We have developed a strategy to specifically immobilize AAV vectors directly onto a substrate to which cells subsequently adhere, thus concentrating the virus for direct contact with the cell. Specifically, we had previously inserted a hexa-histidine (6xHis) onto a physically-exposed loop of the AAV2 (i.e., amino acid 587 position) () and AAV8 capsids, which enabled an efficient, single step viral purification via immobilized metal affinity chromatography (IMAC) 17
. The resulting tagged virus was able to mediate high efficiency gene delivery in vivo
and elicited a macrophage and T-cell immune response equivalent to that of a phosphate buffer control injection 17
. Here, the same 6xHis tag was harnessed to immobilize AAV onto a material surface that presents nickel ions chelated by biotin-nitrilotriacetic acid (biotin-NTA) moieties bound to a streptavidin-coated surface (). Once the AAV vectors were bound to the surface, various cell types – including HEK293T, CHO, HeLa, and B16F10 cells – were plated on these substrates, and the subsequent gene delivery was analyzed and compared to gene expression achieved via the direct addition (i.e., bolus delivery) of AAV vector (with either wild type AAV2 or 6xHis tag capsids) to the cell culture medium.
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 (more ...)
The presence of histidine residues on each of 60 capsid protein subunits likely leads to the binding of a single virus to multiple Ni-NTA groups, resulting in potentially strong multivalent binding to the surface 18
. Tuning virus binding levels and strength could thus potentially optimize downstream transduction.
To modulate the interactions between histidine residues and multiple Ni-NTA groups, chimeric 6xHis-AAV vectors were generated by mixing AAV packaging plasmids encoding the 6xHis mutant (pXX2 His6) or the wild type capsid 2 (pXX2Not) at various mass ratios (i.e., 100%, 75%, 50%, 25%, and 0% (w/w) pXX2 His6). In the case of 100% pXX2 His6 plasmid, all sixty viral protein subunits would display histidine residues on the capsid 17
, and as the proportion of pXX2 His6 is reduced, the number of 6x histidine residues exposed on the viral shells is anticipated to decrease () 19
. This reduced presence of histidine tags on the virus likely decreases the extent of multivalent interaction between a single vector and Ni-NTA, thereby decreasing the overall affinity of the virus for the surface. We anticipate that the capsids should contain sufficient levels of histidine to become effectively immobilized to the surfaces; however, overly strong interactions may inhibit subsequent vector release from the surface and thereby reduce gene delivery. Therefore, low or intermediate histidine levels should both adsorb effectively and subsequently be released in close proximity to adjacent cell surfaces to mediate gene delivery. It is also possible that the binding of the immobilized virus to its cellular receptors may aid vector desorption from the 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 (more ...)
We first analyzed the extent of AAV immobilization to surfaces as a function of histidine content. Interestingly, low levels of 6xHis in the vector resulted in effective AAV immobilization to the surface, and the amount of bound virus decreased with increasing histidine. That is, maximal surface binding occurred for vectors packaged with 25% of 6xHis and 75% wild type AAV2 capsid helper plasmids (i.e., 25% 6xHis AAV vectors, ). The reduced binding with higher levels of 6xHis may be due to free capsid proteins not incorporated into viral particles competing with assembled capsids for binding to the surface. In addition to modulating the capsid, changing the concentration of biotin-NTA on the streptavidin substrate was a major factor that modulated the amount of immobilized 6xHis AAV vector (). Viral binding increased substantially as the level of biotin-NTA was elevated up to 10 pmol, but higher concentrations of biotin-NTA (i.e., 100, 1000 pmol) did not further enhance AAV binding. This binding saturation at 10 pmol was observed for all 6xHis AAV formulations. The increased viral binding with higher biotin-NTA levels may be due to a progressive increase in the number of sterically accessible NTA groups for the virus to bind.
The capacity for localized gene delivery to cells that come into contact with a vector-loaded substrate vector requires that the surfaces not prematurely release the vector. To assess desorption in the absence of cells, we incubated the AAV-laden substrates with cell-culture medium. Approximately 6–14% of bound vector initially dissociated from the surface, but for the subsequent 6 days no additional vector desorbed under any conditions (). To investigate the extent to which cells internalize virus introduced by direct addition vs. substrate-mediated delivery, cell-internalized AAV vector was quantified as a function of time and the concentration of biotin-NTA after plating HEK293 cells on the virus using a previously published approach 20
. Approximately 15–20% of the surface-bound 6xHis AAV vector was associated with cells by 2 days, and the level progressively increased through day 6 (). In contrast, the majority of wild type AAV2 vector and 25% 6xHis AAV vector directly added to the medium was internalized into cells within 2 days of exposure to HEK293T (). The difference in cellular internalization between wtAAV2 and 25% 6xHis AAV upon direct addition may represent differences in the affinities of each vector for cell surface receptors, presumably heparan sulfate proteoglycans (HSPG) 21
, as our previous study indicated that insertion of 6x histidine into aa587 resulted in a slightly reduced affinity for a heparin column compared with wtAAV2 17
. Regardless, the cellular internalization results for the immobilized vector importantly indicate cell contact with the bound vector can lead to localized vector uptake.
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 (more ...)
To analyze substrate-mediated gene delivery, 293 cells were incubated on surfaces bound with AAV at a number of 6xHis formulations, and after 2 days luciferase gene expression was assayed. The 25% 6xHis formulation, which yielded optimal vector binding to the surface (), resulted in the highest substrate-mediated gene expression. This result indicates that the binding capacities of the 6xHis AAV vector are likely an important determinant of gene delivery efficiency ()
We next analyzed whether surface-immobilized AAV could mediate transduction of several additional cell types, including CHO, HeLa, and B16F10 cells. HEK293T and HeLa cells, which are highly permissive to AAV2, were utilized as positive controls, and Chinese hamster ovary (CHO) cells and B16F10 human melanoma cells were chosen to assess delivery to cells known to be non-permissive to AAV2. Infection with AAV carrying a luciferase reporter gene varied as a function of both the levels of histidine residues in the vector and biotin-NTA on the surface, demonstrating the potential for modulating localized gene delivery though engineering and tuning the virus-substrate interactions. Luciferase gene expression following bolus vs. substrate-mediated delivery of 25% 6xHis AAV and wtAAV2 was examined at day 2, 4, and 6. Note that, to enable a comparison with bolus delivery, the level of AAV directly added to the medium was fixed at 1 × 107 viral genomes, equal to the levels of vector immobilized to the substrate prior to addition of cells based on viral binding results ().
The onset of gene expression following bolus infection was more rapid and could be detected by 2 days; however, luciferase expression mediated by substrate-mediated delivery reached that of bolus delivery over time (). Substrate-mediated delivery to 293 cells demonstrated similar or slightly improved gene transfer capabilities as compared with direct addition method. One interesting and important aspect of this substrate-mediated delivery is that comparable gene expression could be obtained even with significantly reduced quantities of internalized vector as compared with bolus delivery (~70% less in ), indicating that substrate-mediated delivery may somehow alter intracellular processing of the vector. In addition to 293 cells, substrate-mediated delivery to HeLa and CHO cells was comparable or slightly reduced compared to bolus delivery. Finally, delivery to B16F10 cells, a human melanoma cell line reported to be non-permissive to AAV transduction 22
, was equivalent or slightly higher for substrate-mediated delivery vs. bolus addition.
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.
In conclusion, this study developed a novel AAV delivery system to mediate local gene delivery, with comparable gene transfer efficiency to a bolus delivery for a variety of cell types. Since one possible rate limiting step for AAV transduction is limitations in binding to the cell surface, we hypothesized that maintaining high local concentration of AAV vectors within the cell microenvironment, as well as increasing the physical contact time with the target cell types, may be a promising approach to mediate localized and efficient AAV vector gene delivery. Importantly, this system yielded comparable gene expression with significantly reduced internalized viral quantities compared to bolus or direct addition to medium. Furthermore, we anticipate that the level of gene expression can be tuned by controlling binding capacities (e.g., bound quantity, strength of binding, etc.), which implies that “smart” gene delivery devices can be developed for controlled release of vector. Finally, since the substrate can be potentially “upgraded” to three dimensional scaffolds, this system may have future application in tissue engineering and regenerative medicine efforts. The AAV vector can be incorporated into such a material or device as the final step, such that complex scaffold fabrication processes would not affect the activity of surface-bound AAV. The development of systems with the capacity for local, efficient gene transfer therefore represents an additional gene delivery mode with the potential for application to a number of disease therapies.