Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery

Abstract

Adeno-associated viral (AAV) vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery because of their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance the potential of AAV in a variety of therapeutic applications by reducing spread outside a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity through a hexa-histidine metal-binding interaction. This immobilized AAV vector system mediates high-efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

Figure 1

Schematic illustration of 6xHis AAV vectors and immobilization of the vectors onto the surface. (a): A view of the trimer at the 3-fold axis of symmetry in the capsid (Rasmol). The heparan sulfate proteoglycan (HSPG) binding site, where the 6xHis insertion occurs, is shaded in black. (b): Binding of 6xHis AAV vectors on Ni-NTA-biotin conjugated on the streptavidin surface.

Figure 2

(a): Schematic illustration of 6xHis AAV vectors formed by varying the mass ratio of pHis to pXX2. (b): Surface-bound quantity of hexa-histidine tagged AAV as a function of both the concentration of biotin-NTA on the surface and the fraction of histidine residues on the viral surface. (c): the amount of 25% 6xHis AAV vectors dissociated from the surface containing no cells.

Figure 3

(a): Quantification of vector internalization into cells after Ni-NTA surface mediated delivery or 25% 6xHis AAV or bolus delivery of vectors. (b): HEK293T cell infection and luciferase gene expression by substrate-bound AAV vectors, which were formulated with different ratios of pHis6/pXX2.

Figure 4

Luciferase expression following substrate-mediated and bolus gene delivery. Luciferase expression for various cell types – including (a) HEK293T, (b) HeLa, (c) CHO, and (d) B16F10 cell lines – were investigated.