Covalent coupling of high-affinity ligands to the surface of viral vector particles by protein trans-splicing mediates cell type-specific gene transfer
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Abstract
We have established a novel approach for the covalent coupling of large polypeptides
to the surface of fully assembled adeno-associated viral gene transfer vector (AAV)
particles via split-intein mediated protein-trans-splicing (PTS). This way, we achieved
selective gene transfer to distinct cell types. Single-chain variable fragments (scFvs)
or designed ankyrin repeat proteins (DARPins), exhibiting high-affinity binding to
cell surface receptors selectively expressed on the surface of target cells, were
coupled to AAV particles harboring mutations in the capsid proteins which ablate natural
receptor usage. Both, the AAV capsid protein VP2 and multiple separately produced
targeting ligands recognizing Her2/neu, EpCAM, CD133 or CD30 were genetically fused
with complementary split-intein domains. Optimized coupling conditions led to an effective
conjugation of each targeting ligand to the universal AAV capsid and translated into
specific gene transfer into target receptor-positive cell types in vitro and in vivo.
Interestingly, PTS-based AAVs exhibited significantly less gene transfer into target
receptor-negative cells than AAVs displaying the same targeting ligand but coupled
genetically. Another important consequence of the PTS technology is the possibility
to now display scFvs or other antibody-derived domain formats harboring disulfide-bonds
in a functionally active form on the surface of AAV particles. Hence, the custom combination
of a universal AAV vector particle and targeting ligands of various formats allows
for an unprecedented flexibility in the generation of gene transfer vectors targeted
to distinct cell types.