Adeno-associated Virus (AAV) Dual Vector Strategies for Gene Therapy Encoding Large Transgenes.

The use of adeno-associated viral (AAV) vectors for gene therapy treatments of inherited disorders has accelerated over the past decade with multiple clinical trials ongoing in varying tissue types and new ones initiating every year. These vectors are exhibiting low-immunogenicity across the clinical trials in addition to showing evidence of efficacy, making it clear they are the current standard vector for any potential gene therapy treatment. However, AAV vectors do have a limitation in their packaging capacity, being capable of holding no more than ~5kb of DNA and in a therapeutic transgene scenario, this length of DNA would need to include genetic control elements in addition to the gene coding sequence (CDS) of interest. Given that numerous diseases are caused by mutations in genes with a CDS exceeding 3.5kb, this makes packaging into a single AAV capsid not possible for larger genes. Due to this problem, yet with the desire to use AAV vectors, research groups have adapted the standard AAV gene therapy approach to enable delivery of such large genes to target cells using dual AAV vector systems. Here we review the AAV dual vector strategies currently employed and highlight the virtues and drawbacks of each method plus the likelihood of success with such approaches.