Transcriptional blocks limit adenoviral replication in primary ovarian tumor.

PURPOSE: Despite the success of conditionally replicating adenoviruses in tumor models, clinical success has been limited when they are used as a single modality agent. Overcoming the disparity in efficacy between in vivo animal models and human use is a key hurdle for better conditionally replicating adenovirus therapy in humans. We endeavored to identify biological blocks to adenoviral infection and replication in tumor cells. EXPERIMENTAL DESIGN: We hypothesized that the differences in adenoviral replication between ovarian cancer cell lines and patient tumor samples are the result of a block in viral RNA transcription. To test this hypothesis, established ovarian cancer cell lines and purified patient ovarian cancer cells were infected with wild-type adenovirus. RNA for early adenoviral genes E1A and E1B as well as the late transcripts for fiber and hexon were measured using real-time PCR. RESULTS: Established ovarian cancer cell lines treated with wild-type virus had a lower E1A:E1B ratio than the patient samples. Additionally, the levels of fiber and hexon relative to E1A were also decreased in the patient samples compared with the established cell lines. These findings were consistent with an early- to late-phase block in the adenovirus replication cycle. CONCLUSIONS: These data suggest that the biology of abortive infection in the patient samples may be linked to a defect in the production of early and late viral transcripts. Identification of factors leading to abortive infection will be crucial to understanding the low viral replication in patient samples.

Retargeting of gene expression using endothelium specific hexon modified adenoviral vector.

Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter was characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.

A phase I clinical trial of Ad5.SSTR/TK.RGD, a novel infectivity-enhanced bicistronic adenovirus, in patients with recurrent gynecologic cancer.

PURPOSE: Ad5.SSTR/TK.RGD is an infectivity-enhanced adenovirus expressing a therapeutic thymidine kinase suicide gene and a somatostatin receptor (SSTR) that allows for noninvasive gene transfer imaging. The purpose of this study was to identify the maximum tolerated dose (MTD), toxicities, clinical efficacy, and biologic effects of Ad5.SSTR/TK.RGD in patients with recurrent gynecologic cancer. EXPERIMENTAL DESIGN: Eligible patients were treated intraperitoneally for 3 days with 1 × 10(9) to 1 × 10(12) vp/dose of Ad5.SSTR/TK.RGD followed by intravenous ganciclovir for 14 days. Toxicity and clinical efficacy were assessed using Common Toxicity Criteria (CTC) Adverse Events grading and Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Imaging using In-111 pentetreotide was obtained before and after treatment. Tissue samples were obtained to evaluate for gene transfer, generation of wild-type virus, viral shedding, and antibody response. RESULTS: Twelve patients were treated in three cohorts. The most common vector-related clinical toxicities were grade I/II constitutional or pain symptoms, experienced most often in patients treated at the highest dose. MTD was not identified. Five patients showed stable disease; all others experienced progressive disease. One patient with stable disease experienced complete resolution of disease and normalization of CA125 on further follow-up. Imaging detected increased In-111 pentetreotide retention in patients treated at the highest dose. Ancillary studies showed presence of Ad5.SSTR/TK.RGD virus and HSV1-tk expression in ascites samples collected at various time points in most patients treated within the higher dose cohorts. CONCLUSIONS: This study shows the safety, potential efficacy, and possible gene transfer imaging capacity of Ad5.SSTR/TK.RGD in patients with recurrent gynecologic cancer. Further development of this novel gene therapeutic appears to be warranted.

A new generation of serotype chimeric infectivity-enhanced conditionally replicative adenovirals: the safety profile of ad5/3-Δ24 in advance of a phase I clinical trial in ovarian cancer patients.

Conditionally replicative adenoviral (CRAd) virotherapy represents a promising therapeutic approach for cancer. We have demonstrated that a serotype chimeric adenoviral 5/3 fiber-knob modification achieves enhanced ovarian cancer infectivity, conditional replication, and oncolytic activity. This study evaluated the safety of intraperitoneal (IP) Ad5/3-Δ24 in advance of a phase I clinical trial in gynecologic cancers. Syrian hamster cohorts were treated with IP Ad5/3-Δ24 or control buffer for 3 consecutive days and euthanized on study days 8, 17, 57, and 89. Blood and tissue samples were harvested from each animal. For biodistribution studies, presence and quantitation of viral levels within samples were determined via quantitative polymerase chain reaction. For safety studies, animals were assessed for adverse vector-related tissue or laboratory effects. In the biodistribution study, low levels of Ad5/3-Δ24 DNA were noted outside of the abdominal cavity. Viral DNA levels in tissues obtained from the peritoneal cavity peaked at day 8 and declined thereafter. In the safety study, no specific histopathologic changes were attributable to virus administration. Hematologic findings noted in the 1 × 10(11) viral particles (vp)/dose group on Days 4 and/or 8 were indicative of an Ad5/3-Δ24-specific generalized inflammatory response; these findings resolved by day 56. The no observable adverse effect level was determined to be 1 × 10(10) vp/dose. This study elucidates the safety profile of IP administration of the serotype chimeric infectivity-enhanced CRAd, Ad5/3-Δ24, and provides guidance for a planned phase I trial for patients with recurrent gynecologic cancers.

A phase I study of a tropism-modified conditionally replicative adenovirus for recurrent malignant gynecologic diseases.

PURPOSE: To determine the maximum tolerated dose (MTD), toxicity spectrum, clinical activity, and biological effects of the tropism-modified, infectivity-enhanced conditionally replicative adenovirus (CRAd), Ad5-Δ24-Arg-Gly-Asp (RGD), in patients with malignant gynecologic diseases. EXPERIMENTAL DESIGN: Cohorts of eligible patients were treated daily for 3 days through an i.p. catheter. Vector doses ranged from 1 × 10(9) to 1 × 10(12) viral particles per day. Toxicity was evaluated using CTCv3.0. CA-125 and Response Evaluation Criteria in Solid Tumors (RECIST) criteria were used to determine clinical efficacy. Corollary biological studies included assessment of CRAd replication, wild-type virus generation, viral shedding, and neutralizing antibody response. RESULTS: Twenty-one patients were treated. Adverse clinical effects were limited to grade 1/2 fever, fatigue, or abdominal pain. No vector-related grade 3/4 toxicities were noted. No clinically significant laboratory abnormalities were noted. The maximum tolerated dose was not reached. Over a 1 month follow-up, 15 (71%) patients had stable disease and six (29%) had progressive disease. No partial or complete responses were noted. Seven patients had a decrease in CA-125; four had a >20% drop. RGD-specific PCR showed the presence of study vector in ascites of 16 patients. Seven revealed an increase in virus after day 3, suggesting replication of Ad5-Δ24-RGD. Minimal wild-type virus generation was detected. Viral shedding studies showed insignificant shedding in the serum, saliva, and urine. Anti-adenoviral neutralizing antibody effects were prevalent. CONCLUSIONS: This study, the first to evaluate an infectivity-enhanced CRAd in human cancer, shows the feasibility, safety, potential antitumor response, and biological activity of this approach in ovarian cancer. Further evaluation of infectivity enhanced virotherapy approaches for malignant gynecologic diseases is warranted.

Enhanced Gene Delivery to Human Primary Endothelial Cells Using Tropism-Modified Adenovirus Vectors.

Endothelial cells have been noted to have relatively low expression of the native receptor for adenovirus serotype 5 (Ad5), coxsackie and adenovirus receptor (CAR), and are thus refractory to Ad5 infection. In this study, we hypothesize that increases in the infectivity of Ad5 in primary human pulmonary artery (HPAEC), coronary artery (HCAEC) and umbilical vein endothelial cells (HUVEC) can be achieved through genetic capsid modification of Ad5 to bypass CAR-dependent infection. The modifications tested in this study include incorporation of an integrin-binding RGD peptide motif (Ad5.RGD), a poly-lysine motif (Ad5.pK7), a combination of both of these peptide domains (Ad5.RGD.pK7), an adenovirus serotype 3 knob domain (Ad5/3Luc1) and canine adenovirus serotype 1 or 2 knob domains (Ad5Luc1-CK1 and Ad5Luc1-CK2). In HPAEC and HCAEC, the greatest infectivity enhancements were achieved using Ad5/3Luc1 (26-fold and 30-fold respectively). HUVEC was most readily infected by Ad5Luc1-CK1 (213-fold). These results demonstrate that gains in Ad5 infectivity in endothelial cells can be accomplished with genetic capsid modifications.

Covalently linked Au nanoparticles to a viral vector: potential for combined photothermal and gene cancer therapy.

Hyperthermia can be produced by near-infrared laser irradiation of gold nanoparticles present in tumors and thus induce tumor cell killing via a bystander effect. To be clinically relevant, however, several problems still need to be resolved. In particular, selective delivery and physical targeting of gold nanoparticles to tumor cells are necessary to improve therapeutic selectivity. Considerable progress has been made with respect to retargeting adenoviral vectors for cancer gene therapy. We therefore hypothesized that covalent coupling of gold nanoparticles to retargeted adenoviral vectors would allow selective delivery of the nanoparticles to tumor cells, thus feasibilizing hyperthermia and gene therapy as a combinatorial therapeutic approach. For this, sulfo-N-hydroxysuccinimide labeled gold nanoparticles were reacted to adenoviral vectors encoding a luciferase reporter gene driven by the cytomegalovirus promoter (AdCMVLuc). We herein demonstrate that covalent coupling could be achieved, while retaining virus infectivity and ability to retarget tumor-associated antigens. These results indicate the possibility of using adenoviral vectors as carriers for gold nanoparticles.