Thesis title: Development of an efficient lipid nanoparticle-based gene delivery system for the treatment of Duchenne Muscular Dystrophy
Duchenne Muscular Dystrophy (DMD) is a X-linked genetic disorder caused by different kind of mutations in the dystrophin gene, leading to severe muscle wasting and respiratory failure. In recent decades, significant efforts have been made to develop effective gene therapy applications; however, the success of these strategies has been limited by the large size of the DMD gene. To overcome this obstacle, recent advancements have introduced engineered micro-dystrophins (μDys), smaller segments of the dystrophin gene that omit non-essential functional domains. Yet, achieving robust and targeted delivery of the modified gene to skeletal muscle remains challenging. In this context, non‑viral gene delivery platforms represent a compelling alternative to conventional viral vectors, offering superior safety profiles and enhanced biocompatibility. Among these systems, lipid nanoparticles (LNPs) produced through microfluidic manufacturing have led to novel strategies for improved design.
To meet the demand for efficient and protective nucleic acid delivery systems in gene therapy, we developed a library of ten different plasmid DNA-loaded LNPs, initially loaded with a luciferase reporter gene and characterized for their physical-chemical properties. Biological efficacy tests in the C2C12 myoblast cell line identified LNP2 as the most promising candidate. We then enhanced LNP2 with a functional DNA coating, significantly boosting its performance if compared to the unmodified version.
Next, we planned to move to transfect primary muscle cells isolated from the dystrophic mdx4cv mouse model. These cells present an inherent challenge due to rapid differentiation, which hinders plasmid nuclear entry. To overcome this problem, we pre-condensed the DNA with Protamine Sulfate (P*), which naturally contains a Nuclear Localization Signal (NLS). After optimizing the P*-to-DNA weight ratio, we finally synthesized and characterized an LNP encapsulating pre-condensed DNA (hereafter indicated as P*-LNP2). Our subsequent screening revealed that combining pre-condensed DNA with DNA surface coating markedly enhanced the transfection efficiency of P*-LNP2. Confocal microscopy confirmed a significant increase in nuclear localization, a crucial result as efficient gene delivery to the nucleus is essential for effective gene therapy in non-dividing cells. Encouraged by these findings, we synthesized and characterized an LNP2 formulation encapsulating μDys pre-condensed with P*. This complexation improved the physical-chemical properties of the LNPs, which were then tested on primary dystrophic muscle cells. Notably, the formulation markedly elevated dystrophin protein expression. Finally, we validated the optimized P*-LNP2 on a well-established bioengineered, three dimensional vascularized skeletal muscle construct, referred to as eX vivo Muscle Engineered Tissue (X MET). Treating the X-MET with P*-LNP2 not only increased μDys levels but also improved the organoid sarcolemmal stability, the functionality and spontaneous contractile force. In conclusion, our innovative approach consisting in using P*-condensed μDys within functionally coated LNPs has shown substantial potential in enhancing the protein expression in the mdx4cv-derived primary muscle cells and X-MET. Although μDys kjhave recently been shown to lack sufficient efficacy in treating DMD, our results nonetheless endorse continued investigation of this approach in in-vivo models and provide crucial insights for the further optimization of this delivery platform with other gene cargos.