Efficacy Of Adenovirus-mediated Gene Therapy Research Articles

High Plasma Lipid Levels Reduce Efficacy of Adenovirus-Mediated Gene Therapy

Adenoviruses are very efficient vectors for delivering therapeutic genes in preclinical and clinical trials. However, randomized controlled human trials have often been lacking clear clinically relevant results. We hypothesized that high lipid levels and specific lipoproteins could significantly decrease adenoviral transduction efficiency in vivo. Here we demonstrate that mice on a high fat diet have lower transgene expression compared to mice on a regular chow. In addition, on a high fat diet, ApoE−/− mice have much higher plasma transgene levels compared to LDLR-deficient mice. We also found that specific lipoprotein receptors play an important role in adenoviral transduction. These findings suggest that high plasma lipid levels, especially apoE-containing lipoproteins, reduce efficacy of adenoviral transduction in mice, which implies that high cholesterol levels in humans could be protective against viral infections and also lead to insufficient transgene expression in clinical trials using adenoviral vectors.

Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population.

The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment.

176. Novel Molecules That Enhance Adenovirus Transduction of the Airway Epithelium: Reinvigorating Adenovirus-Mediated Gene Therapy

Adenovirus remains the most common vector system used in gene therapy clinical trials worldwide and several adenoviral vectors show promise in phase III clinical trials. One major limitation to the efficacy of adenovirus-mediated gene therapy is low transduction due to the limited primary receptor availability in most tissues. Interventions able to boost adenovirus transduction would have significant implications for both improving transduction and lowering the viral dose below the immunogenic threshold. Most human and several key animal adenoviruses share a common receptor with group B coxsackieviruses named the coxsackievirus and adenovirus receptor (CAR). CAR has several alternatively spliced isoforms. We have recently discovered that the eight-exon isoform (CAREx8) can specifically localize to the apical membrane of polarized primary human airway epithelia where it can mediate apical adenovirus infection. Moreover, we have discovered that CAREx8 is both positively and negatively regulated by the cellular scaffolding protein MAGI-1. We hypothesized that cell permeable decoy peptides targeting the interaction between MAGI-1 and CAREx8 would increase CAREx8 protein levels and increase AdV transduction. Tat-conjugated peptide entry into polarized epithelia was confirmed by mass spectrometry and fluorescence microscopy. Relative to control, peptides increased the levels of CAREx8 at the apical surface of polarized epithelia and significantly increased AdV entry and transduction, as measured by apical surface biotinylation, qPCR, and AdV5-β-Gal transduction. Moreover, AdV5 transduction was increased by 300-500% after intranasal peptide administration in mice demonstrating in vivo activity. To determine the mechanism of action, we followed the synthesis and co-localization of CAREx8 with key molecules in the cellular ER-Golgi-vesicular trafficking pathways. We found that CAREx8 was in compartments distinct from MAGI-1 and spread throughout the apical trafficking pathway and at the apical surface of the epithelium. Removal of the peptides reversed both the amount and localization of CAREx8 and returned the susceptibility of the epithelium to baseline. Taken together, these data indicate that decoy peptides able to block the MAGI-1-CAREx8 interaction transiently increase adenovirus-mediated gene transfer and offer the potential to increase the efficacy of adenovirus-mediated gene therapy.

Readministration of helper-dependent adenoviral vectors to mouse airway mediated via transient immunosuppression

The efficacy of adenovirus-mediated gene therapy is attenuated by the host immune responses to both vector and transgene products. Even for helper-dependent adenoviral (HD-Ad) vectors, which have all viral-coding sequences deleted, the viral capsid proteins still cause immune reactions. In order to improve the efficiency in transgene expression during HD-Ad vector readministration, we administered cyclophosphamide to transiently modulate the mouse immune system. We delivered a high dose (5 × 10(10) vector particles (vp) per mouse) of empty HD-Ad to the mouse airway to induce an initial immune response. After 4 weeks, the mice were readministered with an HD-Ad vector containing either the reporter gene, LacZ, or the gene for the human cystic fibrosis transmembrane conductance regulator (CFTR) (1.5 × 10(10) vp per mouse). We found that the expression of both transgenes was greatly improved by the administration of cyclophosphamide when compared with the expression in mice without the immunosuppressing drug. We also found that the high dose of the empty HD-Ad vector administered intranasally does not induce an acute systemic immune response, but it does elicit an acute local response of proinflammatory cytokine production. Antibodies against Ad vector, including the neutralizing antibodies, were greatly reduced by the presence of cyclophosphamide in vector readministratiton. Moreover, cyclophosphamide reduced the infiltration of inflammatory cells, including total leukocytes, lymphocytes, CD4+ and CD8+T cells. These results indicate that transient administration of immunosuppressive agent can be used to extend transgene expression as well as attenuating immunogenicity to HD-Ad vectors in airway readministration.

Combined p14ARFand Antisense EGFR Potentiate the Efficacy of Adenovirus-Mediated Gene Therapy in Laryngeal Squamous Cell Carcinoma (LSCC)

The tumor suppressor p14(ARF) and protooncogene epidermal growth factor receptor (EGFR) play an important role in the development of laryngeal squamous cell carcinoma (LSCC). We explored the inhibition of proliferation and induction of differentiation in human larynx cancer cells (Hep-2) in vitro when p14(ARF) couples with antisense complementary DNA of EGFR to transfect into Hep-2 cells via the AdEasy-1 vector system. In vitro studies, using standard isobologram analyses, identified whether Ad-antisense EGFR is synergistic with Ad-14(ARF). To evaluate the cytotoxicity of these agents the gold standard clonogenic survival assay was used. Western blotting analyses, 3'(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometer (FCM) analysis was used to detect protein expression, proliferation, and cell cycle distribution of Hep-2 cells, respectively. Meanwhile, empty vector and PBS were set as a control. The activity of proliferation of Hep-2 cells was inhibited markedly by infection of Ad-p14(ARF) combined with Ad-antisense EGFR compared with Ad-p14(ARF) or Ad-antisense EGFR alone (P = 0.001, P = 0.002, respectively), with Ad-sense EGFR (P = 0.0005), with vector control (Ad-Ctrl) (P = 0.0001), and with PBS (P = 0.0001). FCM revealed that the proportion in the G(0)/G(1) phases increased by up to 86.9% when Ad-p14(ARF) was associated with Ad-antisense EGFR to transfect Hep-2 cells. A weakened expression of EGFR protein and P14 (ARF) protein overexpression was observed. Our study in vitro indicated that association of Ad-p14(ARF) with Ad-antisense EGFR remarkably inhibited activity of proliferation and inducted differentiation of Hep-2 cells. Therefore, not only EGFR, but also p14(ARF), plays a major role in the genesis and in modulating cell growth and differentiation of LSCC, and their synergistic effect was obvious. An effective potential target of gene therapy to prevent LSCC proliferation was provided.

862. Improved Efficacy of Adenovirus-Mediated Gene Therapy for GSD-II Disease by Selective Depletion of Kupffer Cells

Glycogen storage disease type II (GSD-II) is a fatal genetic muscle disorder caused by a massive accumulation of glycogen in multiple muscle groups, and is caused by the deficiency of intralysosomal acid alpha glucosidase (GAA). Two approaches to the treatment of GSD-II are currently being pursued in our research group: enzyme replacement therapy and gene therapy. Relative to gene therapy, our previous work demonstrated that a single intravenous administration of advanced generation, [E1-,pol-] adenoviral (Ad) based vectors encoding human GAA (hGAA) resulted in efficient hepatic transduction and secretion of high levels of hGAA, consequently, the correction of all affected muscles in a GAA-KO mouse model as well a quail model of GSD-II occurred. The long-term persistence of the improved vector in liver tissues facilitated significant glycogen reduction in multiple muscles (though, with variable responses in extent and duration among the individual muscle group analyzed) for at least 6 months, despite the fact that hGAA is perceived as a foreign antigen in these animal models. Unfortunately, our most recent work suggests that very high doses of Ad vectors must be intravenously injected to facilitate the large amounts of hepatic hGAA secretion required to correct both cardiac and skeletal muscles in these animal models. To determine if this problem can be overcome, we have begun attempts to pre-emptively suppress the innate immune systems of immunocompetent GAA-KO mice, prior to injection with our viral vectors. Macrophages, in particular hepatic Kupffer cells, play a central role in sequestering Ad vectors, as well in triggering early innate immune responses, and hence have a profound impact on the adaptive immune responses following adenoviral vector administration. In this study we evaluated the effect of selective and transient depletion of Kupffer cells (via pretreatment of mice with clodronate liposomes-dichloromethylene-bisphosphonate) one day prior to vector injection. We intravenously injected into the clodronate liposome pretreated GAA-KO mice different dosages of the vector, to determine how removal of Kupffer cells impacted upon the known efficacy of this approach. We found that clodronate pretreatment allowed lower doses of the vector to show greater efficacy. For example, the GAA activities in the clodronate-treated mice were significantly elevated both in plasma and in tissues such as liver, heart, diaphram and quadriceps, as compared to the non-Kupffer cell depleted mice injected with identical doses of the [E1-,pol-]AdhGAA vector. As a result, the pathogenic glycogen accumulations in clodronate liposome pretreated GAA-KO mice were significantly reduced at lower doses of the Ad vector (10–100 fold less) as compared to Ad treated GAA-KO mice that had not been pretreated with clodronate containing liposomes. Our data also suggests however, that liver toxicities were also now apparent at lower vector dosages, suggesting that Kupffer cells act to prevent liver damage by sequestering Ad particles. Whether or not other “Ad induced” toxicities are prevented by Kupffer cell depletion are studies currently ongoing in our laboratories.

Increased revascularization efficacy after administration of an adenovirus encoding VEGF(121).

Adenovirus-mediated VEGF gene delivery is being evaluated in clinical trials as a treatment for patients with vascular diseases that stem from ischemia, such as diffuse coronary artery disease and peripheral vascular disease. Although adenoviral vectors are one of the most widely utilized vectors to deliver therapeutic genes to cells, they also have a major limitation in that their inherent immunogenicity leads to the production of neutralizing antibodies that block effective repeat administration. Although this may be true of intravenous, intranasal, and other routes of administration, recent studies have indicated that it may be possible to effectively readminister adenovirus to skeletal muscle. The present study found improved efficacy after administration of AdVEGF(121.10), an E1/E3-deleted adenovirus encoding human VEGF(121) under the control of a CMV promoter in a rat hindlimb ischemia model. As expected, repeat administration of adenovirus resulted in a marked increase of circulating neutralizing antibody, yet nanogram quantities of VEGF protein were still detectable within the hindlimb skeletal muscle after a second administration of vector. The amount of VEGF protein produced after repeat administration translated into improved efficacy as evidenced by increased blood flow as measured by laser Doppler, increased vessel number upon post-mortem angiography, and an increased number of CD31-positive vessels. These findings have important implications for increasing the efficacy of adenovirus-mediated gene therapy in the treatment of peripheral vascular disease and coronary artery disease.

Ex Vivo and In Vivo Adenovirus-Mediated Gene Therapy Strategies Induce a Systemic Anti-Tumor Immune Defence in the B16 Melanoma Model

The efficacy of adenovirus-mediated gene therapy for treatment of metastatic B16 melanomas, established in syngeneic C57BL/6 mice, was assessed via an ex vivo cytokine vaccine approach or via an in vivo strategy utilizing combination cytokine/herpes simplex virus-thymidine kinase (HSV-tk) suicide gene delivery and treatment with ganciclovir (GCV). In the ex vivo tumor vaccine approach, B16 melanoma cells, transduced in vitro by adenovirus containing either interleukin (IL)-2, granulocyte-macrophage colony stimulating factor (GM-CSF), or tumor necrosis factor-alpha cytokine genes and gamma irradiated, were subcutaneously injected into the flank and a distant subcutaneous challenge injection of unmodified B16 melanoma cells was performed 15 d later. Significant reductions in challenge tumor volume were observed in the IL-2 group (75% reduction; p = 0.02) and in the GM-CSF group (88% reduction; p = 0.0006), whereas the effect for tumor necrosis factor-alpha was not statistically significant. In the in vivo treatment of established melanomas, this cytokine approach was combined with a suicide gene therapy and subcutaneous B16 melanomas were directly injected with (i) IL-2/recombinant, replication-deficient adenovirus (adv) and thymidine kinase (tk)/adv, (ii) GM-CSF/adv, IL-2/adv, and tk/adv, or (iii) control beta-galactosidase (beta-gal)/adv and tk/adv. After intraperitoneal application of GCV (10 mg per kg) for 6 d, the residual tumor masses were excised and the animals challenged with unmodified B16 cells. Challenge tumor growth was reduced by 56% for the IL-2/tk/adv/GCV treatment (p = 0.041) and by 77% for the GM-CSF/IL-2/tk/adv/GCV treatment p (p = 0.037), in comparison with the beta-gal/tk/GCV control group. These data may hold significant promise for the development of effective ex vivo and in vivo gene therapy modalities to counter the highly metastatic nature of human melanoma.

Adenovirus-mediated gene therapy in an experimental model of breast cancer metastatic to the brain.

We investigated the therapeutic efficacy of adenovirus-mediated gene therapy to treat malignant mammary tumors in vitro and in vivo in the brain. A mammary adenocarcinoma cell line derived from Fischer rats (13762 MAT B III; MAT-B) was used. In vitro studies demonstrated that the MAT-B cells could be efficiently transduced with a replication-defective adenovirus (ADV) vector that carried the herpes simplex virus gene for thymidine kinase (ADV-tk), and that ADV-tk transduction rendered the MAT-B cells sensitive to killing, in a dose-dependent manner, with ganciclovir (GCV). An animal model of a mammary tumor metastatic to the brain was produced by injecting MAT-B cells into the caudate nucleus of Fischer rats. Seven days after MAT-B cell injection, when the tumors were approximately 5 mm2 in cross-sectional size, the tumors were injected with ADV-tk or a control adenovirus vector containing the beta-galactosidase (beta-Gal) gene (ADV-beta gal). After vector injection the animals were treated with GCV or with saline for 6 days. Sixteen days after tumor cell injection, the brains were examined histologically. The rats that were injected with ADV-beta gal and treated with GCV or saline, and those that were injected with ADV-tk and treated with saline had large tumors, whereas the rats that were injected with ADV-tk and treated with GCV had no visible tumor tissue at the site of tumor cell injection. In survival studies animals treated with ADV-tk+GCV survived a significantly longer time than animals treated with ADV-beta gal+GCV. Our results demonstrate that the recombinant adenoviral vector containing the tk gene confers GCV cytotoxic sensitivity to mammary tumor cells in vitro and in the brain, and suggest that this treatment strategy may be useful in treating somatic tumors that metastasize to the brain.