocompatibility.
• MCPAEs successfully delivered CRISPR-Cas9 plasmids into RDEB patient-derived keratinocytes, achieving targeted gene editing.

Conclusions:

• Cyclic PAEs, particularly MCPAEs, represent a promising advancement in non-viral gene delivery vectors.
• MCPAEs offer superior gene transfection performance and biocompatibility.
• MCPAEs show significant potential for therapeutic gene editing applications, including in genetic disorders like RDEB.