Directing adenovirus across the blood-brain barrier via melanotransferrin (P97) transcytosis pathway in an in vitro model

Adenovirus serotype 5 (Ad5) is widely used in the development of gene therapy protocols. However, current gene therapy strategies involving brain are mostly based on intra-cranial injection. A major obstacle for systemically administered vectors to infect brain tissue is the blood-brain barrier (BBB). One strategy to cross the BBB is transcytosis, a transcellular transport process that shuttles a molecule from one side of the cell to the other side. Recently, melanotransferrin (MTf)/P97 was found to be able to cross the BBB and accumulate in brain. We thus hypothesize that re-directing Ad5 vectors to the MTf transcytosis pathway may facilitate Ad5 vectors to cross the BBB. To test this hypothesis, we constructed a bi-specific adaptor protein containing the extracellular domain of the coxsackie-adenovirus receptor (CAR) and the full-length melanotransferrin (sCAR-MTf), and investigated its ability to re-direct Ad5 vectors to the MTf transcytosis pathway. We found this adaptor protein could re-direct Ad5 to the MTf transcytosis pathway in an in vitro BBB model, and the transcytosed Ad5 viral particles retained their native infectivity. The sCAR-MTf-mediated Ad5 transcytosis was temperature- and dose dependent. In addition, we examined the directionality of sCAR-MTf-mediated Ad5 transcytosis, and found the efficiency of apical-to-basal transcytosis was much higher than that of basal-to-apical direction, supporting a role of this strategy in transporting Ad5 vectors towards the brain. Taken together, our study demonstrated that re-directing Ad5 to the MTf transcytosis pathway could facilitate gene delivery across the BBB.     

A model system for the design of armed replicating adenoviruses using p53 as a candidate transgene

Cancer gene therapy endeavors to overcome the low therapeutic index of currently available therapeutic modalities via the efficient and safe delivery of genetic material into tumor cells. However, despite promising preclinical results, replication-deficient viral vectors have demonstrated a limited efficacy in the clinical setting. To increase vector efficiency, replication-competent viruses have been proposed. Clinical trials have shown the safety of locally injected, conditionally replicative adenoviruses (Ads) but have underscored the need for improved potency. To further increase the therapeutic effect of replicating viral vectors, armed therapeutic viruses (ATVs) have recently been used for high-efficiency transgene expression. However, interference with cellular signaling and viral production by constitutive transgene expression may be counterproductive for ATV replication, thereby hindering the therapeutic outcome. Consequently, studies are equivocal with regard to the potential benefits of ATVs. To address this issue, we hypothesized that induction of replication of an Ad expressing p53 may be a useful strategy in the context of ATV because p53 does not interfere with Ad replication and may even increase its cytolytic effect. We show that in our in vitro ATV model system, E1 transcomplementation of a replication-deficient Ad encoding p53 resulted in dramatic augmentation of cell killing and circumvented resistance to apoptosis. Correlation was found between the degrees of cell killing and apoptosis induction, rather than with viral burst. Furthermore, both Ad5 E1B 55kDa and E4 orf6 genes were required to enhance the cell killing. In conclusion, our p53-ATV model system demonstrates the potential utility of therapeutic transgene expression by a replicating Ad after a rational selection of a candidate transgene.     

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.     

Epstein-Barr viral nuclear antigen 1 antisense oligodeoxynucleotide inhibits proliferation of Epstein-Barr virus-immortalized B cells

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is a latent viral protein that is expressed in all EBV-immortalized lymphocytes and plays an essential role in immortalization y EBV. EBNA-1 protein is required for replication and maintenance of the episomal viral genome in latently infected, immortalized cells. Given the essential function of EBNA-1 in immortalization, we have examined the effect of EBNA-1 antisense oligodeoxynucleotides on expression of EBNA-1 protein and proliferation in EBV-immortalized lymphoblastoid cells. We have shown that exposure to unmodified antisense oligodeoxynucleotide of codons 6 through 10 of EBNA-1 partially suppressed EBNA-1 protein expression in EBV-immortalized lymphoblastoid cells relative to untreated cells or cells exposed to two scrambled sequences of the EBNA-1 antisense. Furthermore, EBNA-1 antisense inhibited proliferation of EBV- immortalized cells by at least 50% compared with the scrambled antisense sequences. There was no difference in the effect of antisense and scrambled antisense oligodeoxynucleotides on the proliferation of EBV-negative cells, indicating that the antiproliferative effect of EBNA-1 antisense was EBV-specific. These findings underscore the essential role of EBNA-1 in immortalization and, furthermore, have potential therapeutic implications for EBV-associated neoplastic diseases.     

Adenovirus enhancement of transferrin-polylysine-mediated gene delivery.

Gene transfer may be accomplished by the receptor-mediated endocytosis pathway using transferrin-polylysine conjugates. For some target cells, however, gene transfer by this vector is extremely limited, despite the presence of the appropriate surface receptors, a phenomenon attributed to lysosomal degradation of endosome-internalized conjugate-DNA complexes. To enhance DNA escape from the cell vesicle system and thus augment gene transfer by this route, we have used the capacity of adenoviruses to disrupt endosomes as part of their entry mechanism. Adenoviral infection augmented levels of gene transfer by transferrin-polylysine conjugates in a dose-dependent manner: levels of gene transfer of greater than 2000-fold above baseline were achieved. Use of the adenovirus in this context allowed enhanced levels of gene transfer in a variety of target cells, including cell lines otherwise refractory to gene transfer by transferrin-polylysine conjugates. This augmentation was based on adenoviral-mediated vesicle disruption, a process independent of viral gene expression. Thus, the development of specific mechanisms to effect release from the endosome in combination with gene transfer by the receptor-mediated endocytosis pathway will increase the utility of this delivery system by allowing high levels of gene expression in target cells.     

Retargeting to EGFR enhances adenovirus infection efficiency of squamous cell carcinoma.

BACKGROUND: Adenovirus-mediated gene therapy has been used for squamous cell carcinoma of the head and neck (SCCHN), but the in vivo efficacy has been limited by a lack of tissue specificity and low infection efficiency. We are interested in improving cancer gene therapy strategies using targeted adenovirus vectors. OBJECTIVE: To determine if the infection efficiency of adenovirus-mediated gene transfer to SCCHN cells could be enhanced by retargeting to the epidermal growth factor receptor (EGFR), which is known to be overexpressed in these tumors. DESIGN: Epidermal growth factor receptor retargeting in SCCHN cells was accomplished with a bispecific antibody that recognized the knob domain of adenovirus as well as EGFR. Using this retargeting schema, we compared the infection efficiency and specificity of unmodified and EGFR-retargeted adenovirus. RESULTS: Squamous cell carcinoma of the head and neck cell lines were shown to be infected by adenovirus with low efficiency, which is likely because of the low level of adenovirus receptor expressed in the SCCHN cells. Epidermal growth factor receptor retargeting markedly enhanced transduction in both SCCHN cell lines and primary tumor tissue, as indicated by the elevated levels of reporter gene expression. Furthermore, retargeting enhanced infection of tumor tissue compared with normal tissue from the same patient. CONCLUSIONS: Epidermal growth factor receptor retargeting enhanced adenovirus infection of SCCHN cells and, in doing so, augments the potency of the vector. This modification makes the vector potentially more valuable in the clinical setting.