Non-viral CRISPR activation system targeting VEGF-A and TGF-β1 for enhanced osteogenesis of pre-osteoblasts implanted with dual-crosslinked hydrogel

Abstract

Healing of large calvarial bone defects remains challenge but may be improved by stimulating bone regeneration of implanted cells. The aim of this study is to specially co-activate transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF-A) genes expressions in pre-osteoblast MC3T3-E1 cells through the non-viral CRISPR activation (CRISPRa) system to promote osteogenesis. A cationic copolymer carrying nucleus localizing peptides and proton sponge groups dimethyl-histidine was synthesized to deliver CRISPRa system into MC3T3-E1 cells with high cellular uptake, lysosomal escape, and nuclear translocation, which activated VEGF-A and TGF-β1 genes expressions and thereby additively or synergistically induced several osteogenic genes expressions. A tunable dual-crosslinked hydrogel was developed to implant the above engineered cells into mice calvaria bone defect site to promote bone healing in vivo. The combination of multi-genes activation through non-viral CRISPRa system and tunable dual-crosslinked hydrogel provides a versatile strategy for promoting bone healing with synergistic effect.

Keywords

• Bone regeneration
• Nanomaterials for gene delivery
• CRISPR activation
• Dual cross-linked hydrogel