New treatment for Duchenne muscular dystrophy based on zwitterionic polymers to encapsulate Adeno-Associated Virus vectors

Duchenne Muscular Dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene, leading to progressive muscle degeneration. Current treatments, such as corticosteroids and rehabilitation, only slow disease progression without providing a cure. Gene therapy offers a promising alternative by directly addressing the genetic cause, with adeno-associated viruses (AAVs) being the most effective vectors due to their low toxicity, high stability and long-lasting transgene expression. But despite these advantages, there are also some limits. First, the size of the genome has to be lower than 4,7 kb due to the low capacity of the vector. To overcome this, researchers developed a shortened gene called micro-dystrophin which is small enough to fit inside the vector. Moreover, there is the problem of the host immune response: neutralizing antibodies (nAbs) are present in a large part of the population and can block the transgene delivery. To overcome this, our study aims to develop a coating strategy for AAVs that reduces immune recognition while maintaining viral tropism, aligning with the objectives of the Grup d’Enginyeria de Materials (GEMAT). Initial attempts to coat AAVs with OM-pBAEs via electrostatic interactions lacked stability under physiological conditions. A subsequent approach using NHS-functionalized pBAEs achieved in vitro success but failed in vivo. To address these limitations, we explored click chemistry for covalent AAV coating. This method involves a two-step reaction: first CliCr-Osu binds to AAV surface amines, and then the attached CliCr reacts with a zwitterionic polymer, forming a stable coating. We compared this approach with electrostatic and conventional covalent strategies through transduction studies and zeta potential measurements, evaluating stability and immune evasion. Characterization of the CliCr-Osu linker was essential to confirm its reactivity with AAV and polymers. Fluorescence-based assays validated AAV conjugation, while transduction studies assessed intracellular trafficking of coated versus uncoated vectors. Additionally, we synthesized an azide-modified C6 pBAE polymer, enabling specific binding to the CliCr group, and evaluated its thermal properties and its coating ability. Preliminary results suggest that click chemistry provides a more stable and efficient coating than electrostatic interactions. Cytotoxicity tests confirmed the biocompatibility of modified viral formulations, supporting their potential for in vivo applications. While further studies are needed to optimize coating efficiency and immune response modulation, our findings highlight the potential of Click Chemistry to enhance AAV-based gene therapy for DMD.