Faculty Bibliography

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

Cell penetrating peptides (CPPs) are widely used to deliver proteins and other macromolecules into cells. Generally, CPPs are either synthesized and coupled to the therapeutic payload or expressed in bacteria as fusion proteins followed by various purification steps. However, despite great interest in this technology, difficulties with production, purification and unfavorable pharmacokinetics are still limiting its clinical success. As an alternative approach, expression of CPPs that support intercellular transfer of fused proteins could avoid production and purification needs and serve as a tool to deliver therapeutic proteins. We have previously reported that the basic domain of HIV Tat, fused to fluorescent proteins can support intercellular transfer, but only when coupled to a secretory signal peptide (SP). However, SP-Tat fusion proteins are very inefficiently secreted and transduction seems mostly mediated by fusion proteins released by means other than secretion through the classic pathway. We also demonstrated that a modified Tat-based CPP (Tatm) is secreted much more efficiently, but its transduction activity was greatly reduced compared to Tat. We now show that an elongated 3x repeat sequence of Tatm (Tatm3x) enhances transduction activity. Secretion activity is greatly improved compared to Tat, although somewhat reduced when compared to Tatm. Expressed SP-Tatm3x fusion proteins are localized in the ER and cytoplasm and are secreted mainly via the classical pathway. Fusion proteins that are released through cell lysis have greatly improved transduction activity compared to SP-Tatm. Based on co-culture and mixing experiments, expressed SP-Tatm3x supports intercellular transport of fused fluorescent proteins in vitro. As previously shown for SP-Tatm fusion proteins, travel through the secretory pathway also reduces transduction activity of SP-Tatm3x fusion proteins. In conclusion, expressed SP-Tatm3x displays improved secretion and transduction activity and represents an important step forward in the development of a CPP that supports intercellular transport of fused proteins

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

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

Clinical efficacy of adenovirus-mediated cancer gene therapy has been limited thus far. To improve its oncolytic effect, a replication-competent adenoviral vector was previously constructed to express high levels of p53 at a late time point in the viral life cycle. p53 expression from this vector improved tumor cell killing and viral spread in vitro. However, p53 function is antagonized by cellular mdm2 and adenoviral E1b-55kD, both of which are known to bind to and inactivate p53. Therefore, a new vector (Adp53W23S) that expresses a modified p53 transgene, which does not bind to E1b-55kd and mdm2, was constructed. The modified p53 protein was demonstrated to have a substantially prolonged half-life, and its localization was predominantly nuclear. Viral replication was unaffected by expression of the modified p53 and cancer cell killing was improved in vitro. However, in a xenograft model, efficacy was not significantly different from control virus. In conclusion, expression of a degradation-resistant p53 transgene late in the life cycle of a replication-competent adenovirus improves p53 stability and cancer cell killing in vitro. However, other factors, such as the adenoviral E1b-19kD and E1a proteins, which oppose p53 function, and limitations to viral spread need to be addressed to further improve in vivo efficacy

Successful cancer therapy using replicating viral vectors relies on the spread of virus from infected to uninfected cells. To date, there has been limited clinical success in the use of replicating adenoviruses. In animal models, established xenograft tumors are rarely eliminated despite the persistence of high viral titers within the tumor. Hypoxia is a prevalent characteristic of solid tumors, whereas adenovirus naturally infects tissues exposed to ambient oxygen concentrations. Here, we report that hypoxia (1% oxygen) reduces adenoviral replication in H1299 and A549 lung cancer cells, BxPC-3 pancreatic cancer cells, LNCaP prostate cancer cells and HCT116 colon cancer cells. However, hypoxia does not reduce cell viability or restrict S-phase entry. Importantly, the production of E1a and fiber proteins under hypoxic conditions was substantially decreased at 24 and 48 h compared to room air controls. In contrast, Northern analysis showed similar levels of E1a mRNA in room air and hypoxic conditions. In conclusion, a level of hypoxia similar to that found within solid tumors reduces the replication of adenoviral vectors by reduction of viral protein expression without a reduction in mRNA levels. To further improve oncolytic therapy using a replicating adenovirus, it is important to understand the mechanism through which hypoxia and the virus interact to control expression of viral and cellular proteins, and consequently to develop means to overcome decreased viral production in hypoxic conditions

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.