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Adenoviral vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. For effective vaccine development it is often necessary to express or present multiple antigens to the immune system to elicit an optimal vaccine as observed preclinically with mosaic/polyvalent HIV vaccines or malaria vaccines. Due to the wide flexibility of Ad vectors they are an ideal platform for expressing large amounts of antigen and/or polyvalent mosaic antigens. Ad vectors that display antigens on their capsid surface can elicit a robust humoral immune response, the "antigen capsid-incorporation" strategy. The adenoviral hexon protein has been utilized to display peptides in the majority of vaccine strategies involving capsid incorporation. Based on our abilities to manipulate hexon HVR2 and HVR5, we sought to manipulate HVR1 in the context of HIV antigen display for the first time ever. More importantly, peptide incorporation within HVR1 was utilized in combination with other HVRs, thus creating multivalent vectors. To date this is the first report where dual antigens are displayed within one Ad hexon particle. These vectors utilize HVR1 as an incorporation site for a seven amino acid region of the HIV glycoprotein 41, in combination with six Histidine incorporation within HVR2 or HVR5. Our study illustrates that these multivalent antigen vectors are viable and can present HIV antigen as well as His6 within one Ad virion particle. Furthermore, mouse immunizations with these vectors demonstrate that these vectors can elicit a HIV and His6 epitope-specific humoral immune response.

Type 1 Diabetes is characterized by an absolute insulin deficiency due to the autoimmune destruction of insulin producing β-cells in the pancreatic islets. Akt1/Protein Kinase B is the direct downstream target of PI3 Kinase activation, and has shown potent anti-apoptotic and proliferation-inducing activities. This study was designed to explore whether gene transfer of constitutively active Akt1 (CA-Akt1) would promote β-cell survival and proliferation, thus be protective against experimental diabetes. In the study, a fiber-modified infectivity-enhanced adenoviral vector, Ad5RGDpK7, was used to deliver rat insulin promoter (RIP)-driven CA-Akt1 into β-cells. Our data showed this vector efficiently delivered CA-Akt1 into freshly isolated pancreatic islets, and promoted islet cell survival and β-cell proliferation in vitro. The therapeutic effect of the vector in vivo was assessed using streptozotocin (STZ)-induced diabetes mice. Two means of vector administration were explored: intravenous and intra-bile ductal injections. While direct vector administration into pancreas via bile-ductal injection resulted in local adverse effect, intravenous injection of the vectors offered therapeutic benefits. Further analysis suggests systemic vector administration caused endogenous Akt expression and activation in islets, which may be responsible, at least in part, for the protective effect of the infectivity-enhanced CA-Akt1 gene delivery vector. Taken together, our data suggest CA-Akt1 is effective in promoting β-cell survival and proliferation in vitro, but direct in vivo use is compromised by the efficacy of transgene delivery into β-cells. Nonetheless, the vector evoked the expression and activation of endogenous Akt in the islets, thus offering beneficial bystander effect against STZ-induced diabetes.

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(v)β3/α(v)β5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the "antigen capsid-incorporation" strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response.

A safe and efficacious cancer medicine is necessary due to the increasing population of cancer patients whose particular diseases cannot be cured by the currently available treatment. Adenoviral (Ad) vectors represent a promising therapeutic medicine for human cancer therapy. However, several improvements are needed in order for Ad vectors to be effective cancer therapeutics, which include, but are not limited to, improvement of cellular uptake, enhanced cancer cell killing activity, and the capability of vector visualization and tracking once injected into the patients. To this end, we attempted to develop an Ad as a multifunctional platform incorporating targeting, imaging, and therapeutic motifs. In this study, we explored the utility of this proposed platform by generating an Ad vector containing the poly-lysine (pK), the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK), and the monomeric red fluorescent protein (mRFP1) as targeting, tumor cell killing, and imaging motifs, respectively. Our study herein demonstrates the generation of the triple mosaic Ad vector with pK, HSV-1 TK, and mRFP1 at the carboxyl termini of Ad minor capsid protein IX (pIX). In addition, the functionalities of pK, HSV-1 TK, and mRFP1 proteins on the Ad vector were retained as confirmed by corresponding functional assays, indicating the potential multifunctional application of this new Ad vector for cancer gene therapy. The validation of the triple mosaic Ad vectors also argues for the ability of pIX modification as a base for the development of multifunctional Ad vectors.

Despite the many potential advantages of Ad vectors for vaccine application, the full utility of current Ad vaccines may be limited by the host anti-vector immune response. Direct incorporation of antigens into the adenovirus capsid offers a new and exciting approach for vaccination strategies; this strategy exploits the inherent antigenicity of the Ad vector. Critical to exploiting Ad in this new context is the placement of antigenic epitopes within the major Ad capsid protein, hexon. In our current study we illustrate that we have the capability to place a range of antigenic epitopes within Ad5 capsid protein hexon hypervariable regions (HVRs) 2 or 5, thus producing viable Ad virions. Our data define the maximal incorporation size at HVR2 or HVR5 as it relates to identical antigenic epitopes. In addition, this data suggests that Ad5 HVR5 is more permissive to a range of insertions. Most importantly, repeated administration of our hexon-modified viruses resulted in a secondary anti-antigen response, whereas minimal secondary effect was present after administration of Ad5 control. Our study describes antigen placement and optimization within the context of the capsid incorporation approach of Ad vaccine employment, thereby broadening this new methodology.

Human ovarian cancer is a highly lethal malignant neoplasm in woman with no effective treatment if conventional chemotherapy fails. In this regard, conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution to the development of CRAds was the introduction of tumor-selective viral replication to restrict amplification to the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells, killing the cells by cytolysis, leaving normal cells unaffected. However, to date, there have been limitations to the clinical application of these CRAd agents i.e. poor viral infectivity, poor tumor specificity and high toxicity. Here, we report the in vitro and in vivo comparison of four CRAd agents developed for ovarian cancer application, specifically, Ad-Delta24.F5/3, CRAd-C.F5/3, CRAd-M.F5/3 and CRAd-S.F5/3. All CRAd agents contained fiber knob chimeras of adenovirus serotype 3, which enhanced the viral infectivity at the transductional level via a non-Coxsackie-Adenovirus Receptor alternative pathway. In addition, these CRAds embodied distinct mechanisms for the achievement of replication specificity. Tumor cell killing was assessed by using an oncolytic assay and a cell viability assay (MTS) in vitro, while tumor growth was examined in a xenograft model in vivo by using a bioluminescent imaging assay. In addition, the replication rates of the CRAd agents were determined in human liver slices. Both the Ad-Delta24.F5/3 and CRAd-S.F5/3 were demonstrated to have higher tumor killing effects in tumor cells and a lower viral replication rate in human liver. These agents are thus excellent candidates for clinical trials of CRAd agents against human ovarian cancer.

Metallic nanoparticles (NPs) can be used for the diagnosis, imaging, and therapy of tumors and cardiovascular disease. However, targeted delivery of NPs to specific cells remains a major limitation for clinical realization of these potential treatment options. Herein, a novel strategy for the specific coupling of NPs to a targeted adenoviral (Ad) platform to deliver NPs to specific cells is defined. Genetic manipulation of the gene-therapy vector is combined with a specific chemical coupling strategy. In particular, a high-affinity interaction between a sequence of six-histidine amino acid residues genetically incorporated into Ad capsid proteins and nickel(II) nitrilotriacetic acid on the surface of gold NPs is employed. The selective self-assembly of gold NPs and Ad vectors into multifunctional platforms does not negatively affect the targeting of Ad to specific cells. This opens the possibility of using Ad vectors for targeted NP delivery, thereby providing a new type of combinatorial approach for the treatment of diseases that involves both nanotechnology and gene therapy.

Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector. To achieve highly efficient and specific gene delivery, it is often necessary to re-direct Ad5 tropism. Because the capsid protein fiber plays an essential role in directing Ad5 infection, our laboratory attempted to re-target Ad5 through fiber modification. We have developed two strategies in this regard. One is a bi-specific adaptor protein strategy, in which the adaptor protein is designed to bind both the Ad5 fiber and an alternative cell-surface receptor. Another is genetic modification, in which alternative targeting motifs are genetically incorporated into the fiber knob domain so that the Ad5 vectors can infect cells through the alternative receptors. In this chapter, we will focus on the genetic fiber modification strategy and provide a detailed protocol for generation of fiber-modified Ad5 vectors. A series of techniques/procedures used in our laboratory will be described, which include the generation of fiber-modified Ad5 genome by homologous recombination in a bacterial system, rescuing the modified Ad5 viruses, virus amplification and purification, and virus titration.

Adenoviral (Ad) vectors demonstrate several attributes of potential utility for glioma gene therapy. Although Ad infection is limited in vitro by low expression levels of the coxsackie-adenoviral receptor (CAR), in vivo studies have shown the efficacy of Ad vectors as gene delivery vectors. To evaluate the in vivo utility of CAR-independent, infectivity-enhanced Ad vectors, we employed genetically modified Ad vectors in several experimental models of human gliomas. We used three capsid-modified Ad vectors: (1) a chimeric Ad vector with a human Ad backbone and a fiber knob of a canine Ad, (2) an Ad vector with a polylysine motif incorporated into the fiber gene, and (3) a double-modified Ad vector incorporating both an RGD4C peptide and the polylysine motif. These three modified Ad vectors target, respectively, the putative membrane receptor(s) of the canine Ad vector, heparan sulfate proteoglycans (HSPGs), and both integrins and HSPGs. Our in vitro studies indicated that these retargeting strategies all enhanced CAR-independent infectivity in both established and primary low-passage glioma cells. Enhancement of in vitro gene delivery by the capsid-modified vectors correlated inversely with the levels of cellular CAR expression. However, in vivo in orthotopic human glioma xenografts, the unmodified Ad vector was not inferior relative to the capsid-modified Ad vector. Although genetic strategies to circumvent CAR deficiency in glioma cells could reproducibly expand the cellular entry mechanisms of Ad vectors in cultured and primary glioma cells, these approaches were insufficient to confer in vivo significant infectivity enhancement over unmodified Ad vectors. Other factors, probably the extracellular matrix, stromal cells, and the three-dimensional tumor architecture, clearly play important roles in vivo and interfere with Ad-based gene delivery into glioma tumors.

An advantage of the adenoviral vector is its molecular flexibility, which allows for vector tropism modifications for the purpose of cell targeting. In addition to targeting ligands, the capacity to incorporate heterologous peptides has allowed capsid incorporation of other functionalities. We have defined the minor capsid protein IX (pIX) as a locus capable of presenting incorporated ligands on the virion surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we sought to expand the potential utility of our capsid labeling strategy by developing simultaneous imaging capacity for dedicated small animal positron emission tomography and bioluminescence imaging on a single adenoviral vector. Therefore, we constructed an adenovirus that incorporates a fusion protein of herpes simplex virus type 1 thymidine kinase and firefly luciferase (Luc) (TK-Luc) into adenovirus capsid pIX. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional TK-Luc as a component of their capsid surface. Most importantly, Ad-pIX-TK-Luc retained dual enzymatic functions in vitro and in vivo. This dual-modality approach will allow dynamic or real-time imaging analysis of adenovirus-based interventions with maximized analytic flexibility and enhanced resolution potential.

Purpose: Metastatic gastric cancer remains a common and devastating disease without curative treatment. Recent proof-of-concept clinical trials have validated gene therapy with adenoviruses as an effective and safe modality for the treatment of cancer. However, expression of the primary coxsackie-adenovirus receptor is variable in advanced cancers, and therefore, the use of heterologous receptors could be advantageous., Experimental Design: Here, we used capsid-modified adenoviruses for increasing the transduction and subsequent antitumor efficacy. 5/3 chimeric viruses have a serotype 3 knob which allows binding to a receptor distinct from coxsackie-adenovirus receptor. The fiber of Ad5lucRGD is modified with an integrin-targeted motif. Polylysine motifs, pK7 and pK21, bind to heparan sulfates. Oncolytic adenoviruses replicate in and kill tumor cells selectively. Gastric cancer cell lines and fresh clinical samples from patients were infected with transductionally targeted viruses. Capsid-modified oncolytic adenoviruses were used in cell killing experiments. To test viral transduction and therapeutic efficacy in vivo, we developed orthotopic mouse models featuring i.p. disseminated human gastric cancer, which allowed the evaluation of biodistribution and antitumor efficacy in a system similar to humans., Results: Capsid modifications benefited gene transfer efficiency and cell killing in gastric cancer cell lines and clinical samples in vitro and in vivo. Modified oncolytic adenoviruses significantly increased the survival of mice with orthotopic gastric cancer., Conclusions: These preclinical data set the stage for the clinical evaluation of safety and efficacy in patients with disease refractory to current modalities.

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.

Background: New strategies for improving durable functional islet mass will be instrumental in facilitating islet transplantation as a cure for type 1 diabetes mellitus. The ability to transfer immunoregulatory or cytoprotective genes into pancreatic islets may enhance survival. Adenoviral vectors (Ad5) have been used widely to deliver therapeutic genes to different tissues. Limitations associated with the use of Ad5 for gene therapy are related to the reliance of the virus on the presence of its primary receptor, the transient nature of the transgene expression, and the immediate inflammatory and immune response elicited by the infection. Because the arginine-glycine-aspartame (RGD) and polylysine (pK7) motifs have been shown to enhance Ad5 infection through an Ad5 receptor-independent pathway, we hypothesized that they could act additively to improve infectivity and reduce toxicity to isolated human pancreatic islets (IHPI)., Methods: Hand-picked IHPI were infected with nonmodified Ad5, single-modified Ad5 with RGD (Ad5RGD) or pK7 (ad5pK7), and Ad5RGDpK7. Transfection efficiency was evaluated by green fluorescent protein and luciferase expression. Apoptosis was assessed using a quantitative assay, activation of caspase 3 by a colorimetric assay, nuclear factor (NF)-kappaB nuclear translocation using a promoter-luciferase NF-kappaB responsive construct, regulated on activation normal T-cell expressed and secreted (RANTES) by enzyme-linked immunosorbent assay. In vivo functionality was evaluated after transplantation into diabetic nonobese diabetic severe combined immunodeficiency mice., Results: Compared with unmodified and singly-modified Ad5 vectors, Ad5RGDpK7 demonstrated the highest infectivity. After the infection of IHPI with adenoviral vectors using the minimal dose required to infect greater than 80% of the islet cells (Ad5, 500 viral particles [VP]/cell; Ad5RGD and Ad5pK7, 10 VP/cell; Ad5RGDpK7, 0.1 VP/cell), islets infected with Ad5RGDpK7 presented a significant reduction in apoptosis, NF-kappaB nuclear translocation, RANTES expression, and higher glucose disposal rate; reduced Ad5-driven specific Th1 and antibody response were also observed., Conclusions: Ad5RGDpK7 exhibited higher transfection efficiency, allowing a significant reduction in the viral dose required to infect greater than 80% of the islet cells. The reduction in the viral dose was associated with reduced toxicity, inflammation, and immune responses related to Ad5 infection. This strategy may thus be used to successfully modify isolated pancreatic islets.

Adenovirus serotype 5 (Ad5) has great potential for gene therapy applications. A major limitation, however, is the host immune response against Ad5 infection that often prevents the readministration of Ad5 vectors. In this regard, the most abundant capsid protein, hexon, has been implicated as the major target for neutralizing antibodies. In this study, we sought to escape the host neutralization response against Ad5 via hexon replacement. We constructed a chimeric adenovirus vector, Ad5/H3, by replacing the Ad5 hexon gene with the hexon gene of Ad3. The chimeric viruses were successfully rescued in 293 cells. Compared to that for the control Ad5/H5, the growth rate of Ad5/H3 was significantly slower and the final yield was about 1 log order less. These data indicate that the Ad3 hexon can encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficacy of Ad5/H3 in HeLa cells was also lower than that of Ad5/H5. Furthermore, we tested the host neutralization responses against the two viruses by using C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the immunized mice did not cross-neutralize each other in the context of in vitro infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of viruses also did not prevent subsequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5. This strategy may therefore be used to achieve the repeated administration of Ad5 in gene therapy applications.

Adenoviral vectors based on serotype 5 (Ad5) have been widely used to deliver therapeutic genes to different organs and tissues. However, many tissues are poorly infected with Ad5 because of low-level expression of its primary receptor, coxsackievirus-adenovirus receptor (CAR). Two motifs, RGD and polylysine (pK7), have been shown to enhance Ad5 infection via CAR-independent pathways when incorporated into fiber separately. Because the two motifs bind to different cell surface proteins (RGD motif binds to integrins, and pK7 binds to heparan sulfate-containing receptors), we hypothesized that the two motifs function additively to improve gene transfer efficiency. In this study, we sought to improve infectivity of Ad5 by incorporating both RGD and pK7 motifs into fiber. We created an Ad5 vector containing an RGD motif in the HI loop and a pK7 motif at the C terminus of fiber (Ad5.RGD.pK7). Compared with unmodified and singly modified Ad5 vectors Ad5.RGD and Ad5.pK7, the doubly modified Ad5 demonstrated the highest infectivity in both CAR-positive and CAR-negative cells. The enhanced infectivity appeared to be mediated by additive effects of the two motifs. More importantly, Ad5.RGD.pK7 lost the natural CAR-dependent pathway while employing novel targeting mechanisms. This strategy thus may be used to overcome CAR deficiency and to achieve vector retargeting.