Characterization of infectivity of knob-modified adenoviral vectors in glioma.

Malignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors,particularly vectors derived from serotype 5 adenoviruses (Ad5). This results from limited cell surface expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on tumor cells. To circumvent this problem, Ad fiber pseudotyping,the genetic replacement of either the entire fiber or fiber knob domain with its structural counterpart from another human Ad serotype that recognizes a cellular receptor other than CAR, has been shown to enhance Ad infectivity in a variety of tumor types,including human glioma. Here, we have extended the paradigm of genetic pseudotyping to include fiber domains from non-human or"xenotype" Ads for infectivity enhancement of human glioma cell populations. In this study, we evaluated the gene transfer efficiency of a panel of Ad vectors which express one of five different "xenotype"fiber knob domains, including those derived from murine,ovine, porcine and canine species, in both human glioma cell lines as well as primary glioma tumor cells from patients. Adenovirus vectors displaying either canine Ad or porcine Ad fiber elements had the highest gene transfer to both glioma cell lines and primary tumor cells. The correlation between the viral infectivity of modified adenovirus vectors and expression of human CAR and CD46(an adenovirus type B receptor) on the surfaces of tumor cells was also analyzed. Taken together, human adenovirus vectors modified with "xenotype" fiber elements could be excellent candidates to target human glioma.

Transductional targeting of adenovirus vectors for gene therapy.

Cancer gene therapy approaches will derive considerable benefit from adenovirus (Ad) vectors capable of self-directed localization to neoplastic disease or immunomodulatory targets in vivo. The ablation of native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and Ad receptor. Herein, we describe advances in Ad targeting that are predicated on a fundamental understanding of vector/cell interplay. Further, we propose strategies by which existing paradigms, such as nanotechnology, may be combined with Ad vectors to form advanced delivery vehicles with multiple functions.

An adenovirus vector with a chimeric fiber incorporating stabilized single chain antibody achieves targeted gene delivery.

Adenovirus (Ad) vectors are of utility for many therapeutic applications. Strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity employing fiber modifications allowing genetic incorporation of targeting motifs. In this regard, single chain antibodies (scFv) represent potentially useful agents to achieve targeted gene transfer. However, the distinct biosynthetic pathways that scFv and Ad capsid proteins are normally routed through have thus far been problematic with respect to scFv incorporation into the Ad capsid. Utilization of stable scFv, which also maintain correct folding and thus functionality under intracellular reducing conditions, could overcome this restriction. We genetically incorporated a stable scFv into a de-knobbed, fibritin-foldon trimerized Ad fiber and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of cells. We have shown that the scFv employed in this study retains functionality and that stabilizing the targeting molecule, per se, is critical to allow retention of antigen recognition in the adenovirus capsid-incorporated context.

Infectivity enhancement for adenoviral transduction of canine osteosarcoma cells.

The full realization of conditionally replicative adenoviruses (CRAds) for cancer therapy has been hampered by the limited knowledge of CRAd function in vivo and particularly in an immunocompetent host. To address this issue, we previously proposed a canine adenovirus type 2 (CAV2)-based CRAd for clinical evaluation in canine patients with osteosarcoma (OS). In this study, we evaluated infectivity-enhancement strategies to establish the foundation for designing a potent CAV2 CRAd with effective transduction capacity in dog osteosarcoma cells. The results indicate that the native CAV2 fiber-knob can mediate increased binding, and consequently gene transfer, in both canine osteosarcoma immortalized and primary cell lines relative to previously reported Ad5 infectivity-enhancement strategies. Gene delivery was further enhanced by incorporating a polylysine polypeptide onto the carboxy terminus of the CAV2 knob. This vector demonstrated improved gene delivery in osteosarcoma xenograft tumors. These data provide the rationale for generation of infectivity-enhanced syngeneic CAV2 CRAds for clinical evaluation in a dog osteosarcoma model.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy.

The development of novel therapeutic strategies is imperative for the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on 'xenotype' knob switching with non-human adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.

Selective induction of tumor-associated antigens in murine pulmonary vasculature using double-targeted adenoviral vectors.

Targeted therapies directed to tumor-associated antigens are being investigated for the treatment of cancer. However, there are few suitable animal models for testing the ability to target these tumor markers. Therefore, we have exploited mice transgenic for the human coxsackie and adenovirus receptor (hCAR) to establish a new model for transient expression of human tumor-associated antigens in the pulmonary vasculature. Systemic administration of Ad in hCAR mice resulted in an increase in transgene expression in the lungs compared to wild-type mice, as determined using a luciferase reporter gene. To reduce transgene expression in the liver, the predominant organ of ectopic Ad localization and transgene expression following systemic administration, we utilized the endothelial-specific flt-1 promoter, which resulted in a further increased lung-to-liver ratio of luciferase expression. Administration of an adenoviral vector encoding the tumor-associated antigen carcinoembryonic antigen (CEA) under transcriptional control of the flt-1 promoter resulted in selective expression of this antigen in the pulmonary vasculature of hCAR mice. Feasibility of targeting to expressed CEA was subsequently demonstrated using adenoviral vectors preincubated with a bifunctional adapter molecule recognizing this tumor-associated antigen, thus demonstrating utility of this transient transgenic animal model.

Mesothelin-mediated targeting of adenoviral vectors for ovarian cancer gene therapy.

Adenoviruses (Ads) are efficient gene transfer vehicles, but Ad-mediated gene therapy for ovarian cancer remains limited in vivo by inefficient and nonspecific gene transfer. Mesothelin (MSLN), a cell surface glycoprotein, is overexpressed in ovarian cancer but not in normal tissues except mesothelial cells. Therefore, MSLN is an attractive candidate for transcriptional and transductional targeting in the context of ovarian cancer gene therapy. We evaluated the expression of MSLN mRNA and MSLN surface protein in ovarian cancer cells. Ads containing the MSLN promoter driving reporter gene expression were created and tested in ovarian cancer cell lines and purified ovarian cancer cells isolated from patients. To evaluate transductional targeting, we used an Ad vector containing an Fc-binding domain within the fiber protein, which served as a docking domain for binding with anti-MSLN immunoglobulins. Both RT-PCR and flow cytometry revealed high MSLN gene and protein expression in ovarian cancer cells. The MSLN promoter was activated in ovarian cancer cells, but showed significantly reduced activity in normal control cells. Transductional targeting of Ads via anti-MSLN antibody increased transgene expression in ovarian cancer cells. This report describes the use of MSLN for transcriptional as well as transductional targeting strategies for ovarian cancer gene therapy.

Transductional and transcriptional targeting of adenovirus for clinical applications.

Adenovirus (Ad) targeting is a novel approach for the design and administration of therapeutic agents wherein the agent is rationally designed to localize and restrict transgene expression to the site of disease in a self-directed manner, usually via exploitation of unique biophysical and genetic properties specific to the diseased tissue. The ablation of promiscuous native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and adenovirus receptor (CAR), to achieve CAR-independent vector tropism. Herein, we describe advances in Ad targeting that are predicated not only on fundamental understanding of vector/cell interplay, but also on the specific transcriptional profiles of target tissues. Further, targeting is discussed in the context of improving the safety and efficacy of clinical approaches utilizing adenoviral vectors and replication competent oncolytic agents. In summary, existing results suggest a critical linkage between targeted agents and increases in therapeutic utility.

Transcriptional regulation of the BCL-X gene by NF-kappaB is an element of hypoxic responses in the rat brain.

Signal transduction pathways that mediate neuronal commitment to apoptosis involve the nuclear factor kappa B (NF-kappaB) transcription factor. The bcl-x gene is a member of the bcl-2 family of genes that regulate apoptosis, and gives rise to two proteins, Bcl-XL and Bcl-XS, via alternative mRNA splicing. BCl-XL protein, like Bcl-2, is a dominant inhibitor of apoptotic cell death, whereas Bcl-XS promotes apoptosis. While there is high expression of Bcl-XL in the developing and adult brain, few transcriptional control elements have been identified in the bcl-x promoter. There are two functional nuclear factor-kappa B (NF-kappaB) DNA binding sites clustered upstream of the brain-specific transcription start site in the upstream promoter region of murine bcl-x. Recombinant NF-kappaB proteins bind to these sites. Also NF-kappaB overexpression, coupled with bcl-x promoter/reporter assays using a series of murine bcl-x promoter and deletion mutants, has identified the downstream 1.1kb of the bcl-x promoter as necessary for basal promoter activity and induction by NF-kappaB in support of the hypothesis that NF-kappaB can act to enhance BCl-XL expression via highly selective interactions with the bcl-x promoter, where NF-kappaB binding and promoter activation are dependent on specific DNA binding site sequences and NF-kappaB protein dimer composition. Hypoxia induces apoptosis in the hippocampus where the NF-kappaB dimers c-Rel/p50 and p50/pS0 bind to the bcl-x promoter NF-kappaB site.

Differential NF-kappa B regulation of bcl-x gene expression in hippocampus and basal forebrain in response to hypoxia.

Cell death often occurs after hypoxic/ischemic injury to the central nervous system. Changes in levels of the anti-apoptotic Bcl-X(L) protein may be a determining factor in hypoxia-induced neuronal apoptosis. The transcription factor NF-kappa B regulates bcl-x gene expression. In this study, we examined the role of NF-kappa B in the regulation of bcl-x in hypoxia-induced cell death. Rat hippocampus and basal forebrain tissues were collected at different time points after hypoxia (7%O(2), 93% N(2) for 10 or 20 min). We found that 1) hypoxia induced apoptosis in the hippocampus and basal forebrain; 2) the NF-kappa B dimers c-Rel/p50 and p50/p50 bound to the bcl-x promoter NF-kappa B sequence (CS4) in the hippocampus, but only p50/p50 bound to the CS4 sequence in the basal forebrain and hypoxia-induced differential binding patterns of c-Rel/p50 and p50/p50 correlated with the bcl-x expression pattern in the hippocampus; 3) the hypoxia-induced patterns of binding of c-Rel/p50 to the bcl-x promoter CS4 sequence were different from those to the IgG-kappa B enhancer sequence, whereas those of p50/p50 were similar to both sequences; 4) nuclear protein levels of c-Rel, but not p50, correlated with the c-Rel/p50 DNA binding patterns to the bcl-x CS4 site; and 5) there were differential responses to hypoxia among the different NF-kappa B protein subunits. These results suggest that there is a tissue-specific regulation of bcl-x gene expression by NF-kappa B in hypoxia-induced cell death in the hippocampus. The absence of these regulating features in the basal forebrain may account for the early appearance of apoptosis in response to hypoxia as compared with that in hippocampus.

Identification and characterization of nuclear factor kappaB binding sites in the murine bcl-x promoter.

Signal transduction pathways that mediate neuronal commitment to apoptosis involve the nuclear factor kappaB (NF-kappaB) transcription factor. Bcl-X(L) is a potent regulator of apoptosis in the CNS and is highly expressed in the developing and adult brain. We identified three putative NF-kappaB DNA binding sequences clustered upstream of the brain-specific transcription start site in the upstream promoter region. Recombinant p50/p50 and NF-kappaB proteins from nuclear extracts bound to these sites as determined by electrophoretic mobility shift assay and biotin-oligonucleotide/streptavidin affinity assays. NF-kappaB overexpression, coupled with bcl-x promoter/reporter assays using a series of murine bcl-x promoter and deletion mutants, has identified the downstream 1.1 kb of the bcl-x promoter as necessary for basal promoter activity and induction by NF-kappaB. The mutagenic removal of NF-kappaB binding sites individually or in combination revealed altered response patterns to p49/p65 and p50/p65 overexpression. These results support the hypothesis that NF-kappaB can act to enhance Bcl-X(L) expression via highly selective interactions, where NF-kappaB binding and bcl-x promoter activation are dependent on both DNA binding site sequence and NF-kappaB subunit composition. Our data suggest that molecular events associated with NF-kappaB promote regulation of neuronal apoptosis in the developing or injured CNS.