Dual-AAV delivery of large gene sequences to the inner ear

Adeno-associated viruses (AAVs) are preferred vectors for gene replacement therapy, as they are non-pathogenic, non-inflammatory, induce stable transgene expression in terminally differentiated cells, and a series of natural and engineered capsid proteins can be employed to target the vectors to specific cells. Only one feature of AAVs is limiting: the low cargo capacity for foreign DNA, restricting their application to coding sequences of <4 kb. In the last decade, splitting larger cDNAs into two AAVs and co-transducing tissue with such dual-AAV vectors has shown to result in the expression of the full-length protein in different tissues like retina, muscle and liver. This is due to the intrinsic capability of the AAV genomes to undergo homologous recombination and/or head-to-tail multimerization in nuclei of target cells. Recently, two groups independently found that a dual-AAV approach successfully delivered the 6 kb full-length otoferlin cDNA into inner hair cells of otoferlin knock-out mice and restored hearing. These pioneering studies pave the way for gene therapeutics that use dual-AAV vectors to restore hearing in forms of deafness caused by mutations in large genes.