Can Adeno-Associated Viral Vectors Deliver Effectively Large Genes?

Abstract

Gene therapy with adeno-associated viral (AAV) vectors has reached the clinical stage for many inherited and acquired diseases. However, due to a cargo capacity limited to <5 kb, AAV-mediated treatment of diseases that require transfer of larger genes still appears elusive. This is a major drawback of a platform that has otherwise been repeatedly found to be safe and effective. Thus, great efforts have been directed toward the identification of strategies to overcome this limitation. Among the most studied approaches is the use of dual vectors, in which a transgene is split across two separate AAV vectors. Mechanisms acting at either the DNA, pre-mRNA, or protein levels have been explored to restore fulllength transgene expression in infected cells. Here, we will review them as well as additional strategies developed to deliver large genes with AAV. We discuss the pros and cons of these strategies and the aspects that still need to be addressed.

CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors

“…The above-mentioned Cas-effector fusion platforms, especially base editors and prime editors, require delivering even larger cargoes that exceed the packaging limit of AAV (Table 2). In general, delivering over-sized transgenes has been a longstanding hurdle in the AAV gene therapy field, and several approaches have been developed to address this challenge (Patel et al, 2019;Tornabene and Trapani, 2020). The principle is to split the large transgene into two or more segments, each packaged into an individual AAV vector.…”

Vectored Immunotherapeutics for Infectious Diseases: Can rAAVs Be The Game Changers for Fighting Transmissible Pathogens?

“…If scAAV vectors are used for faster mAb generation, the heavy and light chains of the mAb would need to be packaged into more than one rAAV vector ( 75 ). When dealing with cargoes that exceed the packaging capacity of AAV vectors, several strategies can be used ( 76 , 77 ): 1) once inside the cell nucleus, the vector DNAs may reconstitute into the full-length transgene through homologous recombination; 2) the mRNAs from each cargo can be engineered to undergo trans-splicing into a single mRNA transcript for full-length transgene expression; and 3) the polypeptide products of the cargos can be joined into full-length proteins via intein-mediated trans-splicing. The DNA and mRNA recombination-based methods would require co-transduction of the same cell by two rAAV vectors, which can be efficiency-limiting.…”

Challenges for Therapeutic Applications of Opsin-Based Optogenetic Tools in Humans

“…Existing serotypes have been “over-packaged” with mixed success and varying reproducibility and the consensus appears to be that AAV can be overpackaged by ~10%, but with a concomitant reduction in both viral titers and in vivo transduction (Chamberlain et al, 2016 ). Trans-splicing is the favored approach currently used to increase the size of transgenes delivered through AAVs (Tornabene and Trapani, 2020 ). This approach relies on the splitting of the gene of interest and its separate packaging in two different vectors followed by their co-infection in the same cell.…”