Over the past few decades, viral vector-mediated gene therapies have been applied in clinical trials targetinga broad range of diseases, including cardiovascular, muscular, metabolic, neurological, hematological, ophthalmological disorders, and cancer. Adeno-associated virus (AAV) has emerged as a promising vector for in vivo gene delivery due to its non-integration into the host genome, tissue-specific tropism, and low immunogenicity. AAV serotypes such as AAV2, AAV5, AAV8, and AAV9 are widely used in clinical studies, with AAV2 remaining the most prevalent due to itswell-established safety and efficacy profile. Since 2015, the increased application of AAV8 and AAV9 in central nervous system (CNS) clinical trials has furtherexpanded the role of AAV vectors in gene therapy.Moreover, novel capsids like AAV-LK03 and SPK-100 havegainedincreasing attention for their potential to enhance therapeutic outcomes.

Recombinant AAV (rAAV) has achieved significant progress in treating genetic autoimmune diseases, hemophilia, and neurodegenerative disorders. However, challenges persist,including the need for high doses, limited tissuespecificity, low delivery efficiency, neutralizing antibody interference, and quality inconsistencies. Current AAV therapiesface limitations in targeting organssuch as the heart, kidneys, and lungs.Addressing these challenges, particularly byreducing liver tropism and evading immune surveillance, have become top priorities in advancing AAV technology.Consequently, the development of novel AAV variants to enhance vector performanceremains acentral focus in the field of gene therapy.

Capsid development approaches can be  broadly categorized into natural discovery, rational design and directed evolution. With the advancementof computer science and bioinformatics, computer-aided design has emerged asan innovative addition to these strategies. While natural discovery and rational design canproduce acertainnumber ofnewcapsid mutants, they areinherently limited bythe incomplete understanding of AAV binding, internalization, trafficking, uncoating, and gene expression. Directed evolution,in contrast, mimics the process of naturalselection, generating genetic variations with unique advantages and biological properties under selective pressure.This approach offers significant benefits, such as enhanced tissue tropism, immune evasion, and improved transgene expression. Notably, directed evolutioncan generate a large number of mutant proteins, even with limitedunderstanding of the underlying molecular mechanisms. The rapid development of next-generation sequencing (NGS) technologies has furtherelevated directed evolution as a powerful tool for discovering novel capsids. Techniques like capsid shuffling, error-prone PCR, and random peptide display have provenhighlyeffective in generatinglarge librariesof unique capsid mutants.

Figure 1. Approaches to rAAV capsid engineering