Combined Genetic Engineering of the Ad5 Hexon (Tat-PTD) and Fiber (f35) Significantly Increases the Transduction Capacity of a Variety of Primary Human Cell Types
Genetic modification of T cells, monocytes, macrophages and DCs are of great interest for immunotherapy and genetic vaccination strategies. We therefore investigated the transduction capacity of our capsid-modified vectors on different subsets of human hematopoietic cells isolated from healthy blood donors. Both non-activated and activated CD3+
T cells from eight donors were transduced at 2000 evg/cell. We found that activated T cells are easier to transduce with Ad5 vectors than non-activated T cells (). The double-modified Ad5PTDf35(GFP) vector with both hexon and fiber modification had significantly higher transduction capability (p<0.001) than the other vectors (), suggesting that Ad5PTDf35-based vectors could be of interest for modification of T cell genomes by zinc-finger and/or TALE nucleases 
. Transduction of CD14+
monocytes, monocytes-derived immature DCs and monocytes-derived macrophages were evaluated in the same manner. The fiber 35-based vectors, i.e.
, Ad5f35(GFP) and Ad5PTDf35(GFP), had significantly higher transduction capacity of monocytes than the fiber 5-based vectors Ad5(GFP) and Ad5PTD(GFP), (). For immature DCs () and macrophages (), the double-modified Ad5PTDf35(GFP) vector was the most efficient (p<0.001). Our data suggest that Ad5PTDf35-based vectors should be evaluated in genetic vaccination strategies for optimal uptake by DCs.
The immunosuppressive properties of mesenchymal stem cells (MSCs) make them attractive in association with allogeneic cell and organ transplantation. They are also known to migrate to tumors, which make them suitable for use as carriers of oncolytic viruses to tumor sites. Human MSCs were transduced with vectors at different evg/cell ratios. Individual modifications of either the Ad5 hexon (Tat-PTD) of fiber (f35) dramatically increased the transduction capacity. However, the double-modified Ad5PTDf35(GFP) vector was the most efficient with transduction levels of up to 99% (). The data suggests that MSCs are potential carriers of oncolytic Ad5PTDf35-based viruses to tumor metastases.
Transplantation of pancreatic islets is today an attractive alternative to whole organ transplantation for a subset of patients with type-1 diabetes 
. Islets can be modified with adenoviral vectors to over-express certain molecules for optimization of islet engraftment and protection from host rejection 
. Genetic modification of exocrine pancreatic cells is useful to evaluate the potential to differentiate exocrine pancreatic cells into insulin-producing islet beta cells. Human pancreatic exocrine cells and islets were transduced with vectors at different evg/cell ratios. The fiber 35-based vectors yielded significantly higher levels of transduction on both cell types compared to the fiber 5-based vectors (p<0.001), (). In addition, Tat-PTD-modification of the Ad5 hexon improves the transduction level of exocrine cells but not islets, which is probably due to the three-dimensional structure of the pancreatic islets, leaving only the outer cell layers of the islet exposed to the viral vectors during transduction. The results suggest that Ad5PTDf35-based vectors are particularly applicable for experimental research on pancreatic cells.
Ad5PTDf35 Shows Significantly Increased Transduction Capacity of Primary Human Tumor Cells
Oncolytic adenoviruses are attractive agents for cancer therapy and they also have the capacity to kill drug- and radiation-resistant cancer cells with stem-cell like properties, also known as tumor initiating cells or cancer stem cells 
. We therefore investigated the vectors on primary human prostatic epithelial cells. The double-modified Ad5PTDf35(GFP) vector showed the highest transduction capacity in of both benign prostatic hyperplasia (BPH) and prostate cancer cells of Gleason (GL) score 8 and 9 (). Importantly, the beneficial effect of the Ad5PTDf35(GFP) vector was particularly pronounced when evaluated for transduction of the CD133+
population of prostate cancer initiating cells 
where the increase was 3-fold (GL9) and 10-fold (GL8) compared to Ad5(GFP), (). Primary high-grade human glioma cells (glioblastoma multiforme grade IV) labeled U3013MG and U3054MG were evaluated in the same manner and we found that the Tat-PTD-modification increased the transduction capacity of both fiber 5-based and fiber-35 based adenoviral vectors (). Taken together, our data suggests that Ad5PTDf35-based oncolytic adenoviruses can efficiently transduce primary glioma and prostate cancer cells and thus may be of therapeutic value for targeting primary cancer cells and more importantly cancer initiating cells, which are often multidrug resistant as well as radiotherapy resistant.
Biodistribution of Surface-modified Virus in Mice After Systemic Administration
Oncolytic adenoviruses are under evaluation as anti-cancer agents by many research groups and companies. Since the tropism of our surface-modified viruses is altered, it was of interest to investigate their biodistribution in mice after systemic administration (tail-vein injection). The wild-type virus vector, Ad5(GFP), was detected in the spleen (2.3×106 copies/mg tissue), liver (2.7×105 copies/mg tissue), lung (3.2×104 copies/mg tissue), kidney (1.2×104 copies/mg tissue) and heart (1.0×104 copies/mg tissue), (). The hexon HVR5-PTD-modified virus, Ad5PTD(GFP), was mainly detected at lower levels in the spleen (9.5×104 copies/mg tissue) and liver (3.8×104 copies/mg tissue). The same was true for the fiber-chimeric virus, Ad5f35(GFP), which was mainly detected at lower levels in the spleen (2.8×104 copies/mg tissue) and liver (2.5×104 copies/mg tissue). The double-modified virus, Ad5PTDf35(GFP), was only detected at low levels in the spleen (2.4×104 copies/mg tissue) and liver (2.0×103 copies/mg tissue).
Biodistribution of surface-modified adenoviruses in mice after intravenous injection.
Ad5 vectors are known to be sequestrated in mouse liver with potential toxicity due to hepatocyte transduction. This can hamper the therapeutic effect of systemic delivery of oncolytic adenovirus to treat metastatic cancers at other sites than the liver, at least in mouse models. Several groups have reported that the Ad5 fiber shaft and knob and/or the capsid (hexon) play an important role in liver cell transduction. The binding of the KKTK domain on the fiber shaft to heparin sulfate proteoglycans (HSPGs) has been shown to mediate liver uptake 
. Blood coagulation factor IX (FIX) and complement C4-binding protein (C4BP) bridge the Ad5 fiber knob to HSPGs and low-density lipoprotein receptor-related protein (LRP) on hepatocytes 
, and coagulation factor X (FX) binds to HVRs of the hexon of the Ad5 virus capsid leading to liver transduction 
. A number of mutations have been reported to completely abolish or compromise CAR-dependent transduction. However, neither a S408E mutation in the fiber knob AB loop 
, a Y477A mutation in the fiber knob DE loop 
nor deletion of TAYT in the fiber knob FG loop 
reduced liver transduction. In contrast, the CAR binding-abolished viruses AdL.F* and dl-K420A-Z possess approximately 10-fold reduced liver transduction capacity, wherein AdL.F* contains four mutations (R412S, A415G, E416G, K417G) in the AB loop of the Ad5 fiber knob plus an insertion in the HI loop of the fiber knob 
and dl-K420A-Z harbors a mutation (K420A) in the Bβ-sheet of the fiber knob 
As previously reported 
, we also detected liver sequestration of wild-type Ad5. However, by either fiber-replacement, Ad5f35(GFP), or hexon HVR5-modification, Ad5PTD(GFP), liver transduction was reduced approximately 10-fold (p<0.05). The reduction in liver transduction by the PTD-modified virus could be explained by sequence alteration in hexon HVR5 site leads a reduced binding of blood factor FX to hexon 
. The reduction in liver uptake of the fiber-replaced virus is also in accordance with other reports 
, which could be explained by that a change in both the fiber shaft and knob leads to a reduction in of binding of HSPGs and blood factors C4BP, FIX and FX. Importantly, the double-modified virus, Ad5PTDf35, reduced liver sequestration by approximately 100 times, indicating a synergistic effect by modifying both the fiber and capsid.
High level of viral genome was present not only in the liver but in the spleen as well, suggesting that splenocytes also play a role in viral clearance and degradation. However due to that the weight of liver (ca. 1000 mg) is higher than that of spleen (ca. 80 mg), liver is still the predominant organ for adenovirus sequestration. In general, our surface-modified viruses were recovered to a lesser extent compared to the wild-type Ad5(GFP), suggesting a faster in vivo clearance of the surface-modified viruses.
Ad5PTDf35(pp65)-modified DCs are Better than Ad5(pp65)-modified DCs for ex vivo Expansion of CMV pp65-specific T Cells
To demonstrate the beneficial effect of using an Ad5PTDf35-based vector for gene delivery we chose to transduce DCs to express an antigen with the intention to use the modified cells to expand antigen-specific T cells. We have previously shown that a population of CMV pp65495–503
-specific T cells can be significantly expanded if T cells from CMV-seropositive, HLA-A2-positive blood donors are stimulated by Ad5(pp65)-transduced autologous DCs 
. However, large amounts of viral vector need to be used to obtain efficient transduction of DCs. Since monocytes and DCs are far more efficiently transduced with the Ad5PTDf35(GFP) than with the Ad5(GFP) vector, as shown in , we speculated that pp65495–503
-specific T cells could be expanded even more efficiently ex vivo
by using an Ad5PTDf35-based vector to modify the stimulator DC cells. We therefore constructed a non-modified Ad5(pp65) and a double-modified Ad5PTDf35(pp65), which both express the full-length CMV pp65 transgene. We transduced monocytes with Ad5(pp65) or Ad5PTDf35(pp65) at a relatively low dosage (100 evg/cell), differentiated them into DCs 
and used them to stimulate and expand autologous pp65-specific T cells. The Ad5PTDf35(pp65)-transduced DC stimulation increased the pp65495–503
-reactive T cells population for all donors, approximately 50–100 fold, while Ad5(pp65)-transduced DC stimulation only increased the pp65495–503
-reactive T cell population 2–8 fold (). These data clearly show that Ad5PTDf35(pp65) would be highly efficient for DC modification to expand CMV pp65-specific T cells ex vivo
. Expanded donor-derived T cells could then be adoptively transferred to transplant patients experiencing CMV complications due to their immune suppressive medication 
. Furthermore, this example serves as an indication that Ad5PTDf35-based vectors are superior to Ad5-based vectors in other settings as well.
Ex vivo expansion of CMV pp65-reactive T cells using adenovirus-transduced DCs.
Construction of AdEasy System-compatible Backbone Plasmids Containing Hexon (Tat-PTD) and/or Fiber (f35) Modifications for Recombinant Ad5 Vector/Virus Production
In order to make our constructs readily available as research tools we adapted the modifications into plasmids compatible with the AdEasy system 
, where recombinant adenovirus DNA is constructed in homologous recombination-proficient Escherichia coli
bacteria (stain BJ5183). The technology is based on two plasmids, pShuttle (cloning plasmid for the transgene of choice) and pAdEasy (virus backbone plasmid). The pShuttle plasmid allows one to easily clone the gene of interest in the adenoviral E1 region. The entire recombinant adenovirus genome with the gene of interest is then constructed by homologous recombination between the pShuttle(trangene) plasmid and the pAdEasy(backbone) plasmid. Adenovirus particles are produced by transfecting the linearized double-stranded DNA virus genome into producer cells such as HEK-293T or 911. Since this is a relatively easy technology to produce recombinant adenoviruses, it is today a widely used system for recombinant adenovirus construction. The standard AdEasy backbone plasmid that is available today will generate Ad5 vectors without any surface-modification. Due to the beneficial effect observed with our surface-modified Ad5 vectors we decided to clone the sequences leading to these modifications into various pAdEasy backbone plasmids to generate user-friendly AdEasy-compatible backbone plasmids with hexon (Tat-PTD) and/or fiber (f35) modifications. All the adenovirus backbone plasmids are listed in . We believe that they will serve as useful tools to advance the development of genetic vaccine vectors, gene delivery vectors and oncolytic adenoviruses.