Optimization of an adeno-associated viral vector for keratinocytes in vitro and in vivo

Background Local gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. Objective To optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. Methods AAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo . Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. Results A novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo . A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. Conclusion The combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro . The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies. ### Competing Interest Statement M.K. has a patent pending for AAV capsids and the tissue-specific promoter. The remaining authors have no conflicts of interest to declare. * KC : keratinocyte AAV : adeno-associated virus PBS : phosphate-buffered saline EDTA : ethylenediaminetetraacetic acid PFA : paraformaldehyde DAPI : 4ʹ,6-diamidino-2-phenylindole GFPNLS : green fluorescent protein with a nuclear localization signal mCherryNLS : mCherry with a nuclear localization signal mASCs : mouse adipose-derived stromal cells hDFs : human dermal fibroblasts K14 : ( KRT14 ), K16P5 ( KRT16 pseudogene 5).