Naked Adenovirus Research Articles

Aptamer-mediated gene therapy enhanced antitumor activity against human hepatocellular carcinoma in vitro and in vivo

A recombinant adenovirus carrying the tumor suppressor gene PTEN (Ad5-PTEN) is an effective antitumor agent against liver cancer. But the application of Ad5-PTEN has been greatly hindered by its auto-immunogenicity, non-specific toxicity to normal tissues, as well as poor stability in blood stream because of neutralizing antibody. Epithelial cell adhesion molecule (EpCAM) is over-expressed in most solid tumors and it has been identified to be a cancer stem cell surface marker in liver cancer. An RNA aptamer EpDT3 could specially bind with EpCAM and target EpCAM-positive cells. Therefore, we hypothesized that developing a novel gene delivery system of EpDT3-modified Ad5-PTEN could overcome the disadvantages of naked Ad5-PTEN and enhance the antitumor effect on hepatocellular carcinoma. We took polyethylene glycol (PEG) as a linker to conjugate EpDT3 with Ad5-PTEN to prepare EpDT3-PEG-Ad5-PTEN (EPAP) by simple chemical synthesis method. We found that the stability of this novel gene delivery system in human blood serum increased about 16-fold compared to the naked adenovirus. Meanwhile, EPAP enhanced gene expression and cellular uptake in HepG2 cells, and showed significant inhibition in cell proliferation and cell migration against hepatocellular carcinoma cells HepG2 while showing no cytotoxicity to normal liver cells L-02, compared with Ad5-PTEN. Importantly, EPAP could induce cell apoptosis and presented superior antitumor activity against aggressive HepG2 xenograft in nude mice but showed no obvious toxicity to the tested mice at the therapy concentration. In conclusion, EpCAM aptamer EpDT3 could significantly enhance the antitumor effect of Ad5-PTEN with high binding ability to EpCAM-positive cells HepG2.

Gene Therapy with Recombinant Adenovirus Encoding Endostatin Encapsulated in Cationic Liposome in Coxsackievirus and Adenovirus Receptor-Deficient Colon Carcinoma Murine Models

Adenovirus (Ad)-based antiangiogenesis gene therapy is a promising approach for cancer treatment. Downregulation or loss of coxsackievirus and adenovirus receptor (CAR) is often detected in various human cancers, which hampers adenoviral gene therapy approaches. Cationic liposome-complexed adenoviral vectors have been proven useful in CAR-deficient cells to enhance therapeutic gene transfer in vivo. Here, we investigated the antitumor effects of recombinant adenovirus encoding endostatin (Ad-hE) encapsulated in cationic liposome (Ad-hE/Lipo) on CAR-deficient CT26 colon carcinoma murine models. In vitro, Ad-hE/Lipo enhanced adenovirus transfection in CAR-deficient cells (CT26), and endostatin gene expression was measured by both qualitative and quantitative detection. In addition, an antibody neutralizing assay indicated that neutralizing serum inhibited naked adenovirus 5 (Ad5) at rather higher dilution than the complexes of Ad5 and cationic liposomes (Ad5-CL), which demonstrated that Ad5-CL was more capable of protecting Ad5 from neutralization. In vivo, Ad-hE/Lipo treatment in the murine CT26 tumor model by intratumoral injection resulted in marked suppression of tumor growth and prolonged survival time, which was associated with a decreased number of microvessels and increased apoptosis of tumor cells. In conclusion, recombinant endostatin adenovirus encapsulated with cationic liposome effectively inhibited CAR-deficient tumor growth through an antiangiogenic mechanism in murine models without marked toxicity, thus showing a feasible strategy for clinical applications.

Anionic Liposomes Enhance and Prolong Adenovirus-Mediated Gene Expression in Airway Epithelia in Vitro and in Vivo

Adenoviral vector mediated gene therapy has received extensive attention in airway disease treatment. However, the lack of the requisite coxsackie-adenovirus receptor (CAR) on the apical surface of airway epithelium and the host immune response to adenoviruses limit their in vivo application. In our study, we developed for the first time a novel formulation composed of anionic liposomes and adenoviruses (AL-Ad5) using a calcium-induced phase change method. The obtained formulation was employed to enhance the transduction efficiency of airway gene delivery. Our results indicated that primary cultured airway epithelial cells infected by AL-Ad5 displayed higher LacZ gene expression compared to naked adenovirus. Importantly, AL-Ad5 significantly improved and prolonged LacZ gene expression in murine airway tissues when delivered in vivo by intratracheal instillation. Additionally, it was found that anionic liposomes provided immunoprotection to the adenovirus from neutralizing antibody, thus slowing down the elimination of Ad5 particles meanwhile reducing the inflammatory reaction caused by the Ad5 vector. These results suggested that the combination of anionic liposomes with adenovirus may be a useful strategy to deliver therapeutic genes into the airway epithelia and is promising in clinical application.

Improvement of adenoviral vector-mediated gene transfer to airway epithelia by folate-modified anionic liposomes

Despite remarkable progress in the development of both viral and nonviral gene delivery vectors for airway disease treatment, poor gene transfer efficiency to the airway epithelium is a major obstacle in clinical application. To take advantage of the unique features of viral and nonviral vectors, we have developed complexes of adenovirus vector and anionic liposomes (AL-Ad5) by the calcium-induced phase change method. In the current study, based on the fact that there are overexpressed folate receptors on the surface of airway epithelia, we further modified the AL-Ad5 complexes with folate (F-AL-Ad5) to improve the transduction ability of Ad5 in airway epithelia. The transduction efficiencies of the obtained F-AL-Ad5 and AL-Ad5 complexes were assessed in primary-cultured airway epithelia in vitro. Our results indicated that compared with naked adenovirus vector, both AL-Ad5 and F-AL-Ad5 could significantly enhance the gene transduction efficiency of adenovirus vector in primary-cultured airway epithelial cells. Moreover, the enhancement mediated by F-AL-Ad5 was more dramatic than that by AL-Ad5. These results suggested that F-AL-Ad5 may be a useful strategy to deliver therapeutic genes to the airway epithelia and is promising in clinical application.

Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE

We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB–DOPE liposomes to form adenovirus–liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of β-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) ( P < 0.05). The tumor to non-tumor ratio of β-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes ( P < 0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more β-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. Conclusions Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

26. Microsphere Delivery of Adenoviral-Liposome Complexes Reduces Adenoviral Immunogenicity and Allows Successful Re-Administration of the Virus without Loss of Gene Expression Efficiency

The successful use and repeat administration of adenoviral vectors for gene therapy is hampered by the virus's immunogenicity and poor tissue localization. Following initial exposure to the virus, antibody production occurs often limiting the therapeutic benefit of secondary administration. Further, the small size and the ubiquitous expression of the adenoviral receptor limits the ability of the virus to remain localized within a treatment site. In order to overcome these problems we examined a combination of liposomes and microspheres for the delivery of adenovirus. We found that conjugation of liposomes to adenovirus can provide shielding from neutralizing antibodies, and that binding the adenovirus-liposome complexes to microspheres increased the level of protection. We examined microsphere-adenovirus-liposome (MAL) complexes in vivo and compared the antibody production, transgene expression and localization of gene expression to that of naked adenovirus (NA) and adenovirus complexed to liposomes (AL complexes). Our results showed that the MAL complexes induced significantly lower antibody titers than either the AL complexes or NA. In animals pre-exposed to adenovirus, NA induced 14-fold greater antibody levels than the MAL complexes (P < 0.05). Similarly, AL complexes induced significantly higher levels of antibodies when compared to the MAL complexes with 10-fold more antibodies induced (P < 0.05). With respect to transgene expression, animals pre-exposed to adenovirus and treated with MAL complexes exhibited 99% greater transgene expression than those treated with NA and 35% more expression than those treated with AL complexes (P < 0.05). When comparing the level of gene expression in animals not exposed to adenovirus to those pre-exposed, we showed that expression with both the AL complexes and NA decreased significantly while expression in animals treated with MAL complexes remained constant indicating that re-administration of the adenovirus without loss of expression was achievable using the MAL complexes. In addition to the benefits of reduced immunogenicity and an enhanced ability to re-dose the animals, we also show the MAL complexes provided greater localization of gene expression than either the AL complexes or NA. After an intratumoral injection of MAL complexes up to 90% of gene expression was retained in the tumor. This was significantly greater than the levels observed in the tumor following treatment with NA (65%) or AL complexes (75%). Conclusion: The use of MAL complexes to deliver adenovirus provides a simple method to enhance vector localization and inhibit neutralization by the immune system while maintaining high transfection efficiency.

450. Anti-CD22 Immunolipoplexes Enhance Transduction of Adenovirus in B Lymphoma Cells Independent on Adenovirus' Receptor but Do Not Enhance the Cytotoxicity of Oncolytic Adenovirus

Adenovirus vectors have several features that make them attractive for potential use in gene therapy, and attention has focused particularly on replication-competent adenoviruses for cancer applications. These have been evaluated extensively in epithelial tumours but only recently in lymphoid malignancies, mainly due to the known resistance of the lymphoid lineage to adenovirus infection. However, we and other groups have shown that this resistance is not absolute and that adenovirus can infect primary chronic lymphocytic leukaemia (CLL) and B-cell lymphoma lines, and that infection with replication-competent human adenovirus results in cytotoxicity that varies among different patients and different lymphoma cell lines. We found that the cytotoxicity of Adenovirus to Burkitt's lymphoma cells correlates with the infectability of these cells to adenovirus, which depends on the expression of adenovirus receptors such as CAR and heparan sulfate glycosaminoglycans. In a panel of lymphoma cell lines including Jiyoye, Raji, Daudi/ICRF, Daudi/100k, P3HR1, BL-74 and CA-46, only Jiyoye and Raji showed a significant cytotoxicity and Adenovirus receptor expression. Most of these cell lines show a very low or no cytotoxicity to replication-competent adenovirus due to low expression of adenovirus receptors. Most lymphoma cells exhibit high expression of CD22, a membrane protein that is endocytosed when anti-CD22 antibody binds the protein. Based on these observations, an adenovirus-retargeting system was developed for CD22-mediated cell entry: adenovirus-containing immunolipoplexes formed with monoclonal anti-CD22 antibody were tested for enhanced transduction of adenovirus receptor-negative lymphoma cell lines (BL-74 and CA-46). The results demonstrate that the method increased adenovirus transduction by 95.4% (anti-CD22-immunolipoplexes vs naked adenovirus) and 62.3% (anti-CD22-immunolipoplexes vs lipid-adenovirus complex) in CA-46 cells, and 38.2% (anti-CD22-immunolipoplexes vs naked adenovirus) and 27.1% (anti-CD22-immunolipoplexes vs lipid-adenovirus complex) in BL-74 cells, respectively. However, the method did not increase the cytotoxicity of replication-competent adenovirus to these cells. In conclusion, this study demonstrates that anti-CD22 immunolipoplexes represent an efficient strategy to enhance adenovirus transduction independent of adenovirus receptor status. This protocol may have an impact on cancer therapy for lymphomas.