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Supplementary Data from Datopotamab Deruxtecan, a Novel TROP2-directed Antibody–drug Conjugate, Demonstrates Potent Antitumor Activity by Efficient Drug Delivery to Tumor Cells

Education reform helps ensure that the education in a given country is of the highest possible quality and is a key area of focus for many developed countries. Japan's education system rates highly and the evolution of education reform is key to ensuring this high level is sustained. School principals play a key role in delivering high-quality education and, indeed, a school principal's leadership correlates with the quality of education available. This is an area of interest for Professor Hirofumi Hamada, School Management Laboratory in the Faculty of Human Sciences, University of Tsukuba, Japan, who is currently exploring the institutional and organisational conditions that affect the leadership of principals. The goal of this research is to help shape education reform in Japan. Hamada believes it is necessary to create an environment of independent and collaborative learning and to value the individuality of children. In addition, problem situations among children are diverse and complex and how schools respond influences the quality of education. Given that the principal is in charge of how a school is run, they play a vital role in assuring the quality of education. Key to Hamada's work is the idea that principals can share their knowledge and leadership with teachers and this creates an environment of shared leadership. He believes that empowering teachers and encouraging them to take on leadership duties is essential. He is working to inform educators that schools require the leadership of principals and for principals to promote a distributed approach to leadership.

Herpes simplex virus (HSV) is a promising tool for developing oncolytic virotherapy. We recently reported a platform for receptor-retargeted oncolytic HSVs that incorporates single-chain antibodies (scFvs) into envelope glycoprotein D (gD) to mediate virus entry via tumor-associated antigens. Therefore, it would be useful to develop an efficient system that can screen antibodies that might mediate HSV entry when they are incorporated as scFvs into gD. We created an HSV-based screening probe by the genetic fusion of a gD mutant with ablated binding capability to the authentic HSV entry receptors and the antibody-binding C domain of streptococcal protein G. This engineered virus failed to enter cells through authentic receptors. In contrast, when this virus was conjugated with an antibody specific to an antigen on the cell membrane, it specifically entered cells expressing the cognate antigen. This virus was used as a probe to identify antibodies that mediate virus entry via recognition of certain molecules on the cell membrane other than authentic receptors. Using this method, we identified an antibody specific to epiregulin (EREG), which has been investigated mainly as a secreted growth factor, but not necessarily for its precursor that is expressed in a transmembrane form. We constructed an scFv from the anti-EREG antibody for insertion into the retargeted HSV platform and found that the recombinant virus entered cells specifically through EREG expressed by the cells. This novel antibody-screening system may contribute to the discovery of unique and unexpected molecules which might be used for the entry of receptor-retargeted oncolytic HSVs.IMPORTANCEThe tropism of the cellular entry of herpes simplex virus (HSV) is dependent upon the binding of the envelope glycoprotein D (gD) to one of its authentic receptors. This can be fully retargeted to other receptors by inserting single-chain antibodies into gD with appropriate modifications. In theory, upon binding to the engineered gD, receptors other than authentic receptors should induce a conformational change in the gD, which activates downstream mechanisms required for viral entry. However, prerequisite factor(s) for receptors to be used as targets of a retargeted virus remain poorly understood and it is difficult to predict which molecules might be suitable for our retargeted HSV construct. Our HSV-based probe will allow unbiased screening of antibody-antigen pairs that mediate virus entry and might be a useful tool to identify suitable pairs for our construct and enhance our understanding of virus-cell interactions during infection by HSV and possibly other viruses.

Trophoblast cell surface antigen 2 (TROP2) is highly expressed on various epithelial tumors and correlates with poor prognosis. We developed the novel TROP2-directed antibody–drug conjugate (ADC), datopotamab deruxtecan (Dato-DXd, DS-1062a), with a potent DNA topoisomerase I inhibitor (DXd), and evaluated its antitumor activity and safety profiles in preclinical models. The pharmacologic activity and mechanism of action of Dato-DXd were investigated in several human cancer cell lines and xenograft mouse models including patient-derived xenograft (PDX) models. Safety profiles were also assessed in rats and cynomolgus monkeys. Dato-DXd bound specifically to TROP2 and was internalized into tumor cells followed by intracellular trafficking to lysosome and DXd release, which induced DNA damage and apoptosis in TROP2-expressing tumor cells in vitro. Dato-DXd exhibited in vivo antitumor activity with DNA damage induced by the accumulated DXd in TROP2-expressing xenograft tumors, but neither isotype control IgG-ADC nor anti-TROP2 antibody had this effect. Dato-DXd also showed potent antitumor activity with tumor regression in several TROP2-expressing xenograft tumors including NSCLC PDX models. Safety profiles of Dato-DXd in rats and cynomolgus monkeys were acceptable. Dato-DXd demonstrated potent antitumor activity against TROP2-expressing tumors by efficient payload delivery into tumors and acceptable safety profiles in preclinical models. These results suggest Dato-DXd could be a valuable treatment option for patients with TROP2-expressing tumors in the clinical setting.

Oncolytic virotherapy is a novel therapeutic modality for malignant diseases that exploits selective viral replication in cancer cells. Herpes simplex virus (HSV) is a promising agent for oncolytic virotherapy due to its broad cell tropism and the identification of mutations that favor its replication in tumor over normal cells. However, these attenuating mutations also tend to limit the potency of current oncolytic HSV vectors that have entered clinical studies. As an alternative, vector retargeting to novel entry receptors has the potential to achieve tumor specificity at the stage of virus entry, eliminating the need for replication-attenuating mutations. Here, we summarize the molecular mechanism of HSV entry and recent advances in the development of fully retargeted HSV vectors for oncolytic virotherapy. Retargeted HSV vectors offer an attractive platform for the creation of a new generation of oncolytic HSV with improved efficacy and specificity.

Malignant melanoma is one of the untreatable cancers in which conventional therapeutic strategies, including chemotherapy, are hardly effective. Therefore, identification of novel therapeutic targets involved in melanoma progression is urgently needed for developing effective therapeutic methods. Overexpression of interleukin-13 receptor α2 (IL13Rα2) is observed in several cancer types including glioma and pancreatic cancer. Although IL13Rα2 is implicated in the progression of various types of cancer, its expression and roles in the malignant melanoma have not yet been elucidated. In the present study, we showed that IL13Rα2 was expressed in approximately 7.5% melanoma patients. While IL13Rα2 expression in human melanoma cells decreased their proliferation in vitro, it promoted in vivo tumour growth and angiogenesis in melanoma xenograft mouse model. We also found that the expression of amphiregulin, a member of the epidermal growth factor (EGF) family, was correlated with IL13Rα2 expression in cultured melanoma cells, xenograft tumour tissues and melanoma clinical samples. Furthermore, expression of amphiregulin promoted tumour growth, implicating causal relationship between the expression of IL13Rα2 and amphiregulin. These results suggest that IL13Rα2 enhances tumorigenicity by inducing angiogenesis in malignant melanoma, and serves as a potential therapeutic target of malignant melanoma.

Antibody-drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb-DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.

Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors. IMPORTANCE Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

Oncolytic viruses (OVs) are novel cancer therapeutics with great promise, but host antiviral immunity represents the hurdle for their efficacy. Immunosuppression by cyclophosphamide (CP) has thus been shown to enhance the oncolytic efficacy of many OVs, but its effects on OVs armed with therapeutic genes remain unknown. We have previously reported on the efficacy of AxE1CAUP, an oncolytic adenovirus (OAd) expressing uracil phosphoribosyltransferase (UPRT), an enzyme that markedly enhanced the toxicity of 5-fluorouracil (5-FU), in immunodeficient, Ad-nonpermissive nude mice. Here we explored the efficacy and safety of intratumoral (i.t.) AxE1CAUP/5-FU therapy and of its combination with CP for syngenic HaP-T1 pancreatic cancers in immunocompetent, Ad-permissive Syrian hamsters. AxE1CAUP infected, replicated, expressed UPRT, and increased the sensitivity to 5-FU in HaP-T1 cells in vitro. I.t. AxE1CAUP/5-FU treatment inhibited the growth of subcutaneous HaP-T1 allografts. The combination with high-dose CP inhibited serum Ad-neutralizing antibody formation, increased intratumoral AxE1CAUP replication and UPRT expression, and resulted in further enhanced therapeutic effects with 5-FU. Neither body weight nor histology of the liver and lung changed during these treatments. A clinically-approved, intermediate-dose CP also enhanced the efficacy of i.t. AxE1CAUP/5-FU treatment in these hamsters, which was not affected by preexisting immunity to the vector. These data demonstrate the excellent antitumor efficacy and safety of an OAd armed with a suicide gene in combination with CP for treating syngenic tumors in immunocompetent, Ad-permissive animals, indicating the efficacy of CP in overcoming the hurdle of antiviral immunity for effective OV-mediated gene therapy.

Background Multiple factors affect the in vivo antitumor activity of antibody-based therapeutics; however, the influence of cell surface retention on antibody-dependent cellular cytotoxicity (ADCC) is not fully understood. Here we evaluated the importance of cell surface antibody retention in antitumor activity mediated by ADCC in vivo. Materials and methods Two mAbs against tumor-associated calcium signal transducer 2 (TACSTD2/TROP2), AR47A6.4.2 and Pr1E11, were used. Antitumor activities against BxPC3 and Colo205 cells were investigated through in vitro and in vivo assays. Results Pr1E11 showed better cell surface retention than AR47A6.4.2 in vitro although Pr1E11 and AR47A6.4.2 showed equivalent ADCC activity. Complement-dependent cytotoxicity and antiproliferative activity were not observed for either antibody. Pr1E11 exhibited higher antitumor activity than AR47A6.4.2 in vivo. Conclusion Our results suggest that high cell surface retention can result in potent ADCC activity in vivo. This observation could provide novel insight into how effectively screen for antibodies with strong in vivo antitumor activity.

A critical issue in adenovirus (Ad)-based cancer gene therapy is to improve the specificity of gene delivery to cancer cells for better efficacy and safety. We explored methods of retargeting Ad vectors for selective gene therapy of human biliary cancers using the Ad incorporating an IgG Fc-binding motif (Z33) from the Staphylococcus protein A (Ad-FZ33) combined with tumor-specific antibodies. Flow cytometry analysis revealed high-expression levels of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) on human biliary cancer cells. Ad-FZ33 expressing LacZ combined with antibodies against EpCAM or EGFR, followed by β-gal assay, demonstrated highly efficient gene transduction in these biliary cancer cells, compared to the treatment with control antibody or without antibody. Ad-FZ33 expressing uracil phosphoribosyl transferase (UPRT), an enzyme which greatly enhances the toxicity of 5-fluorouracil (FU), combined with antibodies against EpCAM or EGFR, remarkably enhanced the sensitivity of biliary cancer cells to 5-FU. By contrast, the treatment did not affect the 5-FU sensitivity of the cells not expressing EpCAM or EGFR including normal hepatocytes. Finally, treatments with the UPRT-expressing Ad-FZ33 with antibodies against EpCAM or EGFR, followed by 5-FU administration, significantly suppressed the growth of biliary cancer xenografts in nude mice. These results indicate that the gene therapy mediated by the Z33 fiber modified Ad with anti-EpCAM or anti-EGFR antibodies offers a potentially effective therapeutic modality against biliary cancers.

To evaluate the effects of neck-muscle vibration for 5 min before occupational therapy (OT) on unilateral spatial neglect (USN).In this multiple-baseline design study for 6 weeks (A(1)-B-A(2) design: A(1), A(2); conventional OT without neck-muscle vibration, B; neck-muscle vibration before OT together with conventional OT), we examined 11 right brain-damaged patients in the post-acute phase of stroke who showed USN. Sessions A(1) and A(2): conventional OT for 40 min once daily for 5 days a week. Session B: the left posterior neck muscles of the patient were subjected to vibration for 5 min, without confirming the appearance of a kinaesthetic illusion, immediately before OT, and then the same OT programme as in sessions A(1) and A(2) was performed. Each session lasted 2 weeks. USN and activities of daily living (ADL) were evaluated at 2-week intervals by the Behavioural Inattention Test (BIT) and Functional Independence Measure (FIM), respectively.Significant increases in the total scores in both the conventional subtest and behavioural subtest of the BIT were only seen during session B. FIM scores increased significantly during both sessions A(1) and B.The application of neck-muscle vibration before OT may have positive effects on USN, but the specific effect on the improvement of ADL is not clear.

Sendai viral vector (SeV) is emerging as a promising vector for gene therapy. However, little information is available regarding the combination of SeV-mediated gene and mesenchymal stem cell (MSC) therapy in dealing with ischemic diseases. In this study, we infected SeV to the MSCs in vitro; and injected MSCs modified with SeV harboring human angiopoietin-1 gene (SeVhAng-1) into the ischemic limb of rats in vivo. We found SeV had high transductive efficiency to the MSCs. Both MSCs and SeVhAng-1-modified MSCs improved the blood flow recovery and increased the capillary density of the ischemic limb, compared with the control. However, in contrast to MSCs, SeVhAng-1-modified MSCs had a better improvement of blood flow recovery in the ischemic limb. We further found the ischemic limb injected with SeVhAng-1-modified MSCs had strong expression of p-Akt, which improved survival of MSCs injected into the ischemic limb. This indicated SeVhAng-1 modification enhanced angiogenetic effect of MSCs by both angiogenesis and cell protection. We conclude that SeVhAng-1-modified MSCs may serve as a more effective tool in dealing with ischemic limb disease.

Vascular endothelial growth factor (VEGF) administration has recently been assessed as a therapeutic strategy for ischemic diseases including brain ischemia because of its angiogenic effect. However, VEGF also causes detrimental adverse effects by increasing vascular permeability. This study examined whether plasmid human VEGF (phVEGF) administration induced angiogenic effects in the rat brain ischemia model caused by permanent ligation of both common carotid arteries, and investigated the occurrence of adverse effects. Administration of various doses (0-200 microg) of phVEGF in the temporal muscle was followed by encephalo-myo-synangiosis. Thirty days after treatment, the numbers and areas of capillaries per field in the extracted brains were analyzed with the National Institutes of Health Image software program. The maximal angiogenic effect occurred with a 100 microg dose of phVEGF in the numbers and areas of capillaries in the VEGF-treated brains. Histological examination showed no apparent adverse effects in the brain parenchyma even at the highest administration dose (200 microg) of phVEGF. The maximal angiogenic effect at the optimal dose of phVEGF can be considered under the threshold to cause serious adverse effects in the rat brain.

Although intravenous delivery of mesenchymal stem cells (MSCs) prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat middle cerebral artery occlusion models, there are few reports of MSC treatment in global cerebral ischemia. We utilized a global cerebral ischemia model induced by arresting the heart with a combination of hypovolemia and intracardiac injections of a cold potassium chloride solution in order to study the potential therapeutic benefits of human mesenchymal stem cells (hMSCs) on global cerebral ischemia. hMSCs were intravenously injected into the rats 3 h after resuscitation from cardiac arrest. The effects on structural and functional outcome of hMSC were assessed at 5 h and 1, 3, and 7 days using magnetic resonance spectroscopy (MRS), histology, and cognitive functional analysis. Intravenous delivery of hMSCs reduced the Lac/Cr ratios, nuclear DNA fragmentation, neuronal loss, and elicited functional improvement compared with the control sham group. Enzyme-linked immunosorbent assay (ELISA) of the hippocampus revealed an increase in BDNF in hMSC-treated group. These data suggest that intravenous delivery of hMSC may have a therapeutic effect in global cerebral ischemia.

Transplantation of mesenchymal stem cells (MSCs) derived from bone marrow has been shown to improve functional outcome in spinal cord injury (SCI). We transplanted MSCs derived from human bone marrow (hMSCs) to study their potential therapeutic effect in SCI in the rat. In addition to hMSCs, we used gene-modified hMSCs to secrete brain-derived neurotrophic factor (BDNF-hMSCs). After a dorsal transection lesion was induced at T9, cells were microinjected on each side of the transection site. Fluorogold (FG) was injected into the epicenter of the lesion cavity to identify transected corticospinal tract (CST) neurons. At 5 weeks after transplantation, the animals were perfused. Locomotor recovery improvement was observed for the BDNF-hMSC group, but not in the hMSC group. Structurally there was increased sprouting of injured corticospinal tract and serotonergic projections after hMSC and BDNF-hMSC transplantation. Moreover, an increased number of serotonergic fibers was observed in spinal gray matter including the ventral horn at and below the level of the lesion, indicating increased innervation in the terminal regions of a descending projection important for locomotion. Stereological quantification was performed on the brains to determine neuronal density in primary motor (M1) cortex. The number of FG backfilled cells demonstrated an increased cell survival of CST neurons in M1 cortex in both the hMSC and BDNF-hMSC groups at 5 weeks, but the increase for the BDNF-hMSC group was greater. These results indicate that transplantation of hMSCs hypersecreting BDNF results in structural changes in brain and spinal cord, which are associated with improved functional outcome in acute SCI.

Background The prognosis of bile duct cancer is quite poor because of the low resection rate and the tolerance of the cancer to chemotherapy and radiotherapy. We investigated the feasibility of an oncolytic adenovector with two suicide genes for the treatment of bile duct cancer. Materials and Methods We developed a new conditionally replicating adenovirus (AxE1CAUT) with the uracil phosphoribosyltransferase (UPRT) gene and the herpes simplex virus thymidine kinase (HSV-tk) gene, and compared its antitumor effects with a replication defective adenovector (AxCAUT) that has both the UPRT and HSV-tk genes. We evaluated the effects of these adenoviruses with 5-fluorouracil (5-FU) and/or ganciclovir (GCV) on human cholangiocarcinoma cells (HuCCT1, with mutant p53) in vitro and in vivo. Results The drug sensitivity of HuCCT1 cells to 5-FU and/or GCV was increased with an increase in the multiplicity of infection (MOI). The antitumor effect increased when 5-FU and GCV were given at the same time. Subcutaneous tumors of nude mice directly injected with AxCAUT showed a higher response to 5-FU/GCV than 5-FU or GCV alone, but there was no difference between AxCAUT and AxE1CAUT. However, AxE1CAUT with 5-FU/GCV produced a decrease in tumor weight and better survival than AxCAUT in a peritoneal dissemination model infected by intraperitoneal administration of the adenovectors. Conclusion Oncolytic double suicide gene therapy is effective against human cholangiocarcinoma cells in nude mouse models.

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions in experimental models of ischemia, tumors, and neurodegenerative diseases and to ameliorate functional deficits. In this study, we attempted to evaluate the therapeutic potential of MSCs for treating prion diseases. Immortalized human MSCs (hMSCs) that express the LacZ gene were transplanted into the unilateral hippocampi or thalami of mice, and their distributions were monitored by the expression of β-galactosidase. In mice infected with prions, hMSCs transplanted at 120 days postinoculation (dpi) were detected on the contralateral side at 2 days after transplantation and existed there even at 3 weeks after transplantation. In contrast, few hMSCs were detected on the contralateral side for mock-infected mice. Interestingly, the migration of hMSCs appeared to correlate with the severity of neuropathological lesions, including disease-specific prion protein deposition. The hMSCs also migrated to a prion-specific lesion in the brain, even when intravenously injected. Although the effects were modest, intrahippocampal and intravenous transplantation of hMSCs prolonged the survival of mice infected with prions. A subpopulation of hMSCs in the brains of prion-infected mice produced various trophic factors and differentiated into cells of neuronal and glial lineages. These results suggest that MSCs have promise as a cellular vehicle for the delivery of therapeutic genes to brain lesions associated with prion diseases and, furthermore, that they may help to regenerate neuronal tissues damaged by prion propagation.

In this study, we regenerated skin and its appendages by transplanting cultured normal dermal fibroblasts, into which morphogen genes had been introduced. We cultured normal dermal fibroblasts obtained from Fisher 344 rats on the surface of hydroxyapatite beads, and then adsorbed them onto the surface of a collagen sponge, which was transplanted into a full-thickness skin defect prepared on the backs of rats. Before transplantation, genes were introduced into the dermal fibroblasts via adenovirus vector (ad)-bone morphogenetic protein 2 and ad-wingless int 3 genes in addition to fibroblast growth factor-2 protein. By Week 4, the appearance of follicle germs or primitive hair germs was observed only in the ad-bone morphogenetic protein 2+ad-wingless int 3 combined with the fibroblast growth factor-2 protein group. By Week 16, in that same group, hair follicles having mature pilosebaceous systems with equally spaced localization had formed in the ulcer wound.

Intravenous transplantation of human mesenchymal stem cells (hMSCs) expanded from adult bone marrow ameliorates functional deficits in rat cerebral infarction models. Several hypotheses to account for the therapeutic mechanisms have been suggested, but angiogenesis is thought to be of critical importance. Recently, we have reported the therapeutic benefits of hMSCs which have been transfected with the angiopoietin-1 gene in a rat permanent middle cerebral artery occlusion (MCAO) model. To potentially enhance the therapeutic effects of angiopoietin-1 gene-modified hMSC (Ang-hMSC), we transfected hMSCs with the angiopoietin-1 gene and the VEGF gene, and investigated whether the combination of Ang-1 and VEGF gene-modified hMSCs (Ang-VEGF-hMSC) contribute to functional recovery in a rat MCAO model. We induced MCAO using intraluminal vascular occlusion, and hMSCs, Ang-hMSCs, VEGF-hMSCs or Ang-VEGF-hMSCs were intravenously infused 6 h later. MRI and behavioral analyses revealed that rats receiving Ang-VEGF-hMSCs showed the greatest structural-functional recovery as compared to the other groups. These results suggest that intravenous administration of hMSCs transfected with the angiopoietin-1 and VEGF gene using a fiber-mutant adenovirus vector may represent a new strategy for the treatment of ischemia.

Combination therapy with replicative oncolytic viruses is a recent topic in innovative cancer therapy, but few studies have examined the efficacy of oncolytic adenovirus plus replication-deficient adenovirus carrying a suicide gene. We aim to evaluate whether an E1A, E1B double-restricted oncolytic adenovirus, AxdAdB-3, can improve the efficacy for gallbladder cancers (GBCs) of the replication-deficient adenovirus-based herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) therapy directed by the carcinoembryonic antigen (CEA) promoter. Cytopathic effects of AxdAdB-3 plus AxCEAprTK (an adenovirus expressing HSVtk directed by CEA promoter) or AxCAHSVtk (an adenovirus expressing HSVtk directed by a nonspecific CAG promoter) with GCV administration were examined in several GBC lines and normal cells. Efficacy in vivo was tested in severe combined immunodeficiency disease mice with GBC xenografts. Addition of AxdAdB-3 (1 multiplicity of infection, MOI) significantly enhanced the cytopathic effects of AxCEAprTK (10 MOI)/GCV on GBC cells. The augmented effect was attributable to the replication of the AxCEAprTK and also to the enhanced CEA promoter activity, which was presumably transactivated by E1A. In normal cells, AxdAdB-3 (20 MOI) plus AxCEAprTK (200 MOI)/GCV was not cytopathic, whereas AxdAdB-3 (1 MOI) plus AxCAHSVtk (10 MOI)/GCV was significantly toxic. Low-dose AxdAdB-3 (2 x 10(7) PFU, plaque-forming unit) plus AxCEAprTK (2 x 10(8) PFU)/GCV significantly suppressed the growth of GBC xenografts as compared with either AxdAdB-3 (2 x 10(7) PFU)/GCV or AxCEAprTK (2 x 10(9) PFU)/GCV alone. E1A, E1B double-restricted replicating adenovirus at low dose significantly augmented the efficacy of CEA promoter-directed HSVtk/GCV therapy without obvious toxicity to normal cells, suggesting a potential use of this combination for treating GBC and other CEA-producing malignancies.

Pancreatic islet transplantation has the potential to maintain normoglycemia in patients with established type 1 diabetes, thereby obviating the need for frequent insulin injections. Our previous study showed that recombinant IL-12p40-producing islets prevented the recurrence of NOD diabetes. First, to see which immunomodulating molecule-secreting islet grafts can most powerfully prevent diabetes development in NOD mice without immunosuppressant, NOD islets were transfected with one of the following adenoviral vectors: Ad.IL-12p40, Ad.TGF-beta, Ad.CTLA4-Ig, or Ad.TNF-alpha after which they were transplanted under the renal capsule of acutely diabetic NOD mice. The immunomodulating molecules produced by these adenovirus-transfected islets in vitro were 74+/-19ng, 50+/-4ng, 821+/-31ng, and 77+/-18ng/100 islets, respectively. Transplantation of IL-12p40, TNF-alpha, and CTLA4-Ig but not TGF-beta-secreting islets displayed enhanced survival and delayed diabetes recurrence in recent-onset diabetic recipients. IL-12p40-producing islet grafts most powerfully prevented recurrent diabetes in NOD mice. In addition, local production of TNF-alpha and CTLA4-Ig significantly prolonged islet graft survival. In second series of experiment, these manipulated islets were transplanted under the renal capsule of 10-week-old NOD recipients and were also transplanted subcutaneously into 2-week-old NOD recipients. Transplantation of these islets into 2- or 10-week-old pre-diabetic mice failed to protect them from developing diabetes; in fact, transplantation of Ad.TNF-alpha-transfected islets into 2-week-old mice actually accelerated diabetes onset. Taken together, this approach was ineffectual as a prophylactic protocol. In conclusion, this study showed comparisons of the immunomodulating effects of 4 different adenoviral vectors in the same transplantation model and local production of IL-12p40, TNF-alpha and CTLA4-Ig significantly prevented recurrent diabetes in NOD mice.

We previously reported that transferrin (Tf)-modified liposomes (Tf-L) additionally modified with a cholesterylated pH-sensitive fusogenic peptide (Chol-GALA) can release an encapsulated aqueous phase marker to cytosol via endosomal membrane fusion. However, further obstacles need to be overcome to bring the Tf-L to the level of a viral-like gene delivery system. In this study, we developed a novel packaging method to encapsulate condensed plasmid DNA into PEgylated Tf-L (Tf-PEG-L) to form a core-shell-type nanoparticle. The most difficult challenge was to provide a mechanism of escape for the condensed core from endosome to cytosol in the presence of polyethylene glycol (PEG). We hypothesized that a membrane-introduced Chol-GALA and a PEgylated GALA would interact synergistically to induce membrane fusion between liposome and endosome. By simultaneously incorporating Chol-GALA into the membrane of Tf-PEG-L and GALA at tips of PEG chains, a condensed core was released into cytosol, and transfection activity increased 100-fold. We concluded that topological control was responsible for the synergistic effect of GALA derivatives introduced on Tf-PEG-L.

Background Neuron-selective gene transfer is an attractive therapeutic strategy for neurological disorders. However, optimal targets and gene delivery systems remain to be determined. Methods Following immunization of mice with PC12 cells, hybridomas were screened by β-Gal reporter gene assay using FZ33 fiber-modified adenovirus vectors. Subsequently, the efficacy and specificity of monoclonal antibody (mAb)-mediated gene transfer via FZ33 and FdZ adenovirus vectors were evaluated by flow cytometry, chemiluminescent β-Gal reporter gene assay, and immunocytochemistry. Finally, the antigen recognized by the mAb was identified by mass spectrometry and transfection analysis. Results A hybridoma clone 6E3 producing monoclonal antibody, mAb6E3, was screened. Flow cytometry, chemiluminescent β-Gal reporter gene assay, and immunocytochemistry with mAb6E3 and the fiber mutant adenovirus demonstrated efficient gene transfer into the PC12 cells. Treatment of neuron–glia cocultures with mAb6E3 and FdZ adenovirus resulted in neuron-selective gene transfer. Immunohistochemical images of rat spinal cord tissue showed that mAb6E3 reacts specifically with neurons. Finally, Na,K-ATPase β1 was identified as the antigen of mAb6E3. Conclusions Hybridoma screening using FZ33 fiber-modified adenovirus vectors serves as an efficient approach to detect antigens in mAb-targeted gene transfer. Neuronal tropism in the central nervous system through mAb6E3 represents an important initial step towards neuron-selective gene transfer in the treatment of local neurological disorders, such as spinal cord injury. Copyright © 2008 John Wiley & Sons, Ltd.

CD154 (CD40-ligand) is a critical transmembrane molecule with potent immune-stimulatory properties that is used in clinical applications of gene therapy for leukemia and lymphoma. However, CD154 is cleaved into a soluble form, and high levels of sCD154 contribute to systemic inflammatory and cardiovascular diseases, suggesting a deleterious side effect of CD154 gene therapy. In this study, we engineered noncleavable mutants of human CD154 with point mutations to develop a potentially less toxic molecule in vivo. In contrast to wild-type CD154 (CD154-WT) subsequently released as sCD154, both mutants CD154-M3 and CD154-M4 were resistant to cleavage in tumor cells. Also, CD40-expressing leukemia B cells transfected with CD154-M3 mutant were highly effective stimulators in a mixed lymphocyte-leukemia reaction, indicating that CD154-M3 mutant did not lose biologic activity. In mice transplanted with tumors expressing CD154-WT, we found increased plasma levels of human sCD154 followed by various systemic inflammatory reactions such as glomerulonephritis and an increased number of infiltrating mononuclear cells in the liver. However, CD154-M3 mutant did not induce any systemic inflammatory effects in vivo. As such, the noncleavable mutant of CD154 is fully capable of inducing the immune response with less toxic properties and is a useful tool for CD154 immune gene therapy.

Adult neural stem and progenitor cells (NSPCs) are important autologous transplantation tools in regenerative medicine, as they can secrete factors that protect the ischemic brain. We investigated whether adult NSPCs genetically modified to secrete more glial cell line-derived neurotrophic factor (GDNF) could protect against transient ischemia in rats. NSPCs were harvested from the subventricular zone of adult Wistar rats and cultured for 3 weeks in the presence of epidermal growth factor. The NSPCs were treated with fibre-mutant Arg-Gly-Asp adenovirus containing the GDNF gene (NSPC-GDNF) or enhanced green fluorescent protein (EGFP) gene (NSPC-EGFP; control group). In one experiment, cultured cells were transplanted into the right ischemic boundary zone of Wistar rat brains. One week later, animals underwent 90 min of intraluminal right middle cerebral artery occlusion followed by magnetic resonance imaging and behavioural tests. The NSPC-GDNF group had higher behavioural scores and lesser infarct volume than did controls at 1, 7 and 28 days postocclusion. In the second experiment, we transplanted NSPCs 3 h after ischemic insult. Compared to controls, rats receiving NSPC-GDNF had decreased infarct volume and better behavioural assessments at 7 days post-transplant. Animals were killed on day 7 and brains were collected for GDNF ELISA and morphological assessment. Compared to controls, more GDNF was secreted, more NSPC-GDNF cells migrated toward the ischemic core and more NSPC-GDNF cells expressed immature neuronal marker. Moreover, the NSPC-GDNF group showed more effective inhibition of microglial invasion and apoptosis. These findings suggest that NSPC-GDNF may be useful in treatment of cerebral ischemia.

A replication-competent adenoviral vector deficient for expression of the early E1B55K protein has been applied in clinical studies. The vector, however, was not fully effective for the treatment of human cancer. In this study, the E1A gene (which encodes an Rb-binding domain protein) of the adenoviral vector AxE1AdB was further engineered with a point mutation designed to abolish binding to Rb protein (pRb) and arrest the cell cycle (AxdAdB-3). The difference in the cytotoxicity of these vectors in two cancer cell lines was observed in association with differences in replication, infection efficiency, and expression levels of adenovirus receptors. Relative to the parent vector (AxE1AdB), which worked in a manner similar to ONYX-015, AxdAdB-3 with the mutated pRb-binding motif demonstrated increased cytotoxicity against p53-mutant human esophageal cancer cell lines EC-GI-10 and T.Tn. AxdAdB-3 showed a greater oncolytic effect than AxE1AdB in vivo despite almost the same replication efficiency in vitro. Unexpectedly, cell cycle arrest in AxdAdB-3-infected cells was less efficient than that in cell lines infected with AxE1AdB. However, AxdAdB-3 strongly reduced NF-kappaB activity and thereby enhanced apoptosis more than AxE1AdB did. These data demonstrate that the Rb-binding domain of E1A can regulate NF-kappaB activity and that modifications to this domain may lead to advances in gene therapies for the treatment of human cancers.

Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of cardiac hypertrophy and that p53 accumulation is essential for the transition from cardiac hypertrophy to heart failure. Pressure overload initially promoted vascular growth in the heart by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors, and inhibition of angiogenesis prevented the development of cardiac hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results indicate that the anti-angiogenic property of p53 may have a crucial function in the transition from cardiac hypertrophy to heart failure.

Objective Chronic lymphocytic leukemia (CLL) B cells from most patients express both membrane-bound CD27 (mCD27) and soluble CD27 (sCD27). Expression of sCD27 inhibits CD27-dependent T-cell or CLL-cell activation mediated by its ligand, CD70. In this study, we evaluated whether protease inhibitors could inhibit the release of sCD27 from CLL cells and enhance T-cell activation mediated by CD27-CD70 interaction. Methods CLL cells exposed to hydroxamic acid-based matrix metalloprotease (MMP) inhibitors were evaluated for the release of sCD27 by sandwich enzyme-linked immunosorbent assay and immunoprecipitation. We examined for phenotypic changes in CLL cells treated with MMP inhibitors by flow cytometry and T-cell activation by CLL cells was assessed by [ 3 H] thymidine incorporation assay and the production of interferon-gamma. Results Treatment of CLL cells with MMP inhibitors blocked the release of sCD27 to the culture supernatant. In contrast, a non–hydroxamic acid control compound or inhibitors of other proteases, including serine, cysteine, and aspartyl proteases, were ineffective. Furthermore, CLL cells treated with MMP inhibitors expressed significantly higher levels of accessory molecules, such as CD54, CD80, and CD95. Consistent with such changes, we found that CLL cells treated with MMP inhibitors, but not control treated cells, could stimulate allogeneic and autologous T cells in mixed lymphocyte reactions. Conclusion These data reveal that metalloprotease inhibitors can block production of sCD27, which can interfere with mCD27-CD70 interactions that induce expression of immune costimulatory molecules on CLL B cells. Conceivably, treatment of CLL cells with metalloprotease inhibitors may enhance their potential for stimulating cellular immune recognition of leukemia-associated antigens.

BACKGROUND Focusing on Adv-FZ33, a modified adenovirus in which a synthetic 33-amino-acid immunoglobulin G-binding domain was inserted into the adenoviral fiber protein, we tried to identify suitable target molecules for prostate cancer-specific gene therapy. METHODS Hybridomas were established from mice immunized with prostate cancer cell lines. The hybridomas were screened using Adv-FZ33 to create monoclonal antibodies (mAbs) that induced high gene transfer efficiency for PC-3 cells. Furthermore, we identified target antigens of the mAbs by immunoprecipitation and mass spectrometry, and investigated the expression of target molecules by flow cytometry and immunocytochemistry. RESULTS Using Adv-FZ33, we established four different mouse mAbs that increased transduction efficiency for PC-3. The target antigens identified were Ep-CAM, CD155, HAI-1, and Na,K-ATPase β1. These antigens were expressed in several cancer cell lines, including prostate cancer. Human prostatic myofibroblast cells lacked expression of Ep-CAM and HAI-1. CONCLUSIONS We established anti-Ep-CAM mAb and anti- HAI-1 mAbs. Gene transduction via Ep-CAM and HAI-1 may be a novel strategy for treatment of prostate cancer. Prostate 67: 1163–1173, 2007. © 2007 Wiley-Liss, Inc.

筆者らは，プロテインA由来の33個ペプチド配列をファイバー部に組み込んだアデノウイルス（Adv-FZ33）を作製しその生物活性を検討してきた．さまざまなヒト腫瘍標的細胞と結合し，このファイバー変異型アデノウイルスを介した遺伝子導入効率を増強できる標的化抗体の網羅的な樹立法の開発に成功した．

TROP-2 is a type Ⅰ transmembrane glycoprotein that is highly expressed in various epithelial cancer cells, and its increased expression correlates with poor prognosis. Although several anti-TROP-2 antibodies have been described, they were found unsuitable for antitumor therapy use in vivo as naked antibodies. In this study, we established a novel anti-TROP-2 antibody, designated Pr1E11, from mice immunized with primary prostate cancer cells. Antibody screening was based on the infection activity of Adv-LacZ-FZ33, which displays an immunoglobulin G binding domain in the adenoviral fiber protein. We found that Pr1E11 specifically binds to TROP-2 with high affinity and recognizes diverse epithelial cancer cell lines and primary pancreatic cancer tissues. Epitope analysis using TROP-2 deletion mutants revealed that binding site of Pr1E11 is a cysteine-rich domain, a unique epitope compared with other available anti-TROP-2 antibodies. In addition, Pr1E11 exhibited low internalization activity, which may make it suitable for naked antibody therapeutics. Our results suggest that Pr1E11 may stimulate different biological activities from other anti-TROP-2 antibodies based on its unique binding epitope, and is a potential candidate for naked antibody therapeutics for various epithelial cancer treatments.

In the present investigation, we generated platelets (PLTs) from cord blood (CB) CD34(+) cells using a three-phase culture system. We first cultured 500 CB CD34(+) cells on telomerase gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL), and thrombopoietin (TPO) for 14 days. We then transferred the cells to hTERT stroma and cultured for another 14 days with fresh medium containing interleukin-11 (IL-11) in addition to the original cytokine cocktail. Subsequently, we cultured the cells in a liquid culture medium containing SCF, FL, TPO, and IL-11 for another 5 days to recover PLT fractions from the supernatant, which were then gel-filtered to purify the PLTs. The calculated yield of PLTs from 1.0 unit of CB (5 x 10(6) CD34(+) cells) was 1.26 x 10(11) - 1.68 x 10(11) PLTs. These numbers of PLTs are equivalent to 2.5-3.4 units of random donor-derived PLTs or 2/5-6/10 of single-apheresis PLTs. The CB-derived PLTs exhibited features quite similar to those from peripheral blood in morphology, as revealed by electron micrographs, and in function, as revealed by fibrinogen/ADP aggregation, with the appearance of P-selectin and activated glycoprotein IIb-IIIa antigens. Thus, this culture system may be applicable for large-scale generation of PLTs for future clinical use.

Purpose: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy.Experimental Design: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti–carcinoembryonic antigen (CEA) monoclonal antibody, C2-45.Results: In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC50 against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01).Conclusions: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.

Mouse glioma 261 (Gl261) cells are used frequently in experimental glioblastoma therapy; however, no detailed description of the Gl261 tumor model is available. Here we present that Gl261 cells carry point mutations in the K-ras and p53 genes. Basal major histocompatibility complex (MHC)I, but not MHCII, expression was detected in Gl261 cells. The introduction of interferon-γ-encoding genes increased expression of both MHCI and MHCII. A low amount of B7-1 and B7-2 RNA was detected in wild-type cells, but cytokine production did not change expression levels. Gl261 cells were transduced efficiently by adenoviral vectors; the infectivity of retroviral vectors was limited. Low numbers of transplanted Gl261 cells formed both subcutaneous and intracranial tumors in C57BL/6 mice. The cells were moderately immunogenic: prevaccination of mice with irradiated tumor cells 7 days before intracranial tumor challenge prevented tumor formation in approximately 90% of mice. When vaccination was carried out on the day or 3 days after tumor challenge, no surviving animals could be found. In vitro-growing cells were radiosensitive: less than 2 Gy was required to achieve 50% cell mortality. Local tumor irradiation with 4 Gy X-rays in brain tumor-bearing mice slowed down tumor progression, but none of the mice were cured off the tumor. In conclusion, the Gl261 brain tumor model might be efficiently used to study the antitumor effects of various therapeutic modalities, but the moderate immunogenicity of the cells should be considered. (Cancer Sci 2006; 97: 546 – 553)

The pentaspan molecule CD133 has been shown to be a marker of more primitive hematopoietic progenitors in mobilized peripheral blood (PB). Our objective was to assess the efficacy of PB CD133(+) cells in our coculture system using human telomerized stromal (HTS) cells.Five thousand PB CD133(+) cells or conventional cord blood (CB) CD34(+) cells were expanded with or without HTS cells in the presence or absence of stem cell factor, thrombopoietin, and Flk-2/Flt-3 ligand.The coculture was significantly superior in expanding PB clonogenic cells as compared with the stroma-free culture (CFU-C, 2 +/- 0 vs 111 +/- 15-fold of initial cell number, p0.01), and the fold increase of PB clonogenic cells was comparable to that for CB cells after two weeks of coculture (BFU-E, 54 +/- 3 vs 56 +/- 4-fold; CFU-GM, 156 +/- 26 vs 83 +/- 9-fold; CFU-Mix, 30 +/- 11 vs 80 +/- 36-fold). However, proliferation of CFU-Mk from PB on coculture with HTS cells was modest as compared with stroma-free culture. Concomitantly, multiple hematopoietic cells transmigrated below the stromal layer and formed cobblestone areas (CAs). The production of hematopoietic progenitor cells from CAs after coculture with PB was significantly lower than that seen in cells cocultured with CB for four weeks (CFU-Mix, 0 +/- 0 vs 9 +/- 5-fold on day 28, p0.01), although a similar number of CAs derived from PB and CB were observed.PB CD133(+) cells proliferated efficiently above the stromal layer, while the characteristics of PB CD133(+) cells underneath the human stromal layer were likely to be maintained, even after long-term hematopoietic-stromal interaction.

In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.

Object. Vascular endothelial growth factor (VEGF) is a secreted mitogen associated with angiogenesis. The conceptual basis for therapeutic angiogenesis after plasmid human VEGF gene (phVEGF) transfer has been established in patients presenting with limb ischemia and myocardial infarction. The authors hypothesized that overexpression of VEGF using a gene transfer method combined with indirect vasoreconstruction might induce effective brain angiogenesis in chronic cerebral hypoperfusion, leading to prevention of ischemic attacks. Methods. A chronic cerebral hypoperfusion model induced by permanent ligation of both common carotid arteries in rats was used in this investigation. Seven days after induction of cerebral hypoperfusion, encephalomyosynangiosis (EMS) and phVEGF administration in the temporal muscle were performed. Fourteen days after treatment, the VEGF gene therapy group displayed numbers and areas of capillary vessels in temporal muscles that were 2.2 and 2.5 times greater, respectively, in comparison with the control group. In the brain, the number and area of capillary vessels in the group treated with the VEGF gene were 1.5 and 1.8 times greater, respectively, relative to the control group. Conclusions. In rat models of chronic cerebral hypoperfusion, administration of phVEGF combined with indirect vasoreconstructive surgery significantly increased capillary density in the brain. The authors' results indicate that administration of phVEGF may be an effective therapy in patients with chronic cerebral hypoperfusion, such as those with moyamoya disease.

An adenovirus (Adv) retaining normal E1A but lacking the 55 kDa E1B protein replicates preferentially in TP53-deficient cancer cells including pancreatic cancer cell lines, resulting in the oncolysis of the tumor. When tumor cells are exposed to hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is stabilized and activated to promote the transcription of several genes such as vascular endothelial growth factor (VEGF), but in the presence of E1A hypoxia-induced VEGF m-RNA synthesis is inhibited by E1A binding to p300. In this study, we demonstrated that the cancer cells infected with a mutant Adv in which the p300 binding site in E1A was partially deleted induced a higher expression level of VEGF as compared to those of Adv with normal E1A. An immunoprecipitation study for E1A confirmed that mutant E1A had a reduced binding capacity for p300. Although the expressions of HIF-1alpha m-RNA were almost the same in both cancer cells infected with the mutant Adv and those with the wild Adv, the amount of HIF-1alpha protein in cancer cells infected with the wild E1A Adv was lower than in those infected with the mutant E1A type Adv. In vivo, in contrast to the angiogenesis treated with mutant E1A, wild-E1A inhibited tumor angiogenesis significantly. These results suggested that E1A suppressed the production of VEGF and inhibited tumor angiogenesis by binding with p300, resulting in the inhibition of the HIF-1alpha-mediated transcription of genes through binding to HRE. This study demonstrates, for the first time, the effect of an oncolytic replication-competent Adv in inhibiting tumor angiogenesis.

Thrombopoietin (TPO) is the growth factor for megakaryocytes and platelets, however, it also acts as a potent regulator of stem cell proliferation. To examine the significance of TPO expression in proliferation of hepatic oval cells, the effect of adenovirus-mediated TPO gene transfer into livers of the Solt-Farber model, which mimics the condition where liver regeneration is impaired, was examined. Hepatic TPO mRNA peaked its expression at 2 days after gene transduction and then gradually decreased. The peripheral platelet number began to increase at 4 days (P0.05) and reached its plateau at 9 days (P0.01). Oval cells expressed c-Mpl, a receptor for TPO as well as immature hematopoietic and hepatocytic surface markers such as CD34 and AFP. The proliferating cell nuclear antigen-positive oval cells in rats into which adenovirus-TPO gene was transferred at 7 and 9 days were significantly greater than those in adenovirus-LacZ gene transferred (P0.05, each), and the total numbers of oval cells in the adenovirus-TPO gene transferred at 9 and 13 days were also significantly greater than those in adenovirus-LacZ gene transferred (P0.05, each). Expression of SCF protein was increased at 4, 7, and 9 days by TPO gene administration and that of c-Kit was increased at 4 and 7 days. These data suggest that adenovirus-mediated TPO gene transfer stimulated oval cell proliferation in liver as well as increasing peripheral platelet counts, emphasizing the significance of the TPO/c-Mpl system in proliferation of hepatic oval cells.

Background Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. Methods Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. Results The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. Conclusions The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world. Copyright © 2005 John Wiley & Sons, Ltd.

Vascular endothelial growth factor (VEGF) has been shown to display neuroprotective effects on dopaminergic (DA) neurons. Here, we investigated the neurorescue effects of VEGF on 6-hydroxydopamine (6-OHDA)-treated DA neurons in vitro and in vivo. Initially, we examined in vitro whether 1, 10, or 100 ng/ml of VEGF administration at 2 or 4 h after 6-OHDA treatment rescued DA neurons derived from E14 murine ventral mesencephalon. The earlier treatment of VEGF suppressed 6-OHDA-induced loss of DA neurons more than the delayed treatment. Next, we examined whether the continuous infusion of VEGF had neurorescue effects in a rat model of Parkinson's disease. We established a human VEGF secreting cell line (BHK-VEGF) and encapsulated the cells into hollow fibers. The encapsulated cells were unilaterally transplanted into the striatum of adult rats at 1 or 2 weeks after 6-OHDA lesions, and animals subsequently underwent behavioral and immunohistochemical evaluations. Compared to lesioned rats that received BHK-Control capsules, lesioned rats transplanted with BHK-VEGF capsules showed a significant reduction in the number of amphetamine-induced rotations, a significant preservation of TH-positive neurons in the substantia nigra pars compacta, and a remarkable glial proliferation in the striatum, with the earlier transplantation exerting much more benefits than the delayed transplantation. Parallel studies revealed that the observed in vitro and in vivo neurorescue effects were likely mediated by VEGF's angiogenic and glial proliferative effects, as well as its direct effects on the neurons. Our results suggest that VEGF is a highly potent neurorescue molecule for Parkinson's disease therapy.

Hepatic transdifferentiation of bone marrow cells has been previously demonstrated by intravenous administration of donor cells, which may recirculate to the liver after undergoing proliferation and differentiation in the recipient's bone marrow. In the present study, to elucidate which cellular components of human bone marrow more potently differentiate into hepatocytes, we fractionated human bone marrow cells into mesenchymal stem cells (MSCs), CD34+ cells, and non-MSCs/CD34- cells and examined them by directly xenografting to allylalcohol (AA)-treated rat liver. Hepatocyte-like cells, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), albumin (Alb), cytokeratin 19 (CK19), cytokeratin 18 (CK18), and asialoglycoprotein receptor (AGPR), and by reverse transcription-polymerase chain reaction (RT-PCR) for expression of AFP and Alb mRNA, were observed only in recipient livers with MSC fractions. Cell fusion was not likely involved since both human and rat chromosomes were independently identified by fluorescence in situ hybridization (FISH). The differentiation appeared to follow the process of hepatic ontogeny, reprogramming of gene expression in the genome of MSCs, as evidenced by expression of the AFP gene at an early stage and the albumin gene at a later stage. In conclusion, we have demonstrated that MSCs are the most potent component in hepatic differentiation, as revealed by directly xenografting into rat livers. (Blood. 2005;106:756-763)

Background— Apoptosis is an important cause of early graft loss after heart transplantation. Bcl-xL was reported to protect the heart against normothermic ischemia and reperfusion injury. In this study, we determined whether overexpression of Bcl-xL could inhibit tissue injury resulting from prolonged cold preservation followed by warm reperfusion of heart transplants. Methods and Results— Lewis rat hearts were transduced with an adenovirus vector harboring Bcl-xL cDNA (AxCAhBclxL) 4 days before collection of tissue. After preservation in University of Wisconsin solution at 4°C for 24 hours, the heart was either perfused with a Langendorff device ex vivo or used for heterotopic heart transplantation in vivo. Bcl-xL gene transfer significantly reduced the infarct size (23.0±2.6% versus 47.7±7.0% in saline control and 48.6±6.1% in vector control, P P c release from the mitochondria; it also significantly decreased cardiac cell apoptosis and improved graft survival rate after long cold preservation, followed by warm reperfusion. Conclusions— Bcl-xL gene transfer inhibited the translocation of Bax and prolonged the cold preservation time of cardiac transplants. This may be a potential therapeutic method in clinical practice.

We analyzed the effects of the Janus kinase 3 (Jak3)-specific inhibitor WHI-P131 (4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) and the Jak3/Syk inhibitor WHI-P154 (4-(3′-bromo-4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) on the antigen-induced activation of mast cells. In the rat mast cell line RBL-2H3, both WHI-P131 and WHI-P154 inhibited the antigen-induced degranulation and phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK and c-Jun N-terminal kinase (JNK). The phosphorylation of Gab2, Akt and Vav was also inhibited by WHI-P131 and WHI-P154, indicating that these inhibitors suppress the activation of phosphatidylinositol 3-kinase (PI3K). In bone marrow-derived mast cells (BMMCs) from Jak3-deficient (Jak3−/−) mice, degranulation and activation of MAPKs were induced by the antigen in almost the same extent as in BMMCs from wild-type mice. In addition, the antigen-induced degranulation and activation of MAPKs were inhibited by WHI-P131 and WHI-P154 in both groups of BMMCs, indicating that these compounds inhibit a certain step except for Jak3. The antigen-induced increase in the activity of Fyn, a probable tyrosine kinase of Gab2, was also inhibited by WHI-P131 and WHI-P154 in RBL-2H3 cells. In BMMCs from Jak3−/− mice, the antigen stimulation induced tyrosine phosphorylation of Fyn, which was inhibited by WHI-P131, as well as in BMMCs from wild-type mice and in RBL-2H3 cells. These findings suggest that Jak3 does not play a significant role in the antigen-induced degranulation and phosphorylation of MAPKs, and that WHI-P131 and WHI-P154 inhibit the PI3K pathway by preventing the antigen-induced activation of Fyn, thus inhibiting the antigen-induced degranulation and phosphorylation of MAPKs in mast cells. Keywords: Mast cells, WHI-P131, WHI-P154, p44/42 MAP kinase, p38 MAP kinase, c-Jun N-terminal kinase, phosphatidylinositol 3-kinase, Fyn Introduction Mast cells contribute to inflammatory responses by releasing preformed mediators such as histamine and serine proteases, and generating eicosanoids and cytokines (Williams & Galli, 2000). The early signaling events in antigen-stimulated mast cells are initiated by the tyrosine kinases Lyn and Syk (Beaven & Metzger, 1993; Beaven & Ozawa, 1996). The aggregation of the IgE high-affinity receptor I (FcɛRI) induced by the antigen results in the tyrosine phosphorylation of the β- and γ-chains of FcɛRI by Lyn. The phosphorylation of these chains promotes the recruitment of Lyn and Syk to the β-chain and the γ-chain, respectively, resulting in the tyrosine phosphorylation of linker proteins such as linker for activation of T cells (LAT). Furthermore, the tyrosine kinase Fyn is required for mast cell degranulation (Parravicini et al., 2002). Fyn phosphorylates a linker protein Gab2 (Parravicini et al., 2002), and leads to the activation of phosphatidylinositol 3-kinase (PI3K) (Gu et al., 2001; Wilson et al., 2001). PI3K regulates the translocation of Bruton's tyrosine kinase (Btk) (Buhl & Cambier, 1999; Varnai et al., 1999) to membrane, promoting the activation of Btk by Lyn/Syk (Rawlings et al., 1996; Baba et al., 2001). The activation of these tyrosine kinases induces the tyrosine phosphorylation of phospholipase (PL) Cγ (Li et al., 1992) and Vav (Hirasawa et al., 1995b). The former leads to the generation of inositol 1, 4, 5-trisphosphate and diacylglycerol, which induce an increase in the intracellular Ca2+ level and the activation of protein kinase C, respectively. The latter activates low molecular weight G proteins such as Ras and Rac (Gulbins et al., 1994; Han et al., 1998; Abe et al., 2000), resulting in the activation of the mitogen-activated protein kinase (MAPK) family. The activation of MAPKs in mast cells causes the release of arachidonic acid (Hirasawa et al., 1995a) and the production of cytokines such as interleukin (IL)-4 (Hirasawa et al., 2000) and IL-13 (Hirasawa et al., 2003). Janus kinase 3 (Jak3), a member of the Jak family of cytoplasmic nonreceptor tyrosine kinases, is selectively expressed in hematopoietic cells (Johnston et al., 1994; Witthuhn et al., 1994) and associates with the γc-chain of receptors for IL-2, 4, 7, 9, 15 and 21 (Chen et al., 1997; Asao et al., 2001). Jak3 mediates cytokine-induced responses by activating the cytoplasmic latent forms of signal transducers and activators of transcription (STATs) via phosphorylation of a specific tyrosine residue near the SH2 domain (Leonard & O'Shea, 1998). In addition, Jak3 has been suggested to play important roles in the FcɛRI-mediated activation of mast cells (Malaviya & Uckun, 1999; Malaviya et al., 1999, 2000) and T-cell receptor-mediated activation of T cells (Tomita et al., 2001). In Jak3-deficient (Jak3−/−) mice, the anaphylactic reaction was impaired with defective immune responses (Malaviya & Uckun, 1999; Malaviya et al., 1999). Jak3 also plays roles in bacterial clearance and neutrophil recruitment to the sites of infection by regulating the release of tumor necrosis factor-α from mast cells (Malaviya et al., 2001). In addition, stimulation of the rat mast cell line RBL-2H3 with antigen induced the activation of Jak3 and the specific Jak3 inhibitor WHI-P131 (4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) (Sudbeck et al., 1999) inhibited the antigen-induced degranulation, production of tumor necrosis factor-α and increase in the cytosolic Ca2+ level without affecting the activation of Syk (Malaviya et al., 1999). However, the precise role of Jak3 in the antigen-triggered signaling events in mast cells remains to be clarified. In this study, we evaluated the effects of the specific Jak3 inhibitor WHI-P131 and the Jak3/Syk inhibitor WHI-P154 (4-(3′-bromo-4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) (Ghosh et al., 1999) on the IgE/FcɛRI-mediated activation of RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) from Jak3−/− mice and wild-type mice, and found that these inhibitors strongly suppressed the antigen-induced degranulation and phosphorylation of MAPKs in mast cells via the Jak3-independent pathway.

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35 x 10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5. (c) 2005 Elsevier Inc. All rights reserved.

In order to enhance the efficacy of conditionally replicating adenoviruses (CRAd) in the treatment of cancers of the biliary tract, we studied the efficacy in vitro and in vivo of AxE1CAUP, a CRAd vector that carries a gene for uracil phosphoribosyltransferase (UPRT), which converts 5-fluorouracil (5-FU) directly to 5-fluorouridine monophosphate and greatly enhances the cytotoxicity of 5-FU. AxE1CAUP replicated and induced an increased UPRT expression in biliary cancer cells more efficiently than AxCAUP, a nonreplicative adenovirus carrying the UPRT gene. Whereas AxCAUP and AxE1AdB, a CRAd without the UPRT gene, modestly increased the sensitivity of BC cells to 5-FU, AxE1CAUP markedly increased the sensitivity, especially when the timing of 5-FU administration was appropriately chosen. AxE1CAUP replicated much less efficiently in normal WI-38 fibroblasts without any change in the sensitivity to 5-FU. In nude mice with s.c. biliary cancer xenografts, i.t. AxE1CAUP/5-FU therapy inhibited tumor growth significantly more strongly than AxCAUP/5-FU or AxE1AdB/5-FU therapy. Furthermore, in mice with peritoneally disseminated biliary cancer, i.p. AxE1CAUP efficiently proliferated in the tumors, decreased the tumor burden, and prolonged the survival of the mice when 5-FU was started 10 or 15 days after the vector inoculation, whereas earlier initiation of 5-FU resulted in early eradication of the vector and no survival benefit. The present study shows that the CRAd expressing UPRT was a more potent sensitizer of biliary cancer to 5-FU, than was a nonreplicative UPRT-encoding vector or a CRAd without UPRT gene, even at a lower dose of the vector, and that timing of 5-FU administration was a key factor to maximize the efficacy. This gene therapy with appropriately timed administration of 5-FU should be useful in overcoming the resistance of biliary cancers to 5-FU.

Background Bone regeneration therapy using mesenchymal stem cells (MSCs) is beginning to come into clinical use. To overcome the difficulty of healing large bone defects, we previously reported the efficacy of using rat mesenchymal stem cells (rMSCs) carrying a modified adenoviral vector (Adv-F/RGD) with an RGD-containing peptide in the HI loop of the fiber knob domain of adenovirus type 5 (Ad5). Methods Firstly, we evaluated the transduction efficiency of Adv-F/RGD into bone-marrow-derived human MSCs (hMSCs) using a β-galactosidase chemiluminescent assay. Next, we evaluated whether the vector AxCAhBMP2-F/RGD carrying the human bone morphogenetic protein 2 (BMP2) gene could enhance the osteogenic activity of hMSCs in vitro and in vivo (in an ectopic model). In the ectopic model, transduced hMSCs, hMSCs in the presence of recombinant human BMP2 (rhBMP2) or hMSCs alone were implanted into a subcutaneous site of nude mice. We also applied this vector system to an orthotopic model (large bone defect model) using rMSCs. Results The transduction efficiency of Adv-F/RGD into hMSCs was increased 10-fold over the vector containing the wild-type fiber (Adv-F/wt), as assessed by a β-galactosidase chemiluminescent assay. AxCAhBMP2-F/RGD increased the osteogenic activity of hMSCs in vitro. In the ectopic model, AxCAhBMP2-F/RGD-transduced hMSCs were found to induce new bone at 1 week after transplantation, and a greater quantity of new bone was formed than in other groups. Similarly, AxCAhBMP2-F/RGD-transduced rMSCs induced a greater quantity of new bone than other groups (AxCAhBMP2-F/wt-transduced rMSCs, rMSCs in the presence of rhBMP2, rMSCs alone, or scaffolds alone) in the orthotopic model. Conclusions These data suggest that Adv-F/RGD is useful for introducing foreign genes into MSCs and that it will be a powerful gene therapy tool for bone regeneration and other tissue-engineering applications. Copyright © 2005 John Wiley & Sons, Ltd.