Development and Validation of Novel AAV2 Random Libraries Displaying Peptides of Diverse Lengths and at Diverse Capsid Positions

Abstract

Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

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

“…Similarly, insertion of peptides into the viral capsid as well as equipping AAV vectors non-genetically with targeting ligands without destruction or shielding of the natural receptor binding sites will result in an expanded tropism 4,24 . With regard to genetic targeting approaches candidate peptides are commonly selected from phage or AAV peptide display libraries, which-due to technical constraints-contain only short peptides [8][9][10]23,25 . These are unlikely to reach binding affinities comparable to antibodies, because of the entropy loss when a small peptide is bound.…”

“…As of yet, genetic engineering employing DNA shuffling of capsid-encoding genes from different serotypes and directed evolution as means to select mosaic AAV capsids with novel features has demonstrated its promise as powerful strategy to improve, but not to restrict, gene delivery to a tissue of choice [5][6][7] . Similarly, efforts to redirect tropism by insertion of receptor-targeting peptides or by non-genetic targeting approaches were successful, but have so far not resulted in AAVs that do discriminate between defined cell types as specifically as, for example, antibodies or antibody mimetics can do 4,[8][9][10] .…”

Displaying High-affinity Ligands on Adeno-associated Viral Vectors Enables Tumor Cell-specific and Safe Gene Transfer

“…DARPins are high-affinity binding molecules derived from ankyrin repeat proteins that have been developed as alternative to antibody-based scaffolds and which are selected by highthroughput screens from DARPin libraries. 21,22 With 14-17 kDa DARPins largely exceed the size tolerated as insertion within the tight structure of the AAV capsid, 15,23 but did not affect vector packaging when fused to VP2. As VP2 fusion proteins, DARPins were readily displayed on the viral capsid and conferred the AAV vectors with ablated HSPG-binding ability with an as of yet unprecedented level of cell type specificity of vector genome delivery and cell transduction resulting in a restricted biodistribution and the safe delivery of suicide genes following systemic application.…”

Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

“…The AAV peptide display technology represents a powerful tool to screen for viral variants that overcome both, pre-and postentry barriers, since this evolutionary approach selects for mutants capable of fulfilling the whole viral lifecycle, i.e., generate progeny following target cell infection. [19][20][21][41][42][43][44] For selection of AAV variants targeting HK, we used our previous-described library of mutants displaying 7-mer random peptides at amino acid position 587. 20 Using this position for peptide insertion results in natural receptor-blinded mutants as the two main residues of the HSPG binding motif, R585 and R588, become separated.…”