Development of an efficient lipid nanoparticle-based gene delivery system for the treatment of Duchenne Muscular Dystrophy

Biological Issue: Duchenne Muscular Dystrophy (DMD) is an X-linked disorder which prevents dystrophin expression, causing severe muscle degeneration and respiratory failure. Most approved clinical therapies aim only to slow disease progression, while gene therapy approaches have been limited due to the huge size of the DMD gene. To address this, micro-dystrophins (μDys) have been developed as smaller versions that preserve essential functions. To meet the demand for efficient and biocompatible delivery systems, we developed a library of ten different plasmid DNA (pDNA)-loaded LNPs, aiming to identify the most effective formulation in muscular in-vitro model of increasing complexity. Results: after a first physical-chemical characterization, LNPs library was screened for biological efficacy in C2C12 myoblasts cell line, with LNP2 identified as the most promising candidate. To further enhance its performance, we functionalized LNP2 with a pDNA surface coating, achieving a marked improvement. Subsequently, we moved to primary muscle cells isolated from the mdx4cv mouse model, difficult to transfect due to rapid differentiation limiting nuclear entry. To overcome this barrier, pDNA was pre-condensed with Protamine Sulfate, a carrier with intrinsic nuclear localization signals, and subsequently encapsulated in LNP2 (P*LNP2). Screening revealed that combining pre-condensed DNA with surface coating substantially improved transfection efficiency. Thus, we developed an optimized P*LNP2 encapsulating μDys, which showed improved synthetic properties and significantly boosted dystrophin expression in dystrophic muscle cells. Finally, these results were confirmed on a bioengineered 3D vascularized skeletal muscle tissue (X-MET). Conclusions: our innovative approach consisting in using pre-condensed μDys within functionally coated LNPs has shown potential in enhancing the protein expression in multiple in-vitro dystrophic models, providing crucial insights for the further optimization of this delivery platform with other gene cargos.