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

Abstract

Gene transfer vectors derived from the adeno-associated virus (AAV) have recently received increasing attention due to substantial therapeutic benefit in several clinical trials. Nevertheless, their great potential for in vivo gene therapy can only be partially exploited owing to their broad tropism. Current cell surface targeting strategies expanded vector tropism towards transduction of cell types that are inefficiently infected naturally, but failed to restrict or fully re-direct AAV's tropism. Hypothesizing that this limitation can be overcome by equipping natural receptor-blinded AAV vectors with high-affinity ligands, we displayed designed ankyrin repeat proteins (DARPin) as VP2 fusion proteins on AAV capsids ablated for natural primary receptor binding. These second generation targeting vectors demonstrated an as of yet unachieved efficiency to discriminate between target and non-target cells in mono- and mixed cultures. Moreover, DARPin-AAV vectors delivered a suicide gene precisely to tumor tissue and substantially reduced tumor growth without causing fatal liver toxicity. The latter caused death in animals treated with conventional AAV vectors with unmodified capsids, which accumulated in liver tissue and failed to affect tumor growth. This novel targeting platform will be key to translational approaches requiring restricted and cell type-specific in vivo gene delivery.

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

“…14) and only marginally reduced compared with unmodified AAV vector particles. This is in line with our initial finding demonstrating that insertion of DARPins at the N terminus of VP2 does not interfere with capsid assembly or packaging efficiency 11 . DARPins are thus a further example proving the suitability of this insertion site for the display of functionally active polypeptides while keeping basic viral vector functions active 21,22 .…”

“…We recently reported on the development of a new AAVtargeting platform that exploited the N terminus of the viral capsid protein VP2 as insertion site for DARPin-based targeting ligands 11 . To redirect tropism, ligand insertion was combined with mutagenesis to eliminate key residues for natural receptor binding.…”

“…The DARPin scaffold 26 was previously shown to be compatible with insertion at the N terminus of the viral capsid protein VP2, to fold well and to maintain its specificity 11 . Complete redirection of AAV's tropism thus requires a natural receptor-'blinded' capsid as backbone, as we use here, combined with a complete depletion of targeting-domain-deficient particles.…”

“…Reasoning that specificity for a target receptor, and thus for a cell type of choice, is best achieved by displaying a high-affinity targeting domain based on a robust and well-folding scaffold, we recently exploited DARPins as targeting ligands 11 . Such target receptor-specific DARPins with affinities similar or better than those of antibodies can be obtained from libraries by selection approaches against essentially any molecule 12 and fold correctly even in the context of being fused to a virus capsid protein.…”

“…For our proof-of-concept, we had employed a DARPin specific for Her2/neu 13 . Combining insertion of the Her2/neu-specific DARPin-9.29 as fusion to AAV's capsid protein VP2 (viral protein 2) with ablation of natural receptor binding through mutagenesis of two arginine residues in each of the 60 capsid monomers resulted in an AAV vector preparation that was by far more specific for its target cells than any AAV vector developed so far 11 . However, a low, but detectable off-targeting in heart tissue remained.…”