Exploiting high-throughput screens to optimize Adeno-Associated Viral Vectors for gene transfer into primary human keratinocytes

Chronic non-healing wounds such as diabetic ulcers or burns represent a devastating health problem with significant clinical, physical and social implications. The healing can be frustrating and painful for patients. The difficult healing process requires advanced therapeutic strategies such as the use of primary human keratinocytes (HK) as autologous transplants, which may be considered for clinical use. To improve engraftment or to introduce therapeutic genes into primary HK, efficient and safe vectors are required. One of the most promising vector systems today is based on the adeno-associated virus (AAV), a member of the parvovirus family. Recombinant AAV (rAAV) vectors possess a number of attractive properties including low immunogenicity, high stability and the potential to integrate site-specifically without known side-effects. Unfortunately, cell entry into primary HK of rAAV2 is barely detectable and consequentially, HK are poor targets of rAAV2-mediated transductions. As demonstrated in this thesis, primary HK do not express AAV2´s primary receptor heparan sulphate proteoglycan (HSPG), the presence of which, however, is required for binding to AAV2´s internalization receptors. Cell surface targeting allows re-directing the viral vector tropism towards a novel receptor mediating thereby transduction of cells in absence of AAV’s natural receptors. These AAV capsid mutants have displayed improved transduction efficiency in wild-type-AAV non-permissive cells and have provided the opportunity of rAAV-mediated, cell-type-specific gene transfer. As documented in this study, new rAAV vectors were developed as promising tools for modifying primary HK. Using an AAV peptide display library that displayed 7mer peptides of random sequence at capsid position 587; three AAV peptide insertion mutants differing in sequence of inserted ligand (Kera1, Kera2 and Kera3) were selected and subsequently analyzed. To select rAAV targeting vectors with a re-directed tropism, the library was optimized by depleting mutants capable of binding to HSPG prior to selection by heparin affinity chromatography. Furthermore, the selection was performed on primary HK obtained from different donors to target a common receptor and the selection pressure was continuously increased by decreasing the vector genomes per cell ratio to select for the fittest variant. The thereby developed rAAV targeting vectors Kera1 (RGDTATL), Kera2 (PRGDLAP) and Kera3 (RGDQQSL) showed a remarkable change in tropism, transducing primary HK with high efficiency and specificity even in mixed cultures of target and non-target cells. In this study, a novel microarray based bioinformatic approach (comparative gene analysis (CGA)), was used for the identification of the receptor that targeted the mutant that showed the most striking change in tropism, Kera2. Briefly, in cooperation with Giovanni Di Pasquale (NCI/NIH, Bethesda, USA), a screening of the NIH cell line panel was performed, pointing towards the involvement of beta8 integrin subunit for cell transduction by Kera2. Beta8 is unique as it is solely described as heterodimer with alpha V and the integrin αVβ8 could be detected on cell surface of primary human keratinocytes. By blocking experiments with blocking αV- or αVβ8-antibodies experimental evidence was provided that the integrin αVβ8 serves as receptor for Kera2. Finally, this study has shown that the targeting vectors Kera1, Kera2 and Kera3 transduced airlifted differentiated keratinocytes in organotypic 3D cultures. In summary, the three rAAV targeting vectors Kera1, Kera2 and Kera3, selected from an optimized library and using a novel selection strategy, are excellent candidates for successful application in clinical use.