GFP Adeno-Associated Virus ( AAV-Kera2 )

Autologous human keratinocytes (HK) formed into sheet grafts are approved for use as skin substitutes. Genetic engineering of HK is a promising technology that improves engraftment and survival of grafts. Although retroviral/lentiviral vectors are effective in targeted gene transfer in keratinocytes, they may raise safety concerns when applied in regenerative medicine. Adeno-associated virus (AAV)-based vectors have emerged as one of the most promising delivery systems for clinical applications. They have low immunogenicity and can produce and deliver DNA vector genomes at very high titers. However, the application of this vector system in the treatment of genetic skin diseases or wound healing disorders has been hampered by the resistance of primary human keratinocytes (HK) to AAV-mediated transduction. The capsid of AV2 tolerates genetic integration of peptide ligands. It is a 60-mer composed of three different viral proteins, VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa), which share most of their amino acid sequences. This common region ("common VP3 region") is frequently used to insert peptide ligands, as it constitutes the exterior of the capsid. Of particular interest is amino acid position 587, located at the peak of the capsid three-fold symmetry axis. Its exposed position favors receptor binding of inserted peptides. AAV-Kera2 has a specific peptide sequence PRGDLAP inserted into position I587 of the AAV2 capsid. Its ability to selectively target keratinocytes with high specificity and efficiency makes it a promising vector for developing treatments for a variety of skin diseases and for studying epidermal biology.