A ligand‐pseudoreceptor system based on de novo designed peptides for the generation of adenoviral vectors with altered tropism

Abstract

We have established a new system to produce AdVs ablated of natural tropism. This system should permit the retargeting of AdVs by inserting new ligands within the fiber or through the interaction with bispecific adaptors.

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

“…Gene delivery to specific tissues by AdVs relies on their natural tropism being ablated, followed by the introduction of new tropisms for both viral production and specific tissue targeting. We generated an AdV with K-coil fused to the fiber, and demonstrated that this AdV could be propagated in an E-coil-tagged receptor expressing cell line, and that it could be retargeted through the EGF receptor after pre-incubation with E-coil-tagged EGF [11]. Here, we further support this application by showing that an AdV with the K-coil fused at different position in the fiber can be retargeted, after incubation with E-coil-EGF produced in this study, to glioblastoma tumor cells expressing the EGF receptor (Fig.…”

“…Adenovirus vectors (AdVs) expressing Green Fluorescent Protein (GFP) and wild-type fiber (Ad5FiberWt/GFP), or GFP and K5-coil-tagged fiber (Ad5FiberHIK5cDm/GFP), were produced by cleaving plasmids encoding the Adeno genome with PacI followed by transfection of 293E (cell line described in [11]) using polyethylenimine (PEI) [14]. After 21 days, when cytopathic effects were observed, cells were harvested by three cycles of freeze-thawing and the AdVs were propagated using standard methods.…”

“…We have shown previously using SPR-based biosensor studies that E5 or K5-coil-tagged receptor ectodomains (from the transforming growth factor (TGF)-b Types II and III receptors) bind TGF-b similarly to untagged receptor ectodomains, and that the coil tags can be used for receptor capture on the biosensor surface [9,10]. More recently, we used the E/K coiled-coil dimerization system to establish a virus retargeting scheme in which E5-coil-EGF was utilized to retarget K4-coil-expressing adenovirus to EGFR expressing cells [11].…”

In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46

“…The goal of phage and aptamer library screening is to identify variants with the highest affinity, because in in vitro and in vivo studies with single-chain variable fragment fragments and aptamers, higher affinity usually directly translates into more-efficient binding to receptor-positive cells. Along this line, attempts were undertaken to incorporate high-affinity ligands into measles virus (9) and Ad vectors (2,3,40) in order to increase efficacy and specificity of target cell infection in vivo or to establish new receptor-ligand systems for the propagation of vectors.…”

Re-targeted adenovirus vectors with dual specificity; binding specificities conferred by two different Affibody molecules in the fiber

“…10 Others have also showed double targeting by placing one ligand in the HI-loop and the other at the C-terminus of the fiber protein. [23][24][25] These studies were limited to incorporation of shorter peptide ligands and were mainly used to enhance Ad5 infectivity.…”