Impact of capsid modifications by selected peptide ligands on recombinant adeno-associated virus serotype 2-mediated gene transduction

Abstract

Vectors based on adeno-associated virus serotype 2 (AAV2) belong to today's most promising and most frequently used viral vectors in human gene therapy. Like in many other vector systems, the broad but non-specific tropism limits their use for certain cell types or tissues. One approach to screen for transduction-improved vectors is the selection of random peptide libraries displayed directly on the AAV2 capsid. Although the AAV2 library system has been widely applied for the successful selection of improved gene therapy vectors, it remains unknown which steps of the transduction process are most affected and therefore critical for the selection of targeting peptides. Attachment to the cell surface is the first essential step of AAV-mediated gene transduction; however, our experiments challenge the conventional belief that enhanced gene transfer is equivalent to more efficient cell binding of recombinant AAV2 vectors. A comparison of the various steps of gene transfer by vectors carrying a wild-type AAV2 capsid or displaying two exemplary peptide ligands selected from AAV2 random libraries on different human tumour cell lines demonstrated strong alterations in cell binding, cellular uptake, as well as intracellular processing of these vectors. Combined, our results suggest that entry and post-entry events are decisive for the selection of the peptides NDVRSAN and GPQGKNS rather than their cell binding efficiency.

Chondroitin Sulfate is the Primary Receptor for a Peptide-Modified AAV That Targets Brain Vascular Endothelium In Vivo

“…Studies have shown that selecting for capsid modified AAV with improved transduction capacity yields peptides that improve subcellular localization and intracellular processing relative to AAV2. 45 Although the GMN peptide was isolated for endothelial binding and not transduction, it is still possible that it could beneficially modulate restrictive events such as evasion of proteosomal degradation, nuclear trafficking, and capsid uncoating. Furthermore, if the GMN modification does affect these processes, it may provide a partial mechanistic explanation for why AAV-GMN but not AAV2 robustly transduces brain endothelium.…”

Improved Adeno-associated Viral Gene Transfer to Murine Glioma

“…At the highest MOI, ~1.4% of AAV2, ~2.5% of AAV2-TRP and ~8.5% of AAV-ShH19 infected cells tested RFP positive by flow cytometry (Figure 1). Use of an MOI of 10,000 for in vitro transduction was in the range reported by others for different glioma cell lines [38,39,53]. Both rAAV2-TRP and rAAV-ShH19 demonstrated higher transduction efficiency for GL261 cells in vitro compared to rAAV2-WT (2- and 6-fold, respectively).…”

Engineering the AAV capsid to optimize vector–host-interactions

“…These investigations focus as of yet on the AAV2 capsid position 587 located at the tip of the second highest protrusion [13,[40][41][42][43]. This is maybe caused historically as this position was the first that enabled production of AAV targeting vectors with reasonable titers [44].…”