Kimiko Yoshida

Adenovirus-mediated gene transfer of interferon α improves dimethylnitrosamine-induced liver cirrhosis in rat model

Several lines of evidence suggest that interferon (IFN)-α is effective in suppression of liver cirrhosis (LC) as well as hepatitis C virus (HCV) infection, which is a major cause of LC in Japan. However, IFN-α often causes systemic toxicity such as flu-like symptoms, which precludes the IFN-α dose escalation required for clinical efficacy. Since IFN-α is rapidly degraded in the blood circulation, only a small amount of subcutaneously injected IFN-α protein can reach the target organ, the liver. It is expected that on-site IFN-α production in the liver overcomes the limitation of the conventional parenteral IFN-α administration. An adenovirus vector expressing the rat IFN-α gene (AxCA-rIFN) was injected intravenously into rats with dimethylnitrosamine-induced LC. While the subcutaneous IFN-α protein injection led to a transient elevation of the cytokine both in the liver and serum, the vector-mediated IFN-α gene transduction induced a significant amount of IFN-α detected in the liver but not in the serum. The injection of AxCA-rIFN prevented the progression of the rat LC, and improved the survival rate of the treated rats. Although no significant toxicity was noted in the animals, we showed that IFN-α gene expression in the liver can be efficiently downregulated by the Cre/loxP-mediated shut-off system, in case the IFN-α overdose becomes a problem. The study suggested for the first time the advantage and feasibility of IFN-α gene therapy for LC.

Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob

A conditionally replicative adenovirus is a novel anticancer agent designed to replicate selectively in tumor cells. However, a leak of the virus into systemic circulation from the tumors often causes ectopic infection of various organs. Therefore, suppression of naive viral tropism and addition of tumor-targeting potential are necessary to secure patient safety and increase the therapeutic effect of an oncolytic adenovirus in the clinical setting. We have recently developed a direct selection method of targeted vector from a random peptide library displayed on an adenoviral fiber knob to overcome the limitation that many cell type-specific ligands for targeted adenovirus vectors are not known. Here we examined whether the addition of a tumor-targeting ligand to a replication-competent adenovirus ablated for naive tropism enhances its therapeutic index. First, a peptide-display adenovirus library was screened on a pancreatic cancer cell line (AsPC-1), and particular peptide sequences were selected. The replication-competent adenovirus displaying the selected ligand (AdΔCAR-SYE) showed higher oncolytic potency in several other pancreatic caner cell lines as well as AsPC-1 compared with the untargeted adenovirus (AdΔCAR). An intratumoral injection of AdΔCAR-SYE significantly suppressed the growth of AsPC-1 subcutaneous tumors, and an analysis of adenovirus titer in the tumors revealed an effective replication of the virus in the tumors. Ectopic liver gene transduction following the intratumoral injection of AdΔCAR-SYE was not increased compared with the AdΔCAR. The results showed that a tumor-targeting strategy using an adenovirus library is promising for optimizing the safety and efficacy of oncolytic adenovirus therapy.

Interferon-α and antisense K-ras RNA combination gene therapy against pancreatic cancer

Interferon alpha (IFN‐α) is used worldwide for the treatment of a variety of cancers. For pancreatic cancer, recent clinical trials using IFN‐α in combination with standard chemotherapeutic drugs showed some antitumor activity of the cytokine, but the effect was not significant enough to enlist pancreatic cancer as a clinically effective target of IFN‐α. In general, an improved therapeutic effect and safety are expected for cytokine therapy when given in a gene therapy context, because the technology would allow increased local concentrations of this cytokine in the target sites. In this study, we first examined the antiproliferative effect of IFN‐α gene transduction into pancreatic cancer cells. The expression of IFN‐α effectively induced growth suppression and cell death in pancreatic cancer cells, an effect which appeared to be more prominent when compared with other types of cancers and normal cells. Another strategy we have been developing for pancreatic cancer targets its characteristic genetic aberration, K‐ras point mutation, and we reported that the expression of antisense K‐ras RNA significantly suppressed the growth of pancreatic cancer cells. When these two gene therapy strategies are combined, the expression of antisense K‐ras RNA significantly enhanced IFN‐α‐induced cell death (1.3‐ to 3.5‐fold), and suppressed subcutaneous growth of pancreatic cancer cells in mice. Because the 2′,5′‐oligoadenylate synthetase/RNase L pathway, which is regulated by IFN and induces apoptosis of cells, is activated by double‐strand RNA, it is plausible that the double‐strand RNA formed by antisene and endogeneous K‐ras RNA enhanced the antitumor activity of IFN‐α. This study suggested that the combination of IFN‐α and antisense K‐ras RNA is a promising gene therapy strategy against pancreatic cancer. Copyright

Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob.

Targeting of gene transfer at the level of cell entry is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success because proper targeting ligand–receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

Local interferon-α gene therapy elicits systemic immunity in a syngeneic pancreatic cancer model in hamster

The interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis and immunomodulation. We previously examined the two antitumor mechanisms, taking advantage of the fact that IFN‐α did not show cross‐species activity in its in vivo effect. In a nude mouse subcutaneous xenograft model using human pancreatic cancer cells, the expression of human IFN‐α effectively induced cell death of human pancreatic cancer cells, whereas mouse IFN‐α augmented antitumor immunity by stimulation of natural killer cells. Here, we extended our investigation to a syngeneic pancreatic cancer model, so that the integrated antitumor activity of local IFN‐α gene therapy, including the antiproliferative, proapoptotic, antiangiogeneic and immunomodulatory effects, can be evaluated rigorously. When a recombinant hamster IFN‐α adenovirus was injected into syngeneic subcutaneous tumors of hamster pancreatic cancer (PGHAM‐1) cells in Syrian hamster, tumor growth was significantly suppressed due to cell death and T cell‐ and natural killer cell‐mediated antitumor immunity. Moreover, in this case, tumor regression was observed not only for the injected subcutaneous tumors but also for the untreated tumors both in the peritoneal cavity and at distant sites. No significant systemic toxicity was observed in the treated hamsters. Moreover, the subcutaneous rechallenge of PGHAM‐1 cells was rejected in three of four cured hamsters from the initial tumor challenge. This study further demonstrated that local IFN‐α gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its multiple mechanisms of antitumor activity and its lack of significant toxicity. (Cancer Sci 2007; 98: 455–463))

A simple and efficient method for constructing an adenoviral cDNA expression library

cDNA expression cloning is a powerful method for the identification of genes that are able to confer a selectable phenotype on specific cell types. An adenovirus vector is characterized by several advantages over plasmid DNA and retroviral vector-mediated gene transfer, such as broad host range and high infectivity. However, an expression cloning protocol using the adenovirus vector has not been reported. We describe here a simple and efficient method for constructing adenovirus cDNA expression libraries based on Cre-lox-mediated in vitro recombination between adenoviral shuttle plasmid cDNA libraries and adenoviral genomic DNA tagged with terminal protein. In a model experiment, EGFP clones present at the frequency of 0.003% in the shuttle plasmid library could be efficiently converted to adenoviral vector in a 6-cm dish under optimized conditions, indicating that high-complexity libraries harboring low-abundance cDNAs can be produced. The efficiency of this system was demonstrated by the isolation of cDNA for CD2 (frequency less than 1 in 0.3 x 10(4) transcripts in T cells) from the human T cells. This effective and versatile method can facilitate the functional identification of genes for a variety of purposes.

Allogeneic MHC Gene Transfer Enhances an Effective Antitumor Immunity in the Early Period of Autologous Hematopoietic Stem Cell Transplantation

Purpose: In autologous hematopoietic stem cell transplantation (HSCT), lymphopenia-induced homeostatic proliferation of T cells is driven by the recognition of self-antigens, and there is an opportunity to skew the T-cell repertoire during the T-cell recovery by engaging tumor-associated antigens, leading to a break of tolerance against tumors. However, the homeostatic proliferation–driven antitumor responses seem to decline rapidly in association with tumor growth. We hypothesized that a tumor-specific immune response induced by an immune gene therapy could enhance and sustain homeostatic proliferation–induced antitumor immunity. Experimental Design: The antitumor effect of allogeneic MHC (alloMHC) gene transfer was examined at the early phase of the immune reconstitution after syngeneic HSCT. Results: Syngeneic HSCT showed significant tumor growth inhibition of syngeneic colon cancer cells within a period of 30 days; however, the tumor then resumed rapid growth and the survival of the mice was not prolonged. In contrast, when the alloMHC plasmid was intratumorally injected at the early phase after syngeneic HSCT, the established tumors were markedly regressed and the survival of recipient mice was prolonged without significant toxicities, whereas no survival advantage was recognized in recipient mice injected with a control plasmid. This tumor suppression was evident even in the other tumors that were not injected with the alloMHC plasmid. The antitumor response was characterized by the development of tumor-specific T cell– and natural killer cell–mediated cytotoxicities. Conclusion: The results suggest the efficacy and safety of integrating intratumoral alloMHC gene transfer with an autologous HSCT for the treatment of solid cancers.

Development of peritoneal tumor-targeting vector by in vivo screening with a random peptide-displaying adenovirus library.

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.

Intratumoral interferon-α gene transfer enhances tumor immunity after allogeneic hematopoietic stem cell transplantation

One of the major challenges in the treatment of solid cancers by allogenic hematopoietic stem cell transfer (alloHSCT) is the specific enhancement of antitumor immunity. Interferon (IFN) is a cytokine with pleiotropic biological functions including an immunomoduration, and our preclinical studies have shown that an intratumoral IFN-α gene transfer induced strong local tumor control and systemic tumor-specific immunity. In the present study, we examined whether the IFN-α gene transfer could enhance recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. First, when a mouse IFN-α adenovirus vector (Ad-mIFN) was injected into subcutaneous xenografts of syngeneic renal and colon cancer cells, tumor growth was significantly suppressed in a dose-dependent manner. A significant tumor cell death and infiltration of immune cells was recognized in the Ad-mIFN-injected tumors, and the dendrtic cells isolated from the tumors showed a strong Th1-oriented response. The antitumor effect of Ad-mIFN was then examined in a murine model of minor histocompatibility antigen-mismatched alloHSCT. The intratumoral IFN-α gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors which did not receive the vector injection. A cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to IFN-α. Graft-versus-host disease was not exacerbated serologically or clinically in the mice treated with IFN-α. This combination strategy deserves evaluation in future clinical trials for human solid cancers.

Embryonic stem cell transplantation correlates with endogenous neurogenin 3 expression and pancreas regeneration in streptozotocin-injured mice.

Pancreatic β cell regeneration remains poorly understood, yet stimulation of adult β cell neogenesis could lead to therapies for type 1 and type 2 diabetes. We studied the effect of embryonic stem (ES) cell transplantation on pancreas regeneration following β cell injury. Female Balb/c nude mice were treated with streptozotocin to induce hyperglycemia and received an ES cell transplant 24 hr later beneath the renal capsule. Transplantation of ES cells prevented hyperglycemia in a subset of mice, maintaining euglycemia and mild glucose tolerance up to 5 weeks. Pancreata of euglycemic mice showed histological evidence of β cell regeneration and expression of pancreas and duodenum transcription factor-1 (PDX-1) and neurogenin 3 (Ngn3) in ductal epithelium. Cell tracing analysis indicated that significant β cell neogenesis from progenitor cells occurred between 2 to 3 weeks following injury in ES cell-transplanted mice but not in sham-transplanted animals. Significantly, whereas pancreas-localized ES cells or their derivatives were adjacent to sites of regeneration, neogenic pancreatic epithelia, including Ngn3+ cells, were endogenous. In conclusion, transplanted ES cells can migrate to the injured pancreas. Transplantation is associated with enhanced endogenous regeneration characterized by expression of Ngn3 and increased β cell differentiation from endogenous progenitor cells. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.