GM101 in Combination with Histone Deacetylase Inhibitor Enhances Anti-Tumor Effects in Desmoplastic Microenvironment.

Oncolytic adenoviruses (oAds) have been evaluated in numerous clinical trials due to their promising attributes as cancer therapeutics. However, the therapeutic efficacy of oAds was limited due to variable coxsackie and adenovirus receptor (CAR) expression levels and the dense extracellular matrix (ECM) of heterogenic clinical tumors. To overcome these limitations, our present report investigated the therapeutic efficacy of combining GM101, an oAd with excellent tumor ECM degrading properties, and histone deacetylase inhibitor (HDACi). Four different HDACi (suberohydroxamic acid (SBHA), MS-275, trichostatin A (TSA), and valproic acid) candidates in combination with replication-incompetent and GFP-expressing Ad (dAd/GFP) revealed that SBHA and MS-275 exerted more potent enhancement in Ad transduction efficacy than TSA or valproic acid. Further characterization revealed that SBHA and MS-275 effectively upregulated CAR expression in cancer cells, improved the binding of Ad with cancer cell membranes, and led to dynamin 2- and clathrin-mediated endocytosis of Ad. The combination of GM101 with HDACi induced superior cancer cell killing effects compared to any of the monotherapies, without any additional cytotoxicity in normal cell lines. Further, GM101+SBHA and GM101+MS-275 induced more potent antitumor efficacy than any monotherapy in U343 xenograft tumor model. Potent antitumor efficacy was achieved via the combination of GM101 with HDACi, inducing necrotic and apoptotic cancer cell death, inhibiting cancer cell proliferation, degrading ECM in tumor tissue, and thus exerting the highest level of virus dispersion and accumulation. Collectively, these data demonstrate that the combination of GM101 and HDACi can enhance intratumoral dispersion and accumulation of oAd through multifaced mechanisms, making it a promising strategy to address the challenges toward successful clinical development of oAd.

Optimizing Active Tumor Targeting Biocompatible Polymers for Efficient Systemic Delivery of Adenovirus.

Adenovirus (Ad) has risen to be a promising alternative to conventional cancer therapy. However, systemic delivery of Ad, which is necessary for the treatment of metastatic cancer, remains a major challenge within the field, owing to poor tumor tropism and nonspecific hepatic tropism of the virus. To address this limitation of Ad, we have synthesized two variants of folic acid (FA)-conjugated methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-L-glutamate (P5N2LG-FA and P5N5LG-FA) using 5 kDa poly(ethylene glycol) (PEG) with a different level of protonation (N2 < N5 in terms of charge), along with a P5N5LG control polymer without FA. Our findings demonstrate that P5N5LG, P5N2LG-FA, and P5N5LG-FA exert a lower level of cytotoxicity compared to 25 kDa polyethyleneimine. Furthermore, green fluorescent protein (GFP)-expressing Ad complexed with P5N2LG-FA and P5N5LG-FA (Ad/P5N2LG-FA and Ad/P5N5LG-FA, respectively) exerted superior transduction efficiency compared to naked Ad or Ad complexed with P5N5LG (Ad/P5N5LG) in folate receptor (FR)-overexpressing cancer cells (KB and MCF7). All three nanocomplexes (Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA) internalized into cancer cells through coxsackie adenovirus receptor-independent endocytic mechanism and the cell uptake was more efficient than naked Ad. Importantly, the cell uptake of the two FA functionalized nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) was dependent on the complementary interaction of FA-FR. Systemically administered Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA showed exponentially higher retainment of the virus in blood circulation up to 24 h post-administration compared with naked Ad. Both tumor-targeted nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) showed significantly higher intratumoral accumulation than naked Ad or Ad/P5N5LG via systemic administration. Both tumor-targeted nanocomplexes accumulated at a lower level in liver tissues compared to naked Ad. Notably, the nonspecific accumulation of Ad/P5N2LG-FA was significantly lower than Ad/P5N5LG-FA in several normal organs, while exhibiting a significantly higher intratumoral accumulation level, showing that careful optimization of polyplex surface charge is critical to successful tumor-targeted systemic delivery of Ad nanocomplexes.

Synergistic antitumor effect mediated by a paclitaxel-conjugated polymeric micelle-coated oncolytic adenovirus.

Combination treatment consisting of oncolytic adenovirus (Ad) and paclitaxel (PTX) is a promising strategy to achieve synergistic antitumor effect. However, a co-administration approach is subject to inherent limitations due to the poor solubility of PTX and chemoresistance of tumor cells. In order to overcome these limitations, an oncolytic Ad expressing a p53 variant (oAd-vp53) that is resistant to p53 inactivation in the tumor microenvironment was complexed with PEGylated and PTX-conjugated polymeric micelle (APP). This approach generated an oAd-vp53/APP complex (176.4 nm in diameter) that could concurrently deliver both oncolytic Ad and the nanoparticulate drug APP to tumors. APP-complexed replication-incompetent Ad (dAd/APP) exhibited 12-fold higher transduction efficiency than naked dAd in coxsackie adenovirus receptor (CAR)-negative cancer cells. This increased efficiency was attributed to more efficient cellular internalization mediated by charge interactions between APP and anionic cell membranes. Furthermore, oAd-vp53/APP elicited synergistically higher cancer cell killing than naked oAd-vp53, APP, or oAd-vp53 in combination with PTX (oAd-vp53 + PTX); this synergistic effect was shown to be due to superior induction of apoptosis and viral replication. Importantly, oAd-vp53/APP induced more potent and synergistic antitumor effect through both local and systemic administration by enhancing replication of oncolytic Ad and induction of apoptosis in tumor tissue. Further, the APP coating on the surface of Ad markedly attenuated the host immune response against Ad and decreased hepatic sequestration, resulting in minimal hepatotoxicity and a good safety profile. These attributes enabled oAd-vp53/APP to elicit potent antitumor effect over multiple treatment cycles. Altogether, we demonstrate that concurrent delivery of oncolytic Ad and APP as a single nanocomplex is a promising strategy for achieving synergistic antitumor effect.

Human relaxin gene expression delivered by bioreducible dendrimer polymer for post-infarct cardiac remodeling in rats.

In consensus, myocardial infarction (MI) is defined as irreversible cell death secondary to prolonged ischemia in heart. The aim of our study was to evaluate the therapeutic potential of anti-fibrotic human Relaxin-expressing plasmid DNA with hypoxia response element (HRE) 12 copies (HR1) delivered by a dendrimer type PAM-ABP polymer G0 (HR1/G0) after MI on functional, hemodynamic, geometric, and cardiac extracellular matrix (ECM) remodeling in rats. HR1/G0 demonstrated significantly improved LV systolic function, hemodynamic parameters, and geometry on 1 wk and 4 wks after MI in rats, compared with I/R group. The resolution of regional wall motional abnormalities and the increased blood flow of infarct-related coronary artery supported functional improvements of HR1/G0. Furthermore, HR1/G0 polyplex showed favorable post-infarct cardiac ECM remodeling reflected on the favorable cardiac ECM compositions. Overall, this is the first study, which presented an advanced platform for the gene therapy that reverses adverse cardiac remodeling after MI with a HR1 gene delivered by a bioreducible dendrimer polymer in the cardiac ECM.

Hepatoma targeting peptide conjugated bio-reducible polymer complexed with oncolytic adenovirus for cancer gene therapy.

Despite adenovirus (Ad) vector's numerous advantages for cancer gene therapy, such as high ability of endosomal escape, efficient nuclear entry mechanism, and high transduction, and therapeutic efficacy, tumor specific targeting and antiviral immune response still remain as a critical challenge in clinical setting. To overcome these obstacles and achieve cancer-specific targeting, we constructed tumor targeting bioreducible polymer, an arginine grafted bio-reducible polymer (ABP)-PEG-HCBP1, by conjugating PEGylated ABP with HCBP1 peptides which has high affinity and selectivity towards hepatoma. The ABP-PEG-HCBP1-conjugated replication incompetent GFP-expressing ad, (Ad/GFP)-ABP-PEG-HCBP1, showed a hepatoma cancer specific uptake and transduction compared to either naked Ad/GFP or Ad/GFP-ABP. Competition assays demonstrated that Ad/GFP-ABP-PEG-HCBP1-mediated transduction was specifically inhibited by HCBP1 peptide rather than coxsackie and adenovirus receptor specific antibody. In addition, ABP-PEG-HCBP1 can protect biological activity of Ad against serum, and considerably reduced both innate and adaptive immune response against Ad. shMet-expressing oncolytic Ad (oAd; RdB/shMet) complexed with ABP-PEG-HCBP1 delivered oAd efficiently into hepatoma cancer cells. The oAd/ABP-PEG-HCBP1 demonstrated enhanced cancer cell killing efficacy in comparison to oAd/ABP complex. Furthermore, Huh7 and HT1080 cancer cells treated with oAd/shMet-ABP-PEG-HCBP1 complex had significantly decreased Met and VEGF expression in hepatoma cancer, but not in non-hepatoma cancer. In sum, these results suggest that HCBP1-conjugated bioreducible polymer could be used to deliver oncolytic Ad safely and efficiently to treat hepatoma.

Potent antitumor effect of neurotensin receptor-targeted oncolytic adenovirus co-expressing decorin and Wnt antagonist in an orthotopic pancreatic tumor model.

Pancreatic cancer is highly aggressive, malignant, and notoriously difficult to cure using conventional cancer therapies. These conventional therapies have significant limitations due to excessive extracellular matrix (ECM) of pancreatic cancer and poor cancer specificity. The excess ECM prevents infiltration of drugs into the inner layer of the solid tumor. Therefore, novel treatment modalities that can specifically target the tumor and degrade the ECM are required for effective therapy. In the present study, we used ECM-degrading and Wnt signal-disrupting oncolytic adenovirus (oAd/DCN/LRP) to achieve a desirable therapeutic outcome against pancreatic cancer. In addition, to overcome the limitations in systemic delivery of oncolytic Ad (oAd) and to specifically target pancreatic cancer, neurotensin peptide (NT)-conjugated polyethylene glycol (PEG) was chemically crosslinked to the surface of Ad, generating a systemically injectable hybrid system, oAd/DCN/LRP-PEG-NT. We tested the targeting and therapeutic efficacy of oAd/DCN/LRP-PEG-NT toward neurotensin receptor 1 (NTR)-overexpressing pancreatic cancer cells, both in vitro and in vivo. The oAd/DCN/LRP-PEG-NT elicited increased NTR-selective cancer cell killing and transduction efficiency when compared with a cognate control lacking NT (oAd/DCN/LRP-PEG). Furthermore, systemic administration of oAd/DCN/LRP-PEG-NT significantly decreased induction of innate and adaptive immune responses against Ad, and blood retention time was markedly prolonged by PEGylation. Moreover, NTR-targeting oAd elicited greater in vivo tumor growth suppression when compared with naked oAd and 9.5 × 10(6)-fold increased tumor-to-liver ratio. This significantly enhanced antitumor effect of oAd/DCN/LRP-PEG-NT was mediated by active viral replication and viral spreading, which was facilitated by ECM degradation and inhibition of Wnt signaling-related factors (Wnt, ß-catenin, and/or vimentin) in the tumor tissues. Taken together, these results demonstrate that oAd/DCN/LRP-PEG-NT has strong therapeutic potential for systemic treatment of NTR-overexpressing pancreatic cancer due to its NTR-targeting ability, enhanced therapeutic efficacy, and safety.

Polymeric oncolytic adenovirus for cancer gene therapy.

Oncolytic adenovirus (Ad) vectors present a promising modality to treat cancer. Many clinical trials have been done with either naked oncolytic Ad or combination with chemotherapies. However, the systemic injection of oncolytic Ad in clinical applications is restricted due to significant liver toxicity and immunogenicity. To overcome these issues, Ad has been engineered physically or chemically with numerous polymers for shielding the Ad surface, accomplishing extended blood circulation time and reduced immunogenicity as well as hepatotoxicity. In this review, we describe and classify the characteristics of polymer modified oncolytic Ad following each strategy for cancer treatment. Furthermore, this review concludes with the highlights of various polymer-coated Ads and their prospects, and directions for future research.

Enhanced anti-tumor efficacy and safety profile of tumor microenvironment-responsive oncolytic adenovirus nanocomplex by systemic administration.

Oncolytic adenovirus (Ad) holds great promise as a potential gene therapy for cancer. However, intravenously administered Ad may encounter difficulties due to unfavorable host responses, non-specific interactions, and the heterogeneity of the tumor cell population. As an approach to combine the advantages of oncolytic Ad and synthetic polymers and to address the associated difficulties, Ad was physically complexed with a pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine) (mPEG-b-pHis). The in vitro transduction efficiency at an acidic extracellular pH was remarkably enhanced in cancer cells when treated with the Ad expressing green fluorescent protein (GFP) coated with mPEG-b-pHis (c-dE1/GFP) as compared to that of naked Ad (n-dE1/GFP). Time-lapse total internal reflection fluorescence microscopic imaging revealed a significantly enhanced cellular uptake rate of c-dE1/GFP at acidic tumor pH when compared with that at neutral pH or naked cognate Ad (n-dE1/GFP). In addition, c-dE1/GFP remained relatively stable in human serum-containing media, and considerably reduced both the innate and adaptive immune response against Ad. Moreover, the therapeutic efficacy and survival benefit of mPEG-b-pHis-complexed oncolytic Ad (c-H5mT/Luc) by systemic treatment was significantly enhanced compared to that with naked oncolytic Ad (n-H5mT/Luc) in both coxsackie and adenovirus receptor-positive and -negative tumors. Whole-body bioluminescence imaging showed 7.3-fold higher luciferase expression at the tumor site and 23.0-fold less luciferase expression in liver tissue for c-H5mT/Luc relative to that for naked oncolytic Ad (n-H5mT/Luc). Considering the heterogeneity of tumor tissue, these results are important for guiding the development of more potent and specific treatment of devastating metastatic cancers using this viral system. STATEMENT OF SIGNIFICANCE: Although adenoviral systems have shown considerable promise and undergone extensive evaluation attempts to specifically target Ad vectors to cancer cells have met limited success. This shortcoming is due to the strong immune response stimulated by Ad and the hepatotoxicity of the viral particles. To overcome restricted vector issues, we generated Ad/mPEG-b-pHis for tumor microenvironment-targeting hybrid vector systems, an oncolytic Ad coated with a pH-responsive polymer, mPEG-b-pHis. The Ad/mPEG-b-pHis exhibited pH-dependent transduction efficiency and cancer-cell killing effects. Moreover, systemic administration of oncolytic Ad/mPEG-b-pHis led to marked suppression of tumor growth and tumor-specific viral replication. Ad successfully avoided the innate and adaptive immune responses and liver accumulation with the help of mPEG-b-pHis on its surface.

Using a magnetic field to redirect an oncolytic adenovirus complexed with iron oxide augments gene therapy efficacy.

Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited by low coxsackievirus and adenovirus receptor (CAR) expression in tumors and non-specifically targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and directing their migration with an external magnetic field (MGF). We electrostatically complexed GFP-expressing, replication-incompetent Ad (dAd) with PEGylated and cross-linked iron oxide nanoparticles (PCION), generating dAd-PCION complexes. The dAd-PCION showed increased transduction efficiency, independent of CAR expression, in the absence or presence of an MGF. Cancer cell killing and intracellular oncolytic Ad (HmT)-PCION replication significantly increased with MGF exposure. Site-directed, magnetically-targeted delivery of the HmT-PCION elicited significantly greater therapeutic efficacy versus treatment with naked HmT or HmT-PCION without MGF in CAR-negative MCF7 tumors. Immunohistochemical tumor analysis showed increased oncolytic Ad replication in tumors following infection by HmT-PCION using an MGF. Whole-body bioluminescence imaging of tumor-bearing mice showed a 450-fold increased tumor-to-liver ratio for HmT-PCION with, versus without, MGF. These results demonstrate the feasibility and potential of external MGF-responsive PCION-coated oncolytic Ads as smart hybrid vectors for cancer gene therapy.

Tuning Surface Charge and PEGylation of Biocompatible Polymers for Efficient Delivery of Nucleic Acid or Adenoviral Vector.

As an effective and safe strategy to overcome the limits of therapeutic nucleic acid or adenovirus (Ad) vectors for in vivo application, various technologies to modify the surface of vectors with nonimmunogenic/biocompatible polymers have been emerging in the field of gene therapy. However, the transfection efficacy of the polymer to transfer genetic materials is still relatively weak. To develop more advanced and effective polymers to deliver not only Ad vectors, but also nucleic acids, 6 biocompatible polymers were newly designed and synthesized to different sizes (2k, 3.4k, or 5k) of poly(ethylene) glycol (PEG) and different numbers of amine groups (2 or 5) based on methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-l-glutamate (PNLG). We characterized size distribution and surface charge of 6 PNLGs after complexation with either nucleic acid or Ad. Among all 6 PNLGs, the 5 amine group PNLG showed the strongest efficacy in delivering nucleic acid as well as Ad vectors. Interestingly, cellular uptake results showed higher uptake ability in Ad complexed with 2 amine group PNLG than Ad/5 amine group PNLG, suggesting that the size of Ad/PNLGs is more essential than the surface charge for cellular uptake in polymers with charges greater than 30 mV. Moreover, the endosome escape ability of Ad/PNLGs increased depending on the number of amine groups, but decreased by PEG size. Cancer cell killing efficacy and immune response studies of oncolytic Ad/PNLGs showed 5 amine group PNLG to be a more effective and safe carrier for delivering Ad. Overall, these studies provide new insights into the functional mechanism of polymer-based approaches to either nucleic acid or Ad/nanocomplex. Furthermore, the identified ideal biocompatible PNLG polymer formulation (5 amine/2k PEG for nucleic acid, 5 amine/5k PEG for Ad) demonstrated high transduction efficiency as well as therapeutic value (efficacy and safety) and thus has strong potential for in vivo therapeutic use in the future.

Oncolytic Adenovirus Coated with Multidegradable Bioreducible Core-Cross-Linked Polyethylenimine for Cancer Gene Therapy.

Recently, adenovirus (Ad) has been utilized as a viral vector for efficient gene delivery. However, substantial immunogenicity and toxicity have obstructed oncolytic Ad's transition into clinical studies. The goal of this study is to generate an adenoviral vector complexed with multidegradable bioreducible core-cross-linked polyethylenimine (rPEI) polymer that has low immunogenicity and toxicity while having higher transduction efficacy and stability. We have synthesized different molecular weight rPEIs and complexed with Ad at varying molar ratios to optimize delivery of the Ad/polymer complex. The size and surface charge of Ad/rPEIs were characterized. Of note, Ad/rPEIs showed significantly enhanced transduction efficiency compared to either naked Ad or Ad/25 kDa PEI in both coxsackievirus and adenovirus receptor (CAR) positive and negative cancer cells. The cellular uptake result demonstrated that the relatively small size of Ad/16 kDa rPEIs (below 200 nm) was more critical to the complex's internalization than its surface charge. Cancer cell killing effect and viral production were significantly increased when oncolytic Ad (RdB/shMet, or oAd) was complexed with 16 kDa rPEI in comparison to naked oAd-, oAd/25 kDa PEI-, or oAd/32 kDa rPEI-treated cells. This increased anticancer cytotoxicity was more readily apparent in CAR-negative MCF7 cells, implying that it can be used to treat a broad range of cancer cells. Furthermore, A549 and HT1080 cancer cells treated with oAd/16 kDa rPEI had significantly decreased Met and VEGF expression compared to either naked oAd or oAd/25 kDa PEI. Overall, these results demonstrate that shMet expressing oncolytic Ad complexed with multidegradable bioreducible core-cross-linked PEI could be used as efficient and safe cancer gene therapy.

Targeting delivery of tocopherol and doxorubicin grafted-chitosan polymeric micelles for cancer therapy: In vitro and in vivo evaluation.

In this study, we report the development of a novel, redox-sensitive chitosan-based targeted drug delivery system, containing two drugs. We determined whether the synthesized polymeric micelles (HPTOC-DOX) were suitable as a drug carrier. The formation of HPTOC-DOX micelles was confirmed by (1)H NMR. HPTOC-DOX formed micelles of approximately 151.9-311.2nm in size in aqueous solution. Analysis of the drug release profile of HPTOC-DOX in different pH conditions (pH 5.2, 6.2, and 7.4) indicated that DOX was released from HPTOC-DOX micelles at acidic pH (5.2 or 6.2), while almost no DOX was released at pH 7.4. In vitro cell cytotoxicity and hemolysis assays indicated that HPTOC-DOX micelles safely deliver anti-cancer drugs and decrease the cytotoxicity of DOX. In vitro anti-cancer activity assays, confocal laser scanning microscopy analysis of SK-BR-3 cells, and in vivo anti-tumor activity in SK-BR-3-derived tumor-bearing mice were used to evaluate synergistic drug effects and the effect of the targeting peptide (anti-human epidermal growth factor receptor 2 [HER2] target peptide, epitope form; LTVSPWY) on receptor-mediated endocytosis.

Direct observation of interactions of silk-elastinlike protein polymer with adenoviruses and elastase.

Silk-elastinlike protein polymer (SELP) hydrogels have been investigated for sustained local delivery of adenoviral gene carriers to solid tumors. These polymers degrade in the presence of proteases such as elastase. A detailed understanding of the interaction of SELPs with viruses and their degradation in the presence of elastase can provide useful information about mechanisms of sustained gene delivery from these systems. In this work, we investigated the interactions of SELPs with adenoviruses (Ads) and elastase using atomic force microscopy. We observed that viral particles interacted strongly with SELP networks formed by cross-linking of nanofibers. The presence of viruses contributed to enhanced network formation. Incubation of Ad with SELPs in the liquid state induced close packing of the viral colony. Morphological changes of SELP networks cleaved by enzymatic interaction with elastase were investigated. SELP-415K fiber networks were more responsive to temperature changes and were slowly degraded by elastases compared to SELP-47K, a SELP analogue with shorter elastin units in the monomer repeat. These studies provide insight into the influence of SELP structure on degradation and potential mechanisms of increased viral stability.

pH-sensitive oncolytic adenovirus hybrid targeting acidic tumor microenvironment and angiogenesis.

Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine-co-l-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, an oncolytic Ad expressing VEGF promoter-targeting transcriptional repressor (KOX) was used to form complexes. At pH6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH-sensitive polymer-coated Ad complex significantly increases net positive charge upon exposure to hypoxic tumor microenvironment, allowing passive targeting to the tumor tissue. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX.

Dual tumor targeting with pH-sensitive and bioreducible polymer-complexed oncolytic adenovirus.

Oncolytic adenoviruses (Ads) have shown great promise in cancer gene therapy but their efficacy has been compromised by potent immunological, biochemical, and specific tumor-targeting limitations. To take full advantage of the innate cancer-specific killing potency of oncolytic Ads but also exploit the subtleties of the tumor microenvironment, we have generated a pH-sensitive and bio-reducible polymer (PPCBA)-coated oncolytic Ad. Ad-PPCBA complexes showed higher cellular uptake at pH 6.0 than pH 7.4 in both high and low coxsackie and adenovirus receptor-(CAR)-expressing cells, thereby demonstrating Ad-PPCBA's ability to target the low pH hypoxic tumor microenvironment and overcome CAR dependence for target cell uptake. Endocytic mechanism studies indicated that Ad-PPCBA internalization is mediated by macropinocytosis instead of the CAR-dependent endocytic pathway that internalizes naked Ad. VEGF-specific shRNA-expressing oncolytic Ad complexed with PPCBA (RdB/shVEGF-PPCBA) elicited much more potent suppression of U87 human brain cancer cell VEGF gene expression in vitro, and human breast cancer MCF7 cell/Matrigel plug vascularization in a mouse model, when cancer cells had been previously infected at pH 6.0 versus pH 7.4. Moreover, intratumorally and intravenously injected RdB/shVEGF-PPCBA nanocomplexes elicited significantly higher therapeutic efficacy than naked virus in U87-tumor mouse xenograft models, reducing IL-6, ALT, and AST serum levels. These data demonstrated PPCBA's biocompatibility and capability to shield the Ad surface to prevent innate immune response against Ad after both intratumoral and systemic administration. Taken together, these results demonstrate that smart, tumor-specific, oncolytic Ad-PPCBA complexes can be exploited to treat both primary and metastatic tumors.

Safety profiles and antitumor efficacy of oncolytic adenovirus coated with bioreducible polymer in the treatment of a CAR negative tumor model.

Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy.

Sustained local delivery of oncolytic short hairpin RNA adenoviruses for treatment of head and neck cancer.

BACKGROUND: Oncolytic adenovirus (Ad)-mediated gene therapy is a promising approach for suppression of primary tumors. Therapeutic efficacy of Ad-mediated gene therapy has been limited by immunogenicity, rapid dissemination of viral progenies into systemic circulation and short duration of biological activity. Polymeric sustained local delivery can overcome many of these challenges to produce a viable therapy with improved outcomes. METHODS: Silk-elastinlike protein polymer (SELP) hydrogels were used for matrix-mediated delivery of oncolytic Ad, containing short hairpin RNA (shRNA) targeted to C-Met (sh-C-Met), to solid tumors in a nude mouse model of human head and neck cancer. The biological activity of Ad released from SELP hydrogels was examined as a function of time to investigate protective effects on viral activity. Antitumor efficacy and viral distribution were investigated for 3 weeks in tumor-bearing mice. RESULTS: The encapsulation of Ad with SELP hydrogels sustained biological activity longer than Ad alone. Ad in SELP matrix showed 1.5-fold greater antitumor efficacy compared to that of naked Ad in human xenograft tumor models. Histological analysis demonstrated that treatment with Ad in a SELP matrix resulted in apoptosis in a wider area of tumor tissue and higher density of Ad infection compared to Ad administered alone. CONCLUSIONS: Matrix-mediated delivery of Ad-containing shRNA with SELP hydrogels enhances therapeutic efficacy by tumor-selective infection, spatiotemporal control and preservation of biologic activity.

Utilizing adenovirus vectors for gene delivery in cancer.

INTRODUCTION: Adenovirus (Ad) is a promising candidate vector for cancer gene therapy because of its unique characteristics, which include efficient infection, high loading capacity and lack of insertional mutagenesis. However, systemic administration of Ad is hampered by the host's immune response, hepatocytoxicity, short half-life of the vector and low accumulation at the target site. For these reasons, clinical applications of Ad are currently restricted. AREAS COVERED: In this review, we focus on recent developments in Ad nanocomplex systems that improve the transduction and targeting efficacy of Ad vectors in cancer gene therapy. We discuss the development of different Ad delivery systems, including surface modification of Ad, smart Ad/nanohybrid systems and hydrogels for sustained release of Ad. EXPERT OPINION: The fusion of bioengineering and biopharmaceutical technologies can provide solutions to the obstacles encountered during systemic delivery of Ads. The in vivo transgene expression efficiency of Ad nanocomplex systems is typically high, and animal tumor models demonstrate that systemic administration of these Ad complexes can arrest tumor growth. However, further optimization of these smart Ad nanocomplex systems is needed to increase their effectiveness and safety for clinical application in cancer gene therapy.

Therapeutic targeting of chitosan-PEG-folate-complexed oncolytic adenovirus for active and systemic cancer gene therapy.

Adenovirus (Ad)-based cancer therapies have shown much promise. However, until now, Ad has only been delivered directly to primary tumors because the therapeutic efficacy of systemic delivery is limited by the immune response of the host, short blood circulation times, and non-specific liver uptake of Ad. In order to circumvent the issues regarding systemic delivery and to increase the safety and efficacy of Ad therapies, the surface of oncolytic Ad was coated with cationic polymer chitosan via ionic crosslinking (Ad/chitosan), after which polyethylene glycol (PEG) and/or folic acid (FA) was chemically conjugated onto the surface of Ad/chitosan, generating Ad/chitosan-FA, Ad/chitosan-PEG, and Ad/chitosan-PEG-FA nanocomplex. The FA-coordinated Ad nanocomplexes (Ad/chitosan-FA & Ad/chitosan-PEG-FA) elicited folate receptor (FR)-selective cancer cell killing efficacy. In vivo administration of Ad/chitosan-PEG or Ad/chitosan-PEG-FA into mice demonstrated that PEGylation greatly increased blood circulation time, resulting in 9.0-fold and 48.9-fold increases at 24h after injection compared with naked Ad, respectively. In addition, generation of Ad-specific neutralizing antibodies in mice treated with Ad/chitosan-PEG-FA was markedly decreased by 75.3% compared with naked Ad. The quantitative polymerase chain reaction assay results showed a 285.0-fold increase in tumor tissues and a 378-fold reduction of Ad/chitosan-PEG-FA in liver tissues compared with naked Ad. Bioluminescence imaging study further supported the enhanced tumor-to-liver ratio of Ad/chitosan-PEG-FA. Consequently, systemic delivery of Ad/chitosan-PEG-FA significantly inhibited the growth of FR-positive tumor, decreasing 52.8% compared to the naked Ad-treated group. Importantly, PEGylated oncolytic Ad nanocomplexes showed no elevation of both alanine transaminase and aspartate transaminase levels, demonstrating that systemically delivered Ad-related hepatic damage can be completely eliminated with PEG conjugation. In sum, these results demonstrate that conjugation of chitosan-PEG-FA to oncolytic Ad significantly improves antitumor efficacy and safety profiles, suggesting that Ad/chitosan-PEG-FA has potential as a therapeutic agent to target FR-positive cancer via systemic administration.

Enhanced detection of virus particles by nanoisland-based localized surface plasmon resonance.

In this paper, we investigate localized surface plasmon resonance (SPR) detection based on nanoislands. Theoretical calculation performed with rigorous coupled-wave analysis and analytical transfer matrix method using effective medium theory suggests improvement on nanoislands in the limit of detection and sensitivity over conventional thin film-based SPR detection. Experimental results obtained with non-specific detection of ambient adenovirus confirm the improvement by more than one order of magnitude increase in the limit of detection. The enhancement achieved with nanoislands was explored in connection with efficient overlap between target and near-field distribution produced by nanoislands.