Dual and triple AAV delivery of large therapeutic gene sequences into the inner ear

Adeno-associated virus (AAV)-mediated gene therapy has evolved from the bench to the bedside, and is now considered the therapy of choice for certain inherited diseases. AAVs are attractive vectors for several reasons: they are nonpathogenic, result in long-term transgene expression, have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms allowing the targeting of specific cell types. However, one of the greatest limitations of AAVs is the limited genome-packaging capacity of ∼4.7 kb. Given that numerous diseases are caused by mutations in genes with coding sequences exceeding this capacity, packaging into a single AAV capsid is currently unfeasible for larger genes. Taking advantage of the AAV genome's ability to concatemerize, multiple strategies have been explored to overcome the size limit of AAV vectors. One strategy is to split large transgenes into two or three parts, generating dual or triple AAV vectors. Coinfection of a cell with these two or three AAVs will then, through a variety of mechanisms, result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector. This review: 1) documents AAV dual and triple vector strategies currently employed in a variety of tissues, and highlights the advantages and disadvantages of each method; 2) describes the first successful studies using the dual vector approach to restore hearing and prevent deafness in a mouse model of non-syndromic deafness due to absence of the otoferlin protein function, and the implications of these findings for the future of gene therapy in the human inner ear; and 3) highlights additional different deafness genes that could be potential future targets for gene therapy using the dual vector approach.