Expanding the scope of adeno-associated-virus vectors

Babutzka, Sabrina (2023): Expanding the scope of adeno-associated-virus vectors: from retinal gene therapy to next generation vaccination platform. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)

Abstract

Gene therapy of inherited retinal dystrophies (IRDs) using vectors based on adeno-associated viruses (AAVs) has been widely explored to intervene in the progression of retinal degeneration. However, AAVs are further investigated outside the field of gene supplementation approaches allowing for unconventional platforms of use. Here, we introduce novel AAV9-based capsid variants displaying small peptide insertions attempting to alter the natural tropism for effective transduction of retinal cells after less invasive intravitreal (IVT) administration. Previously heparan-sulfate proteoglycan (HSPG) binding has been shown to significantly contribute to retinal cell tropism. Therefore, we introduced an artificial HSPG-binding site with peptide insertions to investigate how this affects penetration of retinal cell layers after IVT administration. A total of ten different AAV9-based variants were characterized on technical aspects as well as in vitro, ex vivo and in vivo tropism and transduction properties in mouse retina. AAV9.NNR and AAV9.GLR outperformed AAV9 wildtype as well as previous AAV9 variants lacking a HSPG motif in vitro and showed strong eGFP expression in vivo. Moreover, AAV9.NNR, AAV9.GLR and AAV9.RAAK mediated efficient transduction of the non-pigmented epithelium (NPCE) of the ciliary body in C57BL/6 mice. Further, we explore the potential of a novel vaccination platform which uses AAV capsids as scaffolds for large immunogenic epitopes to induce strong and specific immune responses using COVID-19 as a model disease to evaluate this approach. To this end, we introduced large immunogenic peptide insertions of approximately 200 amino acids on the surface exposed loop IV of AAV2 and AAV9. Empty, virus-like particles (VLPs) without vector genome were produced and administered subcutaneously in adult rabbits showing potent IgG and IgM responses specific to the presented antigen. Additionally, vector incubation with blood plasma of mRNA-vaccinated human donors resulted in antigen-specific neutralization effects in vitro. In summary, we introduce novel AAV9-based capsid variants with artificial HSPG-binding motifs that show superior transduction efficiency for retinal cells and additional tropism for ciliary body cells. Furthermore, we present a novel platform that expands the toolbox of AAVs as immunogenic scaffolds for the development of next-generation vaccines in the context of diseases where traditional vaccination approaches have so far not succeeded.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Graduate School of Systemic Neurosciences (GSN)
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 12. Dezember 2023
• 1. Bericht­erstatter:in: Michalakis, Stylianos