GFP Adeno-Associated Virus ( AAV2Ald13 )

AAV is a promising gene therapy tool, but known serotypes are generally not suitable for direct clinical use due to their low specificity and immune responses, including cytotoxic lymphocytes and neutralizing antibodies. Several AAV-based drugs have been approved for clinical use, but the serotypes used for gene delivery have a natural tropism for the target tissue: Luxturna, developed for the treatment of Leber congenital amaurosis, is based on the AAV2 serotype with a natural tropism for the retina; Zolgensma utilizes AAV9 to treat spinal muscular atrophy; Glybera is also approved for the treatment of lipoprotein lipase deficiency via muscle transduction, with the serotype AAV1. The development of such therapies should theoretically reduce the cost of treatment, as the expression time of the therapeutic plasmid is relatively long. However, the shortcomings of AAV pose a serious obstacle to the successful implementation of gene therapy in clinical practice, both of which can be overcome through capsid engineering studies.

Capsid engineering strategies involve chemical modification of AAV with the goal of covalently binding specific molecules, such as fluorophores, antibodies, or targeting peptides. Aldehyde tags are introduced into the I587 insertion site. The inserted 13 amino acid peptide LCTPSRAALLTGR contains a cysteine, which contains a thiol group. Subsequent incubation with formylglycine generating enzyme (FGE) results in the conversion of cysteine to formylglycine, which contains an aldehyde. Therefore, the surface of the AAV capsid contains aldehyde groups that can be used for covalent binding to hydrazide- and aminooxy-containing antibodies, proteins, nanoparticles, etc. Chemical conjugation of the RGD motif to the AAV2 capsid resulted in an approximately 3-fold increase in the transduction rate of HeLa cells, and conjugation with anti-HLA mouse antibodies increased the transduction rate of 293T cells from 15.7% to 35.7% compared to wild-type AAV2.