Paul N. Reynolds

Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo.

The promise of gene therapy for health care will not be realized until gene delivery systems are capable of achieving efficient, cell-specific gene delivery in vivo. Here we describe an adenoviral system for achieving cell-specific transgene expression in pulmonary endothelium. The combination of transductional targeting to a pulmonary endothelial marker (angiotensin-converting enzyme, ACE) and an endothelial-specific promoter (for vascular endothelial growth factor receptor type 1, flt-1) resulted in a synergistic, 300,000-fold improvement in the selectivity of transgene expression for lung versus the usual site of vector sequestration, the liver. This combined approach should be useful for the design of other gene delivery systems.

Insertion of an RGD motif into the HI loop of adenovirus fiber protein alters the distribution of transgene expression of the systemically administered vector

Adenoviral vectors are attractive gene delivery vehicles, but their in vivo utility is reduced by lack of cell-specific infection. Tropism modification of the virion by genetic manipulation of capsid proteins is an attractive strategy to achieve targeted transduction. However, no genetic targeting strategies have yet been shown to modify the distribution of transgene expression following systemic administration of vector. This is an essential requirement if such approaches are to form a basis for further vector develop- ment. In this report we present data showing that insertion of a RGD motif into the HI loop of the adenoviral fiber knob results in a significant change in transgene expression profile following intravenous administration. The key finding that a motif in the HI loop is available for cellular interaction when administered systemically means that such modifications can be rationally considered as a foundation upon which further genetic modifications can be superimposed for targeted systemic gene therapy.

Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin-targeted adenoviral vectors.

In this study we analyzed two ways of retargeting of Ad-vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carcinoma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispecific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) genetically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both αv integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-containing peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR-independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma.

Midkine and cyclooxygenase-2 promoters are promising for adenoviral vector gene delivery of pancreatic carcinoma

Midkine (MK), a heparin binding growth factor, and cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandin, are both up-regulated at the mRNA or protein level in many human malignant tumors. Here, we investigated the tumor specificity of both MK and COX-2 promoters in human pancreatic cancer, with the aim to improve the selectivity of therapeutic gene expression. We constructed recombinant adenoviral (Ad) vectors containing either the luciferase (Luc) reporter gene under the control of the COX-2 or MK promoter or the herpes simplex virus thymidine kinase (HSV Tk) gene under the control of the COX-2 promoter and compared the expression with the cytomegalovirus (CMV) promoter. AdMKLuc achieved moderate to relatively high activity upon infection to both primary and established pancreatic carcinoma cells. Of the two COX-2 promoter regions (COX-2M and COX-2L), both revealed a high activity in primary pancreatic carcinoma cells, whereas in the established pancreatic carcinoma cell lines, COX-2L has an approximately equal high activity compared to CMV. In addition, both AdCOX-2M Tk and AdCOX-2L Tk induced marked cell death in response to ganciclovir (GCV) in three of four established pancreatic carcinoma cell lines. From these results, and because it has been reported that AdMKTk and AdCOX-2L Tk in combination with GCV did not reveal significant liver toxicity, we conclude that the MK as well as the COX-2 promoters are promising tumor-specific promoters for Ad vector–based gene therapy of pancreatic cancer. Cancer Gene Therapy (2001) 8, 990–996

A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism

The efficiency of cancer gene therapy with recombinant adenoviruses based on serotype 5 (Ad5) has been limited partly because of variable, and often low, expression by human primary cancer cells of the primary cellular-receptor which recognizes the knob domain of the fiber protein, the coxsackie and adenovirus receptor (CAR). As a means of circumventing CAR deficiency, Ad vectors have been retargeted by utilizing chimeric fibers possessing knob domains of alternate Ad serotypes. We have reported that ovarian cancer cells possess a primary receptor for Ad3 to which the Ad3 knob binds independently of the CAR-Ad5 knob interaction. Furthermore, an Ad5-based chimeric vector, designated Ad5/3, containing a chimeric fiber proteins possessing the Ad3 knob, demonstrates CAR-independent tropism by virtue of targeting the Ad3 receptor. Based on these findings, we hypothesized that a mosaic virus possessing both the Ad5 knob and the Ad3 knob on the same virion could utilize either primary receptor, resulting in expanded tropism. In this study, we generated a dual-knob mosaic virus by coinfection of 293 cells with Ad5-based and Ad5/3-based vectors. Characterization of the resultant virions confirmed the incorporation of both Ad5 and Ad3 knobs in the same particle. Furthermore, this mosaic virus was able to utilize either receptor, CAR and the Ad3 receptor, for virus attachment to cells. Enhanced Ad infectivity with the mosaic virus was shown in a panel of cell lines, with receptor profiles ranging from CAR-dominant to Ad3 receptor-dominant. Thus, this mosaic virus strategy may offer the potential to improve Ad-based gene therapy approaches by infectivity enhancement and tropism expansion.

Chimeric viral vectors--the best of both worlds?

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The vector systems currently available have not allowed the simultaneous provision of both of these goals. Several groups are now developing chimeric viral vector systems that incorporate the favorable attributes of two different viral vectors. These chimeric vectors might allow the goals for specific gene therapy applications to be realized.

Promoter-controlled infectivity-enhanced conditionally replicative adenoviral vectors for the treatment of gastric cancer

BackgroundGastric cancer is the fourth most common malignancy worldwide. Adenoviral vectors (Ads) have been applied for gene therapy of various cancers because of their high transduction efficiency. However, the infectivity of gastrointestinal cancer cells is poor due to the limited expression of the Coxsackie-adenovirus receptor (CAR). In addition, few tumor-specific promoters (TSPs) have been characterized for this type of cancer. To overcome these problems, we proposed TSP-driven conditionally replicating adenoviruses (CRAds) with fiber modification for virotherapy of gastric cancer.MethodsWe assessed the expression profile of eight TSPs in gastric cancer cell lines and evaluated promising candidates in the context of CRAd cytocidal effect. Next, infectivity enhancement by fiber modifications was analyzed in the gastric cancer cell lines. Finally, we combined the TSP-driven CRAds of choice with the fiber modifications to augment the killing effect.ResultsOut of the eight TSPs, the midkine (MK) and cyclooxygenase-2 (Cox-2M and Cox-2L) promoters showed high transcriptional activity in gastric cancer cells. When these promoters were used in a CRAd context, Cox-2 CRAds elicited the strongest cytocidal effect. The greatest infectivity enhancement was observed with adenoviral vectors displaying 5/3 chimeric fibers. Likewise, Cox-2 CRAds with 5/3 chimeric fibers showed the strongest cytocidal effect in gastric cancer cell lines. Therefore, Cox-2 CRAds with 5/3 chimeric fiber modification showed good selectivity and infectivity in gastric cancer cells to yield enhanced oncolysis.ConclusionsCox-2 CRAds with 5/3 chimeric fiber modification are promising for virotherapy of gastric cancer.

Vascular endothelial growth factor promoter-based conditionally replicative adenoviruses for pan-carcinoma application

Treatment of advanced lung cancer is one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and has an extremely poor prognosis. Thus, new therapeutic approaches are needed. Lung tumor formation depends on angiogenesis in which the vascular endothelial growth factor (VEGF) produced by cancer cells plays a pivotal role. Neutralizing VEGF with a soluble VEGF receptor suppresses tumor growth; however, the anticancer effect with this therapy is weakened after the intratumoral vascular network is completed. In this study, we turned the expression of VEGF by tumors to therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 is controlled by the human VEGF promoter. This virus achieved good levels of viral replication in lung cancer cells and induced a substantial anticancer effect in vitro and in vivo. As a further enhancement, the cancer cell killing effect was improved with tropism modification of the virus to express the knob domain of Ad3, which improved infectivity for cancer cells. These VEGF promoter-based CRAds also showed a significant cell killing effect for various types of cancer lines other than lung cancer. Conversely, the VEGF promoter has low activity in normal tissues, and the CRAd caused no damage to normal bronchial epithelial cells. Since tumor-associated angiogenesis via VEGF signalling is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that VEGF promoter-based CRAds have the potential to be an effective strategy for cancer treatment.

Tachykinin-induced bronchoconstriction in sheep is NK-1 receptor mediated and exhibits tachyphylaxis

Objective: Tachykinins are mediators of airway hyper‐reactivity and inflammation. There is in vitro evidence that ovine responses to tachykinins correlate closely to human responses. This study was designed to characterize the effect of intravenously administered tachykinins on sheep lung resistance in vivo to determine the effect of dose timing on reproducibility of responses and the induction of tachyphylaxis. We then used this information to help further characterize the response with several pharmacological agents.

A novel system for mitigation of ectopic transgene expression induced by adenoviral vectors

Adenoviral (Ad) vectors are good candidates for gene therapy in view of their high in vivo gene delivery efficiency. However, greater control over the tissue distribution of transgene expression is required to avoid potentially deleterious effects in non-target organs. In this regard, the liver is particularly at risk due to the high natural tropism of Ad for this organ, where dose limiting toxicity has been seen due to toxic transgene expression. We hypothesized that the cre/loxP system could be utilized to reduce unintended transgene expression at this site. This concept was tested using an Ad vector (AdLCLLL) carrying a reporter gene cassette in which the promoter and luciferase gene were flanked by LoxP sequences. Co-administration of this vector with a second vector carrying the cre recombinase gene in vitro and in vivo resulted in specific down-regulation of transgene expression. This novel approach thus has the potential to improve the safety of gene therapy strategies that rely upon the delivery of genes which may be hepatotoxic.