Isolation of targeted AAV2 vectors from novel virus display libraries

Abstract

Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…A single dose of 2 Â 10 11 vector genomes (vg) of a wt free library 35 was injected into the tail vein of NMRI mice. At 3 days post-infection, 300 mm heart tissue slices were prepared and incubated further for 4 days after super-infection with adenovirus type 5 (Ad5) to induce amplification of heart homing AAV viruses.…”

“…35 Selection of targeted AAV2 from AAV2 display peptide libraries Figure 3). In vitro biopanning of AAV2 peptide display libraries on primary neonatal rat cardiomyocytes was done essentially as described 35 and specified in Supplementary methods.…”

“…35 Selection of targeted AAV2 from AAV2 display peptide libraries Figure 3). In vitro biopanning of AAV2 peptide display libraries on primary neonatal rat cardiomyocytes was done essentially as described 35 and specified in Supplementary methods. For sequencing, DNA was extracted from lysates of cells infected with the selected libraries using the DNeasy tissue kit (Qiagen, Hilden, Germany), amplified by PCR using TOPO primers, subcloned into the pCR2.1-TOPO vector and sequenced.…”

“…Also, the post-entry events may be determined by yet unknown tissue-specific factors. A major step forward to improve the target peptide design has been the development of virus display peptide libraries [33][34][35][36][37] or libraries of random capsid chimeras derived from different AAV serotypes. [8][9][10] In such libraries, a multitude of potentialtargeting motifs is presented within the restrictions of capsid assembly and genome packaging.…”

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

“…Moreover, position 587 of the AAV-2 capsid, previously identified as possible insertion site for His-tags 29 , has recently been exploited to separate DARPin-displaying and DARPindeficient particles 30 . However, since His-tag and DARPin are not physically linked in this setting, recombination, which is a frequent event in packaging cells 31 , can give rise to DARPindeficient but His-tag-displaying particles. Moreover, position 587 is embedded in the natural HSPG receptor binding motif 32,33 .…”