John G. Hay

Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.

We have administered a recombinant adenovirus vector (AdCFTR) containing the normal human CFTR cDNA to the nasal and bronchial epithelium of four individuals with cystic fibrosis (CF). We show that this vector can express the CFTR cDNA in the CF respiratory epithelium in vivo. With doses up to 2 × 109 pfu, there was no recombination/complementation or shedding of the vector or rise of neutralizing antibody titres. At 2 × 109 pfu, a transient systemic and pulmonary syndrome was observed, possibly mediated by interleukin-6. Follow-up at 6–12 months demonstrated no long term adverse effects. Thus, it is feasible to use an adenovirus vector to transfer and express the CFTR cDNA in the respiratory epithelium of individuals with CF. Correction of the CF phenotype of the airway epithelium might be achieved with this strategy.

Intratumoral Spread of Wild-Type Adenovirus Is Limited After Local Injection of Human Xenograft Tumors: Virus Persists and Spreads Systemically at Late Time Points

Oncolytic replicating adenoviruses are a promising new modality for the treatment of cancer. Despite the assumed biologic advantage of continued viral replication and spread from infected to uninfected cancer cells, early clinical trials demonstrate that the efficacy of current vectors is limited. In xenograft tumor models using immune-incompetent mice, wild-type adenovirus is also rarely able to eradicate established tumors. This suggests that innate immune mechanisms may clear the virus or that barriers within the tumor prevent viral spread. The aim of this study was to evaluate the kinetics of viral distribution and spread after intratumoral injection of virus in a human tumor xenograft model. After intratumoral injection of wild-type virus, high levels of titratable virus persisted within the xenograft tumors for at least 8 weeks. Virus distribution within the tumors as determined by immunohistochemistry was patchy, and virus-infected cells appeared to be flanked by tumor necrosis and connective tissue. The close proximity of virus-infected cells to the tumor-supporting structure, which is of murine origin, was clearly demonstrated using a DNA probe that specifically hybridizes to the B1 murine DNA repeat. Importantly, although virus was cleared from the circulation 6 hr after intratumoral injection, after 4 weeks systemic spread of virus was detected. In addition, vessels of infected tumors were surrounded by necrosis and an advancing rim of virus-infected tumor cells, suggesting reinfection of the xenograft tumor through the vasculature. These data suggest that human adenoviral spread within tumor xenografts is impaired by murine tumor-supporting structures. In addition, there is evidence for continued viral replication within the tumor, with subsequent systemic dissemination and reinfection of tumors via the tumor vasculature. Despite the limitations of immune-incompetent models, an understanding of the interactions between the virus and the tumor-bearing host is important in the design of effective therapies.

Wild-Type Adenovirus Decreases Tumor Xenograft Growth, but Despite Viral Persistence Complete Tumor Responses Are Rarely Achieved— Deletion of the Viral E1b-19-kD Gene Increases the Viral Oncolytic Effect

Strategies to target viral replication to tumor cells hold great promise for the treatment of cancer, but even with replicating adenoviruses complete tumor responses are rarely achieved. To evaluate replicating adenoviral vectors, we have used A549 human lung cancer nude mouse xenografts as a model system. Intratumoral injection of wild-type adenovirus (Ad309) significantly reduced tumor growth from day 14 (p = 0.04) onward; however, tumor volumes reached a plateau at day 50. At 100 days, high levels of titratable virus were present within persistent viable tumors. In contrast to viral injection into established tumors, when tumor cells were infected in vitro with wild-type virus and then mixed with uninfected tumor cells, 1% of infected cells was sufficient to prevent tumor establishment. An E1b-19kD-deleted viral mutant (Ad337) was more efficient than Ad309 in this cell-mixing model. Just 1 cell in 1000 infected with Ad337 prevented tumor growth. However, although better than wild-type virus, Ad337 was unable to eradicate established flank tumors. These data suggest that although replicating adenoviruses exhibit significant oncolytic activity, barriers within the established tumor, such as connective tissue and tumor matrix, may limit the spread of virus. Strategies to enhance viral spread through established tumors are therefore likely to greatly improve the therapeutic efficacy of replicating adenoviruses.

Deletion of the Adenoviral E1b-19kD Gene Enhances Tumor Cell Killing of a Replicating Adenoviral Vector

Replicating adenoviral vectors are a promising new modality for cancer treatment and clinical trials with such vectors are ongoing. Targeting these vectors to cancer cells has been the focus of research. However, even if perfect targeting were to be achieved, a vector still must effectively kill cancer cells and spread throughout the bulk of the tumor. The adenoviral E1b-19kD protein is a potent inhibitor of apoptosis and may therefore compromise the therapeutic efficacy of an adenoviral vector. In this study we have investigated if an E1b-19kD gene deletion could improve the ability of a replicating adenoviral vector to spread through and kill cancer cells. In several lung cancer cell lines an E1b-19kD-deleted virus (Ad337) induced substantially more apoptosis than did a wild-type virus (Ad309), and tumor cell survival was significantly reduced in three of four cell lines. In addition, the apoptotic effects of cisplatin or paclitaxel were augmented by Ad337, but inhibited by wild-type virus. The number of infectious virus particles in the supernatant of infected cells was increased with Ad337 compared with wild-type virus, indicating enhanced early viral release. Ad337, in contrast to Ad309, induced significantly larger plaques after infection of A549 cells. This well-described large plaque phenotype of an E1b-19kD mutant virus is likely the result of early viral release and enhanced cell-to-cell viral spread. Loss of E1b-19kD function caused only minor cell line-specific increase or decrease in viral yield. We conclude that deletion of the E1b-19kD gene may enhance the tumoricidal effects of a replicating adenoviral vector.

Targeting the Replication of Adenoviral Gene Therapy Vectors to Lung Cancer Cells: The Importance of the Adenoviral E1b-55kD Gene

It has been proposed that an adenovirus with the E1b-55kD gene deleted has a selective advantage in replicating in cancer cells that have mutations in the p53 gene (Bischoff et al., 1996). We have explored this hypothesis in several lung cancer cell lines, and evaluated potential mechanisms that might regulate the replication of Ad338, an E1b-55kD-deleted virus, with the objective of developing a rational approach for targeting gene therapy to lung tumors. Our data show that Ad338 replicates poorly in three lung cancer cell lines with various p53 mutations (H441, H446, and Calu1), yet this virus replicates to a high level in a lung cancer cell line with wild-type p53 (A549) and in a normal lung fibroblast line (IMR90). Viral DNA replication, expression of viral proteins, and shutoff of host cell proteins were not important variables in limiting the replication of the E1b-55kD-deleted virus. However, the cell lines resistant to host cell protein shutoff were also the most resistant to the cytopathic effect induced by mutant and wild-type virus and the only cells to survive for 8 days following infection. The E1b-55kD protein clearly has an important role in viral replication beyond its interaction with p53. Thus, an E1b-55kD-deleted virus cannot be used to specifically target viral replication to p53-mutated lung cancer cells.

Late Expression of p53 from a Replicating Adenovirus Improves Tumor Cell Killing and Is More Tumor Cell Specific than Expression of the Adenoviral Death Protein

Gene transfer of p53 induces cell death in most cancer cells, and replication-defective adenoviral vectors expressing p53 are being evaluated in clinical trials. However, low transduction efficiency limits the efficacy of replication-defective vector systems for cancer therapy. The use of replication-competent vectors for gene delivery may have several advantages, holding the potential to multiply and spread the therapeutic agent after infection of only a few cells. However, expression of a transgene may adversely affect viral replication. We have constructed a replicating adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle and also contains a deletion of the adenoviral death protein (ADP). Adp53rc-infected cancer cells demonstrated high levels of p53 expression in parallel with the late expression pattern of the adenoviral fiber protein. p53 expression late in the viral life cycle did not impair effective virus propagation. Survival of several lung cancer cell lines was significantly diminished after infection with Adp53rc, compared with an identical p53-negative control virus. p53 expression also improved virus release and spread. Interestingly, p53 was more cytotoxic than the ADP in cancer cells but less cytotoxic than the ADP in normal cells. In conclusion, late expression of p53 from a replicating virus improves tumor cell killing and viral spread without impairing viral replication. In addition, in combination with a deletion of the ADP, specificity of tumor cell killing is improved.

Angiopoietin-1 increases survival and reduces the development of lung edema induced by endotoxin administration in a murine model of acute lung injury.

Objective:To evaluate the effect of angiopoietin-1, an angiogenic growth factor, on lung capillary leakage and survival in a murine model of acute lung injury. Design:Laboratory investigation. Setting:Research laboratory at New York University School of Medicine and Department of Veterans Affairs, NY Harbor Healthcare System. Subjects:C57BL/6 mice weighing 18–20 g, susceptible to endotoxin-induced acute lung injury. Interventions:Acute lung injury was induced in C57BL/6 mice by the intraperitoneal administration of endotoxin. The effects of angiopoietin-1, expressed from a nonreplicating E1a-deleted adenovirus containing the angiopoietin-1 complementary DNA (AdAng1), on survival and lung injury were evaluated. An E1a-deleted adenovirus that does not contain a transgene (Ad312) and phosphate-buffered saline were used as controls. Measurements and Main Results:Angiopoietin-1 protein was detected by immunoblotting in the serum of mice that received an intraperitoneal injection of AdAng1 but not in mice that received the control virus Ad312. When compared with control groups, mice that received AdAng1 5 days before endotoxin administration had improved survival and significantly less protein leakage from the circulation into the lungs, as detected by quantitative spectrophotometric measurements of Evans blue dye. Furthermore, when compared with controls, histopathology and immunostaining of lungs against CD31 and smooth muscle actin suggested preservation of vascular integrity and decreased tissue damage in mice pretreated with AdAng1. When endotoxin administration preceded infection with AdAng1 by 3 hrs, no benefit was observed. Conclusions:These data show that adenoviral mediated expression of angiopoietin-1 can protect against the development of lung capillary protein leak and decrease the mortality induced by endotoxin. However, the timing of AdAng1 administration in relation to the onset of lung injury may be critical.

Dysregulation of angiopoietin-1 plays a mechanistic role in the pathogenesis of cerebral malaria

Angiopoietin-1 is dysregulated in pediatric severe malaria, and targeting it prevents vascular dysfunction and death in a mouse model of cerebral malaria. Bolstering the host to beat malaria Cerebral malaria is a devastating disease associated with high death rates and brain injury despite the use of potent antimalarials. Understanding the role of the host response in determining the ability of the host to survive a severe malaria infection may enable development of host-based therapeutics to prevent infection-induced death. Higgins et al. now demonstrate how the loss of a key host vascular protective protein, angiopoietin-1, is associated with severe and fatal malaria in children and in a mouse model. Administering angiopoietin-1 to mice with cerebral malaria reinforced the blood-brain barrier and improved survival, even when initiated late in disease. Cerebral malaria is a leading cause of global morbidity and mortality. Interventions targeting the underlying pathophysiology of cerebral malaria may improve outcomes compared to treatment with antimalarials alone. Microvascular leak plays an important role in the pathogenesis of cerebral malaria. The angiopoietin (Ang)–Tie-2 system is a critical regulator of vascular function. We show that Ang-1 expression and soluble Tie-2 expression were associated with disease severity and outcome in a prospective study of Ugandan children with severe malaria and in a preclinical murine model of experimental cerebral malaria. Ang-1 was necessary for maintenance of vascular integrity and survival in a mouse model of cerebral malaria. Therapeutic administration of Ang-1 preserved blood-brain barrier integrity and, in combination with artesunate treatment, improved survival beyond that with artesunate alone. These data define a role for dysregulation of the Ang–Tie-2 axis in the pathogenesis of cerebral malaria and support the evaluation of Ang–Tie-2–based interventions as potential adjunctive therapies for treating severe malaria.

Expressed cell-penetrating peptides can induce a bystander effect, but passage through the secretory pathway reduces protein transduction activity.

Despite advances in vector technology, inefficient gene transfer still limits clinical efficacy of cancer gene therapy. Cell-penetrating peptides (CPPs), such as the basic domain of the transactivator of transcription (Tat) protein of HIV-1, are internalized by intact cells and have been used to deliver purified recombinant proteins. A combination of gene therapy with protein transduction technology could induce a strong bystander effect and represent a platform to deliver proteins to target cells. However, whether expressed CPP can facilitate intercellular trafficking, i.e., a bystander effect, is controversial. Our data suggest that expressed fusion proteins that contain the basic domain of Tat do not induce a detectable bystander effect. However, Tat-fusion proteins that also contain a secretory signal peptide (SP) can induce a bystander effect in vitro, although the in vivo effect is small. Surprisingly, despite the presence of a SP, the bystander effect does not seem to be related to secretion of the fusion protein. In fact, Tat-fusion proteins are secreted very inefficiently, and protein transduction seems largely mediated by fusion proteins that are released by cell lysis. Modification of Tat can improve secretion efficacy and prevent cleavage by the endoprotease furin, but passage through the secretory pathway is associated with reduced transduction activity of Tat-fusion proteins.

“Man's best friend”: a new model system for cancer therapeutics?

The use of chemotherapeutic agents has not led to high cure rates in the treatment of most solid tumors, lung cancer and pancreatic cancer being good examples. One major obstacle is the inability to deliver adequate levels of drug to the tumor without giving rise to serious toxic side effects in other organs. Conditionally replicating viruses are therapeutic agents that replicate specifically within the target tumor. Two main strategies are being followed to achieve tumor specific replication of adenoviral gene therapy vectors. The first relies on the use of tumor-specific promoter elements that can be inserted into the virus to limit the expression of viral genes controlling replication to tumor cells. The second strategy limits viral replication to dividing cells, by mutation or deletion of viral genes that encode proteins that interact with cell-cycle regulating proteins. Examples include deletion of the E1b-55kD gene that normally binds to p53, and modification of the E1a gene that interacts with Rb (see1 for review).