TEHC · Towards the Engineering of High-Capacity Viral Vectors for Neuromuscular Diseases

Gene therapies are a fast becoming a realistic treatment for patients suffering from genetic disorders. To date, systemically applied invivo gene therapies exclusively rely on adeno-associated viral (AAV) vectors for gene delivery to affected cells. AAV vectors have manyfeatures that make them successful gene delivery tools and through biomining and bioengineering efforts over the years, AAVvariants are able to transduce a number of clinically relevant targets. However, many cell types, including human muscle cells, are notsufficiently transduced by existing viral vectors. Another challenge is the small genome size of AAV vectors, which is frequently aproblem for neuromuscular disorders where affected genes are larger than in other types of disorders. In the light of these challenges,we are proposing to tackle new avenues for viral vector engineering in two separate, but related, objectives. Objective one is a newway of viral vector re-targeting through incorporation of single domain antibodies (nanobodies) followed by a directed evolutionaffinity maturation step. This approach of adds value to an already published method and should create a capsid-nanobody fusionthat is fit for purpose and potentially also has benefits regarding the evasion of pre-existing antibodies and improved packaging. Thesecond and more ambitious objective of our proposal consists of the adoption of a novel bacteriophage-based viral vector capable oftransducing mammalian cells and delivering up to 171 kilo base pairs of DNA. We are proposing to couple this new vector systemwith our experience in capsid bioengineering and gene therapy, which has not been done before. For this objective, we propose toincorporate antibody-derived targeting molecules such as nanobodies and single-chain variable fragments to highly protruding fibreproteins on the bacteriophage surface and create cell type-specific viral vectors capable of delivering large genes, i.e. humandystrophin.