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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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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．

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.

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.

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.

Mesenchymal stem cells (MSC) were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we previously transfected the telomerized human MSC with the BDNF gene using a fiber-mutant adenovirus vector and reported that such treatment contributed to improved ischemic recovery in a rat transient middle cerebral artery occlusion (MCAO) model. In the present study, we investigated whether other cytokines in addition to BDNF, i.e., GDNF, CNTF, or NT3, might have a similar or greater effect in this model. Rats that received MSC-BDNF (P < 0.05) or MSC-GDNF (P < 0.05) showed significantly more functional recovery as demonstrated by improved behavioral test results and reduced ischemic damage on MRI than did control rats 7 and 14 days following MCAO. On the other hand, rats that received MSC-CNTF or MSC-NT3 showed neither functional recovery nor ischemic damage reduction compared to control rats. Thus, MSC transfected with the BDNF or GDNF gene resulted in improved function and reduced ischemic damage in a rat model of MCAO. These data suggest that gene-modified cell therapy may be a useful approach for the treatment of stroke.

Liver is the most preferential site for metastasis of colon cancer. We, in the present study, constructed a self-replicable adenovirus in which E1A is driven by a CEA promoter and E1B-55K is deleted from the E1B region (AdCEAp/Rep) and examined its effects on multiple metastases of a human colon cancer cell in a mouse xenograft model. We first showed effective replication of the virus in various CEA-producing human colon cancer cells (M7609, HT-29) and subsequent lysis of the infected cells in vitro. We then demonstrated that a single intratumoral injection of the virus (1 x 10(8) PFU/100 microl) induced a complete regression of subcutaneous tumors (M7609) inoculated into nude mice. Further, we demonstrated that systemic administration of the virus (1 x 10(8) PFU/100 microl) through the tail vein to nude mice, which 1 week prior had been inoculated with tumor cells (colon carcinoma cell line HT-29) via the spleen and showed apparent multiple metastases in the liver, effectively suppressed the metastasis formation. The mean survival time of the treated mice was significantly longer than that of the controls. Thus, the systemic administration of AdCEAp/Rep was considered to be effective on multiple liver metastases of CEA-positive colon cancer in a xenograft model.

We produced lethally irradiated retrovirally GM-CSF-transduced autologous renal tumor cell vaccines (GVAX) from six Japanese patients with stage IV renal cell cancer (RCC). Four patients received GVAX ranging from 1.4 × 108 to 3.7 × 108 cells on 6–17 occasions. Throughout a total of 48 vaccinations, there were no severe adverse events. After vaccination, DTH skin tests became positive to autologous RCC (auto-RCC) in all patients. The vaccination sites showed significant infiltration by CD4+ T cells, eosinophils, and HLA-DR-positive cells. The kinetic analyses of cellular immune responses using peripheral blood lymphocytes revealed an enhanced proliferative response against auto-RCC in four patients, and cytotoxicity against auto-RCC was augmented in three patients. T cell receptor β-chain analysis revealed oligoclonal expansion of T cells in the peripheral blood, skin biopsy specimens from DTH sites, and tumors. Western blot analysis demonstrated the induction of a humoral immune response against auto-RCC. Two of the four patients are currently alive 58 and 40 months after the initial vaccination with low-dose interleukin-2. Our results suggest that GVAX substantially enhanced the antitumor cellular and humoral immune responses, which might have contributed to the relatively long survival times of our patients in the present study.

Gene targeting using short interfering RNA (siRNA) has become a common strategy to explore gene function because of its prominent efficacy and specificity. For the application of siRNA technology to gene therapy, however, still more efficient transduction of siRNA into target cells is needed. In this study, we developed an adenoviral vector harboring a tandem-type siRNA expression unit, in which sense and antisense strands composing the siRNA duplex were separately transcribed by two human U6 promoters. Targeting survivin, an antiapoptotic molecule widely overexpressed in malignancies but not detected in terminally differentiated adult tissues, this type of adenoviral vector (Adv-siSurv) successfully exerted a gene knockdown effect and induced apoptosis in HeLa, U251, and MCF-7 cells. These cancer cells, once infected with Adv-siSurv, displayed remarkably attenuated growth potential, both in vitro and in vivo. Moreover, intratumoral injection of Adv-siSurv significantly suppressed tumor growth in a xenograft model using U251 glioma cells. This novel modality may be a promising tool for cancer therapy.

The prognosis of gastric cancer with peritoneal metastasis has not improved. Despite many promising studies, gene therapy has limited clinical application because of the lack of suitable vector systems to enable selective gene transduction to tumor cells. The aim of this study was to clarify whether gene therapy targeted to peritoneal mesothelial cells (PMCs) can inhibit peritoneal dissemination of gastric cancer. In vitro experiments showed that adenovirus expressing LacZ infected human omental tissue-derived PMCs more efficiently than human gastric cancer cell lines MKN1 and MKN45. When adenovirus expressing LacZ was injected into the peritoneal cavity of nude mice, the expression was detected in the peritoneum for at least 4 weeks. Furthermore, when adenovirus expressing soluble Flt-1 (Ad-sFLT-1) was i.p. administered in vivo, a high level of sFlt-1 protein could be detected in peritoneal lavage for 8 weeks. When MKN45 cells were i.p. inoculated 3 days after adenoviral vector injection, Ad-sFLT-1 markedly reduced the number of metastatic nodules larger than 1 mm in diameter on the peritoneal surface, and significantly prolonged the survival of nude mice without any significant side effects. Thus, peritoneal dissemination was significantly suppressed by a single i.p. injection of Ad-sFlt-1. Anti-angiogenic gene therapy targeted to PMCs could be a novel and practical strategy against peritoneal dissemination of gastric cancer, because it does not require tumor-specific gene transfer.

Vascular endothelial growth factor (VEGF) has previously been shown to display neuroprotective effects following ischemia, suggesting that VEGF may potentially be applied as a neuroprotective agent for the treatment of other neurological diseases. In this study, we investigated the neuroprotective capacity of VEGF in a model of Parkinson's disease. VEGF was found to be neuroprotective against cell death of primary E14 murine ventral mesencephalic neurons induced by 6-hydroxydopamine (6-OHDA) treatment in vitro. Further, rats receiving a continuous infusion of VEGF into the striatum via encapsulated hVEGF-secreting cells (baby hamster kidney-VEGF) displayed a significant decrease in amphetamine-induced rotational behavior and a significant preservation of tyrosine hydroxylase-positive neurons and fibers compared with control animals. VEGF likely functions via direct mechanisms by signaling through the neuropilin receptor expressed upon dopaminergic neurons in response to 6-OHDA treatment. Further, VEGF is likely to promote neuroprotection indirectly by activating the proliferation of glia and by promoting angiogenesis. Our results support a potential neuroprotective role for VEGF in the treatment of Parkinson's disease.

Examination of the clinical therapeutic efficacy of using bone marrow stromal cells, including mesenchymal stem cells (MSC), has recently been the focus of much investigation. MSC were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we transfected telomerized human MSC with the BDNF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether these cells contributed to improved functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. BDNF production by MSC-BDNF cells was 23-fold greater than that seen in uninfected MSC. Rats that received MSC-BDNF showed significantly more functional recovery than did control rats following MCAO. Specifically, MRI analysis revealed that the rats in the MSC-BDNF group exhibited more significant recovery from ischemia after 7 and 14 days. The number of TUNEL-positive cells in the ischemic boundary zone was significantly smaller in animals treated with MSC-BDNF compared to animals in the control group. These data suggest that MSC transfected with the BDNF gene may be useful in the treatment of cerebral ischemia and may represent a new strategy for the treatment of stroke.

We developed a new human stromal cell line that could expand human hematopoietic progenitor/stem cells. Primary human bone marrow stromal cells were infected with retrovirus containing the human telomerase catalytic subunit (hTERT) gene, resulting in increased population doubling and the acquisition of cell immortalization. Characteristics of the hTERT-transduced stromal (hTERT-stromal) cells were identical with those of the primary stromal cells in terms of morphologic appearance and expression of surface antigens. Human cord blood (CB) CD34+ cells were expanded by coculture with primary stromal or hTERT-stromal cells in the presence of stem cell factor, thrombopoietin, and Flk-2/Flt-3 ligand under serum-free condition. The degree of expansion of CD34+ cells and total number of colony-forming units in culture (CFU-Cs) after 2 weeks' coculture with the hTERT-stromal cells were nearly the same as those after 2 weeks' coculture with primary stromal cells (CD34+ cells, 118-fold ± 8-fold versus 117-fold ± 13-fold; CFU-Cs, 71-fold ± 5-fold versus 67-fold ± 5-fold of initial cell number). CB expansion on hTERT-stromal cells occurred at a similar rate through 7 weeks. In contrast, the rate of CB expansion on primary stromal cells had drastically declined at 7 weeks. In nonobese diabetic/severe combined immunodeficiency (SCID) mice, the degree of engraftment of SCID-repopulating cells that had been cocultured with hTERT-stromal cells for 4 weeks was significantly higher than that of precocultured CB cells. These results indicate that this hTERT-stromal cell line could be useful for ex vivo expansion of hematopoietic progenitor/stem cells and for analyzing the microenvironment of human bone marrow.

Radiation therapy is an established modality for the treatment of malignant gliomas. Several reports have shown the advantage of additional radiation in combination with gene therapy. In this study, we investigated the ability of radiation therapy to enhance 5-fluorocytosine (5-FC)/cytosine deaminase (CD) plus uracil phosphoribosyltransferase (UPRT) gene therapy in malignant gliomas. In vitro study suggested evidence of a significant cytotoxic interaction between radiation therapy and 5-FC/CD + UPRT gene therapy for glioma cells. In vivo experiments demonstrated that the combination of gene therapy and radiation possessed superior antitumor effect in comparison to single therapy. However, the adverse effects of radiation therapy in combination with the gene therapy were observed with respect to normal brain. This combination therapy may be feasible for the treatment of gliomas, although the radiation dose and area should be reduced in order to prevent side effects.

Non-amyloid beta (Abeta) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Abeta protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC(50) = 5.6 microm) than in astrocytes (IC(50) approximately 50 microm). The neuron-specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre-treatment with the antioxidants, propyl gallate (PG) and N-t-butyl-phenylnitrone (BPN), or overexpression of human Bcl-2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2',7'-dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl-tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF-kappaB) and enhanced its DNA-binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF-kappaB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.

Aims: To evaluate a new delivery system of 5-fluorouracil (5-FU) using 5-fluorocytosine (5-FC) as a prodrug and cytosine deaminase induced in vitro and in vivo. Methods: Fibroblastic cells from rabbit Tenon’s capsule were cultured. The cells were exposed to 5-FU and 5-FC with or without cytosine deaminase induced by recombinant adenovirus. In the in vitro study, cell proliferation and DNA synthesis were assessed by MTS, BrdU assay. The effect of 5-FC removal after the treatment of 5-FC and cytosine deaminase induction was also assayed. In the in vivo study cells with or without cytosine deaminase induction were transplanted into the subconjunctival space of mice, followed by eye drops of 1000 µg/ml of 5-FC three times a day. The mice were sacrificed at days 1, 5, and 10, then the cells transplanted were evaluated. Results: Cell proliferation was inhibited by exposure to 5-FU in a dose dependent manner; however, up to 1000 µg/ml of 5-FC did not affect cell proliferation. Cell proliferation was inhibited by exposure to 5-FC in a time dependent manner with induction of cytosine deaminase following infection of recombinant adenovirus. When 5-FC was removed 3 or 6 days after the treatment, the cells grew again. The effect was reproduced in the in vivo model of subconjunctival cellular proliferation although 5-FC was administrated as eye drops. There were no cases with corneal erosion. Conclusion: Cell proliferation was inhibited by co-exposure of 5-FC and cytosine deaminase. This new delivery system may merit controlled delivery of 5-FU after filtering surgery.

Objective The aim of this study was to establish a new method to overcome the problems of gene therapy targeting hematopoietic cells, namely low transduction efficiency and induction of differentiation during cytokine treatment. Materials and Methods The K- sam gene encoding the receptor for keratinocyte growth factor (KGF) was transduced to three factor-dependent hematopoietic cell lines (Ba/F3, 32Dcl3, and UT-7/GM) using retroviral vector, and their proliferation, differentiation, and intracellular signaling were studied. This gene also was transduced to murine bone marrow cells, and proliferation of colony-forming cells (CFCs) by KGF stimulation was examined. Results Although KGF is known to target only epithelial cells, all of the three cell lines transduced with K- sam proliferated due to KGF stimulation. Morphologic observation showed that KGF induced proliferation but did not cause significant differentiation of 32D/K- sam cells. KGF treatment increased phosphorylation of ERK1/2 but did not activate STAT molecules. Granulocyte colony-stimulating factor transduced the differentiation signal with the phosphorylation of STAT3 without significant ERK1/2 activation. Proliferation by KGF of murine primary bone marrow cells transduced with K- sam then was examined in liquid culture. KGF treatment significantly increased production of CFCs derived from K- sam –transduced bone marrow cells without causing the exhaustion of immature CFCs. Conclusions KGF could efficiently induce proliferation of hematopoietic cells expressing the K- sam gene without obvious induction of differentiation or exhaustion of immature progenitor cells. The in vitro data are important for further preclinical in vivo study.

The utility of recombinant adenoviral vectors (Adv) for gene therapy is limited by their low transduction efficiency and lack of specificity for target cells. The low transduction efficiency is often recognized as due to deficiency of the primary adenoviral receptor, the coxsackievirus-adenovirus receptor (CAR). In this paper, studies of CAR levels on human melanoma cell lines confirmed that low transduction efficiency was closely related to deficiency of the adenoviral receptor. To achieve CAR-independent gene transfer via Adv, we modified viral tropism via genetic alteration of the adenovirus type 5 (Ad5) fiber protein. Insertion of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain allowed the virus to use an alternative receptor, the integrin receptor, during the cell entry process. With this modified vector (Adv-F/RGD) transduction was increased 5- to 96-fold relative to a vector containing wild-type fiber (Adv-F/wt) in five human melanoma cells expressing integrins of the alpha(v)beta(3), alpha(v)beta(5) class, which are recognized by the RGD peptide motif. In contrast, no significant difference in transduction efficiency between Adv-F/RGD and Adv-F/wt was observed in 293 cells, which show high-level expression of CAR. In this study, we attempted to apply Adv-F/RGD for gene therapy for malignant melanoma. At the same multiplicity of infection, melanoma cells infected with Adv-F/RGD carrying human interleukin 2 (AxCAhIL2-F/RGD) produced a higher level of cytokine than cells infected with AxCAhIL2-F/wt. Treatment by intratumoral injection of AxCAhIL2-F/RGD was more effective than intratumoral injection of AxCAhIL2-F/wt in regressing tumors in a melanoma xenograft model. These data suggest that integrin-targeted adenoviral vectors may be a powerful tool in gene therapy for CAR-deficient melanomas.

Mutation of the p53 gene plays a critical role in the development of cancer and response to cancer therapy. To analyze the mechanism of cancer development and to improve cancer therapy, it is important to assess which genes are downstream components of p53 in cancers, and whether the expression levels of these genes affect p53-mediated apoptosis. In this study, we transduced the wild type p53 gene along with the Apaf-1 and caspase-9 genes via adenovirus vectors into U251 and U-373MG glioma cells harbouring a mutated p53, and evaluated the degree of apoptosis. Co-induction of Apaf-1 and caspase-9 genes highly enhanced p53-mediated apoptosis in glioma cells. Induction of wild type p53 enhanced the expression levels of Bax, p21/WAF1, and Fas protein. To determine which gene is activated by wild type p53 induction and, in turn, activates Apaf-1 and caspase-9, we transduced the Bax, p21/WAF1 or Fas gene via adenovirus vector to U251 cells to achieve a similar expression level as that induced by the Adv for p53 in U251 cells. U251 cells transduced with Fas concomitant with the Apaf-1 and caspase-9 genes underwent drastic apoptosis. This suggests that induction of wild type p53 upregulates Fas, which in turn may play a role in the activation of Apaf-1 and caspase-9. These results are important for analyzing the mechanism of tumour development and for predicting the therapeutic effect of p53 replacement gene therapy in a particular patient. British Journal of Cancer (2002) 86, 587–595. DOI: 10.1038/sj/bjc/6600061 www.bjcancer.com © 2002 Cancer Research UK

Using a syngeneic murine model, we investigated the therapeutic efficacy of combined gene therapy using adenoviral vectors expressing murine interleukin-2 (AdmIL-2) and Escherichia coli cytosine deaminase (AdCD). In a subcutaneous tumor model, tumor-bearing mice were treated with an intratumoral injection of adenoviral vectors and received an intraperitoneal administration of 5-fluorocytosine (5-FC). Only the mice treated with AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) survived significantly longer than mice treated with AdCD alone (P < 0.01). Moreover, 40% of these treated mice obtained complete remission from tumor-bearing status. The cytotoxicity of splenocytes obtained from the treated mice was related to the survival period. Tumor-specific cytotoxic T lymphocyte assay showed that the cell-mediated cytotoxic response was specific for parental tumor cells. In a hepatic metastasis model, mice treated with an intravenous administration of both AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) demonstrated the most significant reduction of metastatic foci and the longest survival following a 5-FC administration. These results suggest that gene therapy combined with AdmIL-2 and AdCD may be a promising strategy for clinical application and, in addition, that translation of combined gene therapy from murine models into the clinical setting will require careful attention to the variables of cytokine expression levels in the design of clinical trials and in the evaluation of treatment efficacy.

Intestinal injury is a major side effect of radiation treatment for many malignancies. The present study investigated whether transducing Bcl-xL, a potent anti-apoptotic gene, into intestinal epithelial cells would exert protective effects against radiation-induced acute injuries. Methods: Adenoviral vectors containing the human Bcl-xL gene (AxCABclxL) or β-galactosidase gene (AxCAlacZ) driven by the CAG promoter were generated. To increase transduction efficiency into the mucosal epithelium, the intraluminal space of the small intestine of mice was washed with buffered-saline and mucus components were digested with Pronase MS®. Gene transduction was performed by injecting 2×108 pfu adenoviral vector into the pre-treated small intestine. Transduction efficiency was examined by X-gal staining 24 hours after AxCAlacZ infection. Radiation-induced acute injury of the small intestine was induced by whole body irradiation (15 Gy) performed 24 hours after adenoviral vector infection. Apoptotic epithelial cells were visualized by TUNEL assay. Morphological analysis was assessed by histological examination. Results: Successful transduction after Pronase MS® treatment was achieved in the basal crypt epithelial cells in the ileum, thought to be the location of stem cells, as determined by X-gal staining. The AxCABclxL group demonstrated significantly fewer radiation-induced apoptotic mucosal epithelial cells when compared with the other two groups at 6 hours after irradiation (p

Advanced esophageal cancers are highly malignant and frequently resistant to 5-fluorouracil (5-FU). Escherichia coli uracil phosphoribosyltransferase (UP) is a pyrimidine salvage enzyme that alters 5-FU metabolism and sensitivity. A recombinant adenovirus encoding the UP gene (AxCA.UP) has been applied in gastric cancer gene therapy to sensitize cancer cells to lower concentrations of 5-FU. We have generated a recombinant adenovirus (AxCA.UT) encoding UP and herpes simplex virus thymidine kinase fusion protein (UT) to examine whether it would enhance the antitumor activity of AxCA.UP treatment. AxCA.UT treatment significantly enhanced the sensitivity of human esophageal cancer cells to and significantly enhanced the growth inhibition effects of UP gene therapy in vitro. Moreover, both 5-FU and ganciclovir showed bystander effects on growth inhibition. In an in vivo study, the therapeutic outcome of AxCA.UT treatment significantly enhanced the antitumor activity of AxCA.UP treatment. These observations suggest that AxCA.UT may be useful in esophageal cancer gene therapy.