Ad2 DNA Research Articles

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Development of an Inducible, Replication-Competent Assay Cell Line for Titration of Infectious Recombinant Adeno-Associated Virus Vectors.

An important quality attribute of a recombinant adeno-associated virus (rAAV) as a therapeutic vector is its infectivity. Current assays to quantify infectious rAAV rely on coinfection with a helper virus such as adenovirus (Ad), which requires helper virus preparation and introduces additional variability. A simple method that has high sensitivity and removes the need for helper virus would improve assay consistency and facilitate high-throughput applications such as rAAV producer cell line development. In this study, we describe a stable assay cell line that was generated by integrating the coding sequences for AAV Rep68 and Ad E4orf6 and DNA binding protein under the control of inducible promoters. The Rep68 protein expression was further modulated by a ligand-responsive destabilization domain. In several benchmarks, the cell line gave comparable titers with those obtained using a classical Ad coinfection method. The cell line was also used to titer vectors of multiple rAAV serotypes. This cell line has the potential to serve as an effective and robust tool for quantifying infectious rAAV titers to advance gene therapy vector biomanufacturing.

Hydrogeochemical and isotopic study of the origins of groundwater salinization in the deep confined aquifer of northern Yangtze River

The deep confined water of coastal plain of northern Yangtze River suffers salinization. That results from overexploitation. This work aims to investigate the geochemical processes that lead to the salinization for this aquifer. Multiple environmental tracers of major ions, minor ions and isotopes (18O, 87Sr, 13C) were used to yield reasonable conclusions. The TDS of the aquifer ranges from 387 to 2600 mg/L. The aquifer is mainly composed of fresh water. Brackish water is distributed in the eastern and southern coastal areas, and is scattered in some inland areas. The water chemical type evolves from HCO3-Na to Cl-Na as TDS increases. Groundwater salinization is caused by mixing with saline water of marine origin. The aquifer is also affected by other hydrochemical processes. Silicate weathering and carbonate dissolution add Na+, K+, Ca2+, Mg2+ and HCO3- to groundwater. Sulfate reduction makes sulfate be deficient and HCO3- be enriched. Carbonate minerals equilibrium limits the increase in the concentrations of Ca2+, Mg2+ and HCO3-. Cation exchange reduces the concentration of Ca2++Mg2+ and increases the concentration of Na++K+. The aforementioned processes have a combined influence on the formation of fresh water with HCO3-Na type.

Localization of the kinase Ataxia Telangiectasia Mutated to Adenovirus E4 mutant DNA replication centers is important for its inhibitory effect on viral DNA accumulation

Adenovirus (Ad) type 5 (Ad5) E4 deletion mutants including H5dl1007 (E4-) induce a DNA damage response (DDR) that activates the kinase ataxia-telangiectasia mutated (ATM), which can interfere with efficient viral DNA replication. We find that localization of active phosphorylated ATM (pATM) to E4- viral replication centers (VRCs) is important for its inhibitory effect. ATM is necessary for localization of RNF8 and 53BP1 to E4 mutant VRCs, while recruitment of DDR factors Mre11, Mdc1 and γH2AX is ATM-independent, raising the possibility that ATM may affect viral chromatin at VRCs. We assessed E4- and Ad5 chromatin organization by micrococcal nuclease (MN) digestion. A significant fraction of Ad5 DNA is somewhat resistant to MN digestion, whereas E4- DNA is more susceptible. ATM inhibition increases the fraction of E4- DNA that is resistant to MN digestion. Our results address possible mechanisms through which ATM inhibits E4- DNA replication.

Tridimensional infiltration of DNA viruses into the host genome shows preferential contact with active chromatin

Whether non-integrated viral DNAs distribute randomly or target specific positions within the higher-order architecture of mammalian genomes remains largely unknown. Here we use Hi-C and viral DNA capture (CHi-C) in primary human hepatocytes infected by either hepatitis B virus (HBV) or adenovirus type 5 (Ad5) virus to show that they adopt different strategies in their respective positioning at active chromatin. HBV contacts preferentially CpG islands (CGIs) enriched in Cfp1 a factor required for its transcription. These CGIs are often associated with highly expressed genes (HEG) and genes deregulated during infection. Ad5 DNA interacts preferentially with transcription start sites (TSSs) and enhancers of HEG, as well as genes upregulated during infection. These results show that DNA viruses use different strategies to infiltrate genomic 3D networks and target specific regions. This targeting may facilitate the recruitment of transcription factors necessary for their own replication and contribute to the deregulation of cellular gene expression.

Human adenoviral DNA association with nucleosomes containing histone variant H3.3 during the early phase of infection is not dependent on viral transcription or replication.

Adenovirus (Ad) DNA undergoes dynamic changes in protein association as the virus progresses through its replicative cycle. Within the virion, the Ad DNA associates primarily with the virus-encoded, protamine-like protein VII. During the early phase of infection (∼6 h), the viral DNA showed declining association with VII, suggesting that VII was removed from at least some regions of the viral DNA. Within 6 h, the viral DNA was wrapped into a repeating nucleosome-like array containing the histone variant H3.3. Transcription elongation was not required to strip VII from the viral DNA or for deposition of H3.3. H3.1 did not associate with the viral DNA at any point during infection. During the late phase of infection (i.e., active DNA replication ∼12-24 h), association with H3 was dramatically reduced and the repeating nucleosome-like pattern was no longer evident. Thus, we have uncovered some of the changes in nucleoprotein structure that occur during lytic Ad infection.

Molecular dynamics investigation of surfactant micelles as a potential drug delivery vehicle

A new molecular dynamics study is being initiated in which micellar structures of cetyltrimethylammonium bromide (CTAB) are simulated within computationally constructed physiological conditions. Leading up to the simulation phase, several programs and reference files were used to construct a topology file, initial structure/coordinate files, and parameter files to accurately represent a single molecule of CTAB. This single molecule is utilized in making the initial configuration of a system. All systems begin in one of three configurations: a random assembly of molecules in a box, a pre-formed micelle, or an inverse micelle. These initial configurations, once assembled, are placed under generated physiological conditions by using Visual Molecular Dynamics (VMD) to solvate the system with water and add Na+ and Cl- ions to neutralize the system to 0.15 M NaCl, then using Nanoscale Molecular Dynamics (NAMD) to minimize energy and heat the system to 310 K. The system is then simulated using NAMD until a micelle has formed and stabilized. Following each simulation, quantitative and qualitative analysis is used to characterize the system. The overarching goal of this study is to determine the viability of CTAB as a drug carrier in a targeted drug delivery system. Funding for the project is provided by the College of Science and Technology at Fairmont State University and the NASA WV Space Grant Consortium.

Abstract 192: Persistence of First Generation Adenovirus in the Myocardium: Refuting Old Dogma

Introduction: Adenovirus (Ad) vectors do not integrate into host genomes and thus offer clinical benefits as non-mutagenic, acute-term gene delivery vehicles. One limitation to Ad vector use is the transient duration of transgene expression, supposedly resulting from immune-mediated cytolysis of Ad-infected cells. Our recent observation of Ad-induced transdifferentiation of infected cardiac fibroblasts into induced cardiomyocytes over 28 days in vivo called this premise into question. Therefore, we hypothesized that Ad remains in the myocardium for over 28 days and, to our knowledge, are the first to suggest alternate mechanisms for this persistence. Methods and Results: Ad encoding green fluorescent protein (AdGFP) or mock was administered via intramyocardial injection into Sprague-Dawley rats, and Ad genome and GFP expression were analyzed 3 or 28 days later (n=6 per group, 3 euthanized at each time point). Polymerase chain reaction typing and amplicon sequencing confirmed the presence of Ad DNA in all 6 treated animals. Quantitative real time polymerase chain reaction assays further demonstrated more than a three-fold increase in Ad DNA levels at both days 3 and 28 compared to untreated control animals (3.41±1.40 and 3.37±1.23, respectively). Thus, at 28 days Ad DNA persisted at a high level comparable to that which was present at 3 days. Next, AdGFP expression and host immune response were characterized by fluorescence microscopy and histology. We found that GFP-expressing cells decreased five-fold by 28 days (49±9 v. 3±0 cells). In addition, hematoxylin and eosin staining showed minimal infiltrating mononuclear inflammatory cells in AdGFP and mock-treated animals at both time points. Conclusions: Ad DNA and infected host cell persistence suggests that host-mediated cytolysis may not be responsible for the downregulation of transgene expression, indicating that Ad vectors may consequently be useful for therapies like cellular reprogramming wherein host cell persistence is required.

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection.

The Adenovirus (Ad) genome within the capsid is tightly associated with a virus-encoded, histone-like core protein—protein VII. Two other Ad core proteins, V and X/μ, also are located within the virion and are loosely associated with viral DNA. Core protein VII remains associated with the Ad genome during the early phase of infection. It is not known if naked Ad DNA is packaged into the capsid, as with dsDNA bacteriophage and herpesviruses, followed by the encapsidation of viral core proteins, or if a unique packaging mechanism exists with Ad where a DNA-protein complex is simultaneously packaged into the virion. The latter model would require an entirely new molecular mechanism for packaging compared to known viral packaging motors. We characterized a virus with a conditional knockout of core protein VII. Remarkably, virus particles were assembled efficiently in the absence of protein VII. No changes in protein composition were evident with VII−virus particles, including the abundance of core protein V, but changes in the proteolytic processing of some capsid proteins were evident. Virus particles that lack protein VII enter the cell, but incoming virions did not escape efficiently from endosomes. This greatly diminished all subsequent aspects of the infectious cycle. These results reveal that the Ad major core protein VII is not required to condense viral DNA within the capsid, but rather plays an unexpected role during virus maturation and the early stages of infection. These results establish a new paradigm pertaining to the Ad assembly mechanism and reveal a new and important role of protein VII in early stages of infection.

Engineering Plasmid-Free Klebsiella Pneumoniae for Production of 3-Hydroxypropionic Acid.

Although plasmid-dependent microbial breeding is predominant in manufacturing bio-based chemicals, it shows pitfall of genetic instability and thus hinders industrial production. Alternatively, chromosome engineering free from plasmid enables genetic stability and thus represents the new trend of microbial breeding. 3-Hydroxypropionic acid (3-HP) is an economically important platform compound as the versatile precursor of a suite of C3 compound, such as 1, 3-PDO. Klebsiella pneumoniae is regarded as a promising host strain due to both its dha regulon and exceptional glycerol fermentation ability. To produce 3-HP in K. pneumoniae, the IS1 region in chromosome was replaced with the AD DNA cassette containing aldH gene from E.coli through homologous recombination approach. The engineered recombinant converted glycerol into 3-HPA and then 3-HP when 40g/L of initial glycerol was added. The novelties of this study comprise (i) the genetic stability of plasmid-free strains (ii) without using any inducer and antibiotics and thus more applicable than plasmid-based strains.

A clinical protocol to inhibit the HGF/c-Met pathway for malignant mesothelioma with an intrapleural injection of adenoviruses expressing the NK4 gene.

BackgroundThe hepatocyte growth factor (HGF)/c-Met signal pathway is up-regulated in human mesothelioma and suppression of the HGF/c-Met signaling with a competitive inhibitor, NK4 homologous to HGF in the structure, produced anti-tumor effects to mesothelioma in a preclinical study. Mesothelioma is highly resistant to a number of chemotherapeutic agents but distant metastasis to extra-thoracic organs is relatively infrequent until the late stage.Methods/designWe planned to conduct a clinical study of gene therapy with adenoviruses expressing the NK4 gene (Ad-NK4) to control the local tumor growth. The study is designed to inject Ad-NK4 into the intrapleural cavity with a dose escalation manner from 1010 to 1012 virus particles per patient and to examine safety and possible clinical benefits. The clinical investigation is a first-in-human trial to use the NK4 gene and to block the HGF/c-Met pathway with gene medicine. We conducted in vivo animal experiments to examine the safety level as one of the preclinical studies, and showed that Ad DNA administered in the pleural cavity was detected in many parenchymal organs. Biochemical and pathological analyses showed that liver damages were the major adverse effects with little toxicity to other organs. These studies firstly demonstrated biodistribution and transgene expression after an intrapleural injection of Ad vectors in an animal study, which contrasts with an intravenous injection showing relatively rapid clearance of Ad-NK4.DiscussionThe clinical study can also provide information regarding production of NK4 protein and antibody against NK4, and inhibition levels of the HGF/c-Met pathway by detecting dephosphorylation of c-Met in mesothelioma cells. These data will be crucial to judge whether local production of NK4 molecules can be an anti-cancer strategy.Trial registration: UMIN clinical trials registry, Japan. Register ID: UMIN15771

Valganciclovir inhibits human adenovirus replication and pathology in permissive immunosuppressed female and male Syrian hamsters.

Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate) is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5) infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients.

Ganciclovir inhibits human adenovirus replication and pathogenicity in permissive immunosuppressed Syrian hamsters.

Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.

Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative

The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections.

SPOC1-Mediated Antiviral Host Cell Response Is Antagonized Early in Human Adenovirus Type 5 Infection

Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24–48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming viral DNA would increase Ad vector efficacy and safety for the patient.

Antitumor efficacy of a recombinant adenovirus encoding endostatin combined with an E1B55KD-deficient adenovirus in gastric cancer cells

BackgroundGene therapy using a recombinant adenovirus (Ad) encoding secretory human endostatin (Ad-Endo) has been demonstrated to be a promising antiangiogenesis and antitumor strategy of in animal models and clinical trials. The E1B55KD-deficient Ad dl1520 was also found to replicate selectively in and destroy cancer cells. In this study, we aimed to investigate the antitumor effects of antiangiogenic agent Ad-Endo combined with the oncolytic Ad dl1520 on gastric cancer (GC) in vitro and in vivo and determine the mechanisms of these effects.MethodsThe Ad DNA copy number was determined by real-time PCR, and gene expression was assessed by ELISA, Western blotting or immunohistochemistry. The anti-proliferation effect (cytotoxicity) of Ad was assessed using the colorimetry-based MTT cell viability assay. The antitumor effects were evaluated in BALB/c nude mice carrying SGC-7901 GC xenografts. The microvessel density and Ad replication in tumor tissue were evaluated by checking the expression of CD34 and hexon proteins, respectively.Resultsdl1520 replicated selectively in GC cells harboring an abnormal p53 pathway, including p53 mutation and the loss of p14ARF expression, but did not in normal epithelial cells. In cultured GC cells, dl1520 rescued Ad-Endo replication, and dramatically promoted endostatin expression by Ad-Endo in a dose- and time-dependent manner. In turn, the addition of Ad-Endo enhanced the inhibitory effect of dl1520 on the proliferation of GC cells. The transgenic expression of Ad5 E1A and E1B19K simulated the rescue effect of dl1520 supporting Ad-Endo replication in GC cells. In the nude mouse xenograft model, the combined treatment with dl1520 and Ad-Endo significantly inhibited tumor angiogenesis and the growth of GC xenografts through the increased endostatin expression and oncolytic effects.ConclusionsAd-Endo combined with dl1520 has more antitumor efficacy against GC than Ad-Endo or dl1520 alone. These findings indicate that the combination of Ad-mediated antiangiogenic gene therapy and oncolytic Ad therapeutics could be one of promising comprehensive treatment strategies for GC.

Reduced Infectivity of Adenovirus Type 5 Particles and Degradation of Entering Viral Genomes Associated with Incomplete Processing of the Preterminal Protein

To investigate further the contribution of the adenovirus type 5 (Ad5) E1B 55-kDa protein to genome replication, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected with Ad5 or the E1B 55-kDa-null mutant Hr6. Unexpectedly, all cell types were observed to contain a significantly higher concentration of entering Hr6 than of Ad5 DNA, as did an infectious unit of Hr6. However, the great majority of the Hr6 genomes were degraded soon after entry. As this unusual phenotype cannot be ascribed to the Hr6 E1B frameshift mutation (J. S. Chahal and S. J. Flint, J. Virol. 86:3064-3072, 2012), the sequences of the Ad5 and Hr6 genomes were compared by using high-throughput sequencing. Seven previously unrecognized mutations were identified in the Hr6 genome, two of which result in substitutions in virion proteins, G315V in the preterminal protein (preTP) and A406V in fiber protein IV. Previous observations and the visualization by immunofluorescence of greater numbers of viral genomes entering the cytosol of Hr6-infected cells than of Ad5-infected cells indicated that the fiber mutation could not be responsible for the low-infectivity phenotype of Hr6. However, comparison of the forms of terminal protein present in purified virus particles indicated that the production of mature terminal protein from a processing intermediate is impaired in Hr6 particles. We therefore propose that complete processing of preTP within virus particles is necessary for the ability of viral genomes to become localized at appropriate sites and persist in infected cells.

Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.

Targeted expression of herpes simplex virus thymidine kinase in adenovirus‐infected cells reduces virus titers upon treatment with ganciclovir in vitro

Adenoviruses are a frequent cause of life-threatening infections in immunocompromised patients. Available therapeutics still cannot completely prevent fatal outcomes. By contrast, herpes viruses are well treatable with prodrugs such as ganciclovir (GCV), which are selectively activated in virus-infected cells by virus-encoded thymidine kinases. This effective group of prodrugs is not applicable to adenoviruses and other DNA viruses because they lack those kinases. To render adenoviruses amenable to GCV treatment, we generated an adenoviral vector-based delivery system for targeted expression of herpes simplex virus thymidine kinase (HSV-TK) in wild-type adenovirus 5 (wt Ad5)-infected cells. HSV-TK expression was largely restricted to wt virus-infected cells by transcription of the gene from the Ad5 E4 promoter. Its activity is dependent on the adenoviral E1A gene product which is not produced by the vector but is only provided in cells infected with wt adenovirus. The anti-adenoviral effect of HSV-TK expression and concomitant treatment with GCV was assessed in vitro in four different cell lines or primary cells. E4 promoter-mediated HSV-TK background expression was sufficiently low to prevent cytotoxicity in the presence of low-levels GCV in cells not infected with wt Ad5. However, expression was several-fold increased in wt Ad5-infected cells and treatment with low levels of GCV efficiently inhibited wt Ad5 DNA replication. Genome copy numbers and output of infectious particles were reduced by up to > 99.99% and cell viability was greatly increased. We extended the concept of enzyme/prodrug therapy to adenovirus infections by selectively sensitizing adenovirus-infected cells to treatment with GCV.

Linearized oncolytic adenoviral plasmid DNA delivered by bioreducible polymers

As an effort to overcome limits of adenovirus (Ad) as a systemic delivery vector for cancer therapy, we developed a novel system using oncolytic Ad plasmid DNA with two bioreducible polymers: arginine-grafted bioreducible poly(disulfide amine)polymer (ABP) and PEG5k-conjugated ABP (ABP5k) in expectation of oncolytic effect caused by progeny viral production followed by replication. The linearized Ad DNAs for active viral replication polyplexed with each polymer were able to replicate only in human cancer cells and produce progeny viruses. The non-immunogenic polymers delivering the DNAs markedly elicited to evade the innate and adaptive immune response. The biodistribution ratio of the polyplexes administered systemically was approximately 99% decreased in liver when compared with naked Ad. Moreover, tumor-to-liver ratio of the Ad DNA delivered by ABP or ABP5k was significantly elevated at 229- or 419-fold greater than that of naked Ad, respectively. The ABP5k improved the chance of the DNA to localize within tumor versus liver with 1.8-fold increased ratio. In conclusion, the innovative and simple system for delivering oncolytic Ad plasmid DNA with the bioreducible polymers, skipping time-consuming steps such as generation and characterization of oncolytic Ad vectors, can be utilized as an alternative approach for cancer therapy.