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

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.

A novel cell-penetrating peptide to facilitate intercellular transport of fused proteins.

Cell-based delivery of cell penetrating peptides (CPPs) could represent a new platform for intracellular peptide delivery to local tissues. Expressed CPPs, coupled to a secretory signal peptide (SP), can support intercellular transport. However, low secretion efficiency, which may correlate with the positive charge of most CPPs, has emerged as one of the main impediments for efficient intercellular transport. We have reported that a modified Tat-based CPP (Tatm) with reduced positive charge is secreted efficiently, but its transduction activity was greatly reduced. We now show that a triple repeat of Tatm (Tatm3x) with an elongated α-helical amphipathic structure enhances transduction activity and simultaneously retains its secretion efficacy, although passage through the secretory pathway reduces its cell-penetrating activity. SP-Tatm3x supports intercellular transport of fused fluorescent proteins, as well as cell entry and function of a pro-apoptotic peptide. In addition, SP-Tatm3x largely escapes RNA inhibition, which is identified as another potential impediment to CPP-mediated intercellular transport. Expression of SP-Tatm3x in heparan sulfate proteoglycan-negative cells further improves its transduction activity. These results demonstrate the feasibility of intercellular transport of proteins, but further work is needed to better understand the reduction of cell-penetrating activity associated with secretion of CPP-fusion proteins.

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.

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.

Human matrix metalloproteinase-8 gene delivery increases the oncolytic activity of a replicating adenovirus.

The success of replicating adenoviruses for cancer therapy is limited by inefficient virus delivery and poor distribution within the tumor mass. Stromal matrix within the tumor may hinder the free cell-to-cell spread of the virus. In this study, in vitro cell culture experiments showed that collagen I blocked the passage of an adenoviral vector through a membrane. On the basis of reports of the effective collagen I-degrading activity of matrix metalloproteinase-8 (MMP-8), we constructed an adenovirus to express the MMP-8 transgene (AdMMP8). A549 cells infected in vitro with AdMMP8 did not show altered growth but were able to modify a fibrillar collagen substrate to allow viral diffusion. Further, AdMMP8 did not affect replication of the wild-type virus (Adwt300). Established human A549 lung cancer and BxPC-3 pancreatic cancer xenograft tumors that were injected with Adwt300 together with the non-replicating AdMMP8 virus showed significantly reduced growth compared with control tumors. Histochemical analysis showed reduced amounts of collagen within necrotic areas of MMP-8-injected tumors compared with controls. These results demonstrate that intra-tumoral expression of MMP-8 is a possible strategy for improving viral spread and improving the oncolytic activity of replicating adenovirus.

Drug evaluation: Reolysin--wild-type reovirus as a cancer therapeutic.

Oncolytics Biotech is developing an oncolytic reovirus therapy (Reolysin, Reosyn) for the potential treatment of a variety of Ras-mediated cancers, including glioma and medulloblastoma, pancreatic, prostate, breast, lung, colon, bladder, ovarian and hematological cancers, and melanoma and childhood sarcoma. Phase I/II clinical trials in recurrent malignant glioma began in 2002.

The potential impact of hypoxia on the success of oncolytic virotherapy.

The biological properties of oncolytic viruses, in particular the ability to replicate within a tumor cell and then spread from cell to cell are highly desirable features for cancer therapy. However. several features of the tumor environment may not be conducive for efficient viral spread. Hypoxia is an important feature of solid tumors and the ability of viruses to replicate in hypoxic conditions may be a critical determinant in the success or failure of virotherapy. Turning off protein translation is a central process in the cellular adaptation to many types of stress, including viral infection and hypoxia. How a specific virus has evolved to overcome the cellular mechanisms that regulate translation in the cell under stress may be critical for the success of virotherapy.

Sindbis virus--an effective targeted cancer therapeutic.

Viral therapies for cancer therapy have many potential positive attributes. These include the ability to specifically infect targeted cells, specifically express toxic or immune-enhancing genes, and the ability to specifically replicate within a tumor cell. Despite these biological advantages, efficacy to date has been limited. A recent report demonstrates that the Sindbis virus has remarkable properties in three challenging areas of gene therapy - specificity, efficacy and delivery, suggesting that Sindbis has the potential to become an important gene therapy vector for cancer therapy.

Impact of E1a modifications on tumor-selective adenoviral replication and toxicity.

Replicating adenoviral vectors are capable of multiplying up to a thousandfold in the target cell, a property that might prove to be of tremendous potential for cancer therapy. However, restricting viral replication and toxicity to cancer cells is essential to optimize safety. It has been proposed that modifications of the E1a protein that impair binding to Rb or p300 will prevent S-phase induction in normal cells, resulting in selective viral replication in tumor cells. However, it remains uncertain which of the several possible E1a modifications would be most effective at protecting normal cells without compromising the oncolytic effect of the vector. In this study, we have expressed several E1a-deletion mutants at high levels using the CMV promoter and tested them for their ability to facilitate S-phase induction, viral replication, and cytotoxicity in both normal and cancer cells. Deletion of the Rb-binding domain within E1a only slightly decreased the ability of the virus to induce S phase in growth-arrested cells. The effect of this deletion on viral replication and cytotoxicity was variable. There was reduced cytotoxicity in normal bronchial epithelial cells; however, in some normal cell types there was equal viral replication and cytotoxicity compared with wild type. Deletions in both the N-terminus and the Rb-binding domain were required to block S-phase induction effectively in growth-arrested normal cells; in addition, this virus demonstrated reduced viral replication and cytotoxicity in normal cells. An equally favorable replication and cytotoxicity profile was induced by a virus expressing E1a that is incapable of binding to the transcriptional adapter motif (TRAM) of p300. All viruses were equally cytotoxic to cancer cells compared with wild-type virus. In conclusion, deletion of the Rb-binding site alone within E1a may not be the most efficacious means of targeting viral replication and toxicity. However, deletion within the N-terminus in conjunction with a deletion within the Rb-binding domain, or deletion of the p300-TRAM binding domain, induces a more favorable cytotoxicity profile.

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.

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.