Adenoviruses are widely used for gene transduction and oncolytic therapy. In order to selectively target certain cell types, many groups, including our own, have modified the viral capsid or fiber protein (8
). Most studies report modifications of either the HI loop or the C terminus of the adenovirus fiber. However, tumor selectivity can also be achieved by promoter-controlled E1A expression in tumor tissues or micro RNA target sequences to selectively degrade E1A expression in off-target tissues (7
). The main aim of the present study was to increase viral transduction efficiency and to overcome the fiber-masking problem caused by excessive fiber proteins release from infected cells that blocks CAR on noninfected neighboring cells and prevents progeny virus entry (31
). To achieve this, we decided to keep the targeting agent away from the fiber and to put it on the virus capsid. Although modification of the hexon HVR has been difficult to achieve (50
), several groups have verified that the HVR5 site is tolerant for foreign peptide insertion (37
). Moreover, given the fact that there are 240 hexon trimers expressed on the adenoviral surface versus only 12 fiber trimer molecules and that hexon modification would not affect the native fiber binding, we decided to modify the hexon HVR5 site.
Our targeting peptide of choice is the protein transduction domain of the Tat protein from HIV-1 (Tat-PTD). Kurachi et al. have previously introduced Tat-PTD in the adenovirus fiber knob (23
), and Eto et al. reported a method to modify adenovirus with Tat-PTD by chemical conjugation to lysine residues on exposed viral proteins (15
). However, the chemical conjugation procedure is relatively complex, and the exact ratio of conjugated Tat-PTD peptide per viral particle could not be determined (15
). In our case, the Tat-PTD sequence was flanked by a short α-helix spacer and genetically inserted into the hexon HVR5 region. We hypothesized that the short α-helix spacer would expose the Tat-PTD motif, thereby increasing the virus-cell interactions, thus improving the transduction efficiency. The predicted model of the modified trimerized hexon (b) was obtained by superimposition of the Tet-PTD and linkers on the hexon trimer previously modeled by others. It shows that the Tat-PTD sequence in HVR5 is exposed on the top surface of the hexon, the portion of the protein facing the surrounding.
The transduction efficiency of Ad5PTD(GFP) was dramatically increased for CAR-negative cell lines compared to the unmodified virus Ad5(GFP). Interestingly, up to 90% of the SK-N-SH cells, which are nonpermissive for native adenovirus transduction, could be transduced by the Tat-PTD-modified Ad5PTD(GFP). In all other tested cell lines, the modified vector shows the same or better transduction efficiency than the nonmodified Ad5(GFP) vector. The mechanism of cellular uptake and cell penetration of CPPs has been studied for decades and still remains divergent. Different models have been proposed to describe the mechanism. In general, these models can be categorized as energy-dependent endocytosis and direct translocation via the lipid bilayer (34
). Another suggestion is that CPPs only play a role in “adherence” or “docking” to the cell surface while endocytosis mediates the actual cellular uptake (25
). The secondary structure was also found to be important for different classes of CPPs (11
). In our case, the exact transduction mechanism of the Tat-PTD modified viruses is unclear. We are able to transduce CAR-negative cells with the Tat-PTD-modified viruses and the transduction can only be partly blocked by soluble fiber molecules, which strongly indicates that a CAR-independent pathway is utilized for cellular uptake.
Recent data have demonstrated that the overproduced fiber molecules during the first round of viral infection are released prior to cell lysis and mask the receptor on adjacent uninfected cells and therefore inhibit the following rounds of infection (31
). This phenomenon limits the usage of replicating oncolytic adenoviruses as anticancer agents. In contrast to chemically conjugated Tat-PTD-modified virus (15
) or HI-loop/C-terminus Tat-PTD-modified virus (23
), which would only enhance the first round of infection, we show that our Tat-PTD-modified virus, which utilizes a CAR-independent cellular transduction pathway, can overcome this problem. The plaque formation assay confirmed that Ad5PTD(wt) spreads faster than Ad5(wt) in a two-dimensional model, which suggests that the Tat-PTD-modified virus should spread faster also in three-dimentional structural tumors.
Hexon proteins were reported to play a major role in liver toxicity after intravenous administration of adenovirus (47
). Liver infection, at least in mice, is mediated by binding of FX (Gla domain) to the HVR of the Ad5 hexon surface. The uptake of FX-Ad5 complexes in hepatocytes is mediated through a heparin-binding exosite in the FX serine protease domain. It has also been demonstrated that a single mutation on HVR5 or HVR7 could significantly reduce or totally abolish the FX binding ability (1
). We evaluated the FX de-targeting ability of our HVR5 modified virus. Consistent with other reports, we found that the substitution of HVR5 by the Tat-PTD motif significantly reduces the FX-mediated virus cellular binding activity in two independent assays. In addition, the modification of the hexon removes antigenic epitopes on the virus particle surface, which leads to partial protection from preexisting neutralizing anti-Ad5 antibodies. Surprisingly, the protection from NAbs was not as efficient as was reported for the Tat-PTD chemically conjugated viruses (15
) and the other hexon-modified viruses (32
). This is probably because the relatively small-sized modification cannot remove all natural Ad5 viral capsid epitopes.
We also examined the in vivo
therapeutic effects of the Tat-PTD-modified oncolytic viruses on human neuroblastoma and neuroendocrine tumors. To our knowledge, this is the first study using adenoviruses modified with cell-penetrating peptides as oncolytic agents for cancer therapy. The human neuroblastoma cell line SK-N-SH was chosen for establishing xenografts since it is not transducible by native Ad5. We found that a therapeutic effect on SCID/beige mice with SK-N-SH xenografts can only be achieved by treatment with Tat-PTD-modified viruses, indicating that viral entry is crucial in order to achieve an oncolytic therapeutic effect. Since Ad5PTD(wt) is not tumor selective, we also produced and evaluated the Ad5PTD(D24) virus, along with Ad5(wt) and Ad5PTD(wt). We found that Ad5PTD(D24) is as efficient as Ad5PTD(wt), but not better (a and b). Therefore, only Ad5PTD(D24) was selected for treatment of neuroendocrine CDNT2.5 tumors on NMRI-nude mice. Geoerger et al. reported on an adenovirus AdΔ24-425S11 expressing a bispecific scFv which targets both the adenoviral fiber knob and the epidermal growth factor receptor, to generate higher transduction level on CAR-low neuroblastoma cells (21
). However, the infectivity enhancement of that virus still relies on uptake via CAR at the first round of viral infection in order to produce the 425S11-targeting adapter. In contrast, the infectivity of our Tat-PTD-modified viruses is guaranteed also on CAR-low cells at the first viral infection step and will be carried on to viral progeny. Parikh et al. claimed that treatment of neuroblastoma by wild-type Ad5 was not as efficient as by oncolytic herpes simplex virus due to the lack of Ad5 transduction (29
). We show in the present study that by enhancing Ad5 transduction, a therapeutic effect could be achieved for neuroblastoma.
It has been reported that the therapeutic effect achieved by treatment with oncolytic virus is partially dependent on the host immune response raised by viral infection (5
). We evaluated the therapeutic effect on both nude and SCID/beige mouse models. Nude mice, lacking T cells but with functional B and NK cells, reflect the therapeutic effect from both viral oncolysis and a partially functioning host immune system. SCID/beige mice, deficient for T, B, and NK cells, are severely immunocompromised; thus, any therapeutic effect observed is solely dependent on viral oncolytic activity. Nude mice harboring CNDT2.5 xenografts treated with Ad5(mock) showed delayed tumor growth, indicating that a virally induced host immune response was involved. SCID/beige mice have also reported to have dysfunctional platelets and therefore prolonged bleeding time after needle puncture (18
). All of the SCID/beige mice harboring tumor xenografts got wounds in the tumor area during tumor growth; therefore, the experiment had to be terminated immediately after the last mouse in the Ad5(wt) treatment group was sacrificed.
The changed tropism caused by genetic introduction of Tat-PTD in the Ad5 hexon raises potential safety concerns since the virulence and pathogenicity/transmission in the natural host as well as the host range may have changed. It is therefore important to combine the transductional alteration caused by Tat-PTD with a transcriptional modification in order to restrict virus activity in normal cells. In the present study we chose to combine it with the D24 deletion of E1A. An alternative approach would be to control E1A gene expression with a tissue- or tumor-specific promoter. In either case, the safety and virulence of Tat-PTD-modified Ad5 will have to be further examined and monitored before a clinical trial can be proposed. It should, however, be noted that Ad5 NAbs are also quite efficient in neutralizing Tat-PTD-modified Ad5. Ad5PTD(wt) shows significantly better shielding against Ad5 NAbs only under certain dilutions of sera (1:1,250 to 1:625). This means that under physiological conditions, Ad5 NAbs in the bloodstream will neutralize Tat-PTD-modified Ad5. Nevertheless, it is very important to follow strict guidelines when working with Tat-PTD-modified replicating viruses.
In conclusion, we have developed Tat-PTD-modified oncolytic Ad5-based viruses with elevated infectivity. The viruses circumvent problems caused by excessive production and secretion of virus fiber protein in the first round of infection, fibers that could block receptors on neighboring noninfected cells and slow down subsequent replication rounds. They are particularly promising for the treatment of tumors with low CAR expression, as demonstrated here for experimental neuroblastoma and neuroendocrine tumors.