Sapporo Medical University Dept. Molecular Medicine Latest renewal date of this page was August 27, 2004. RESEARCH SUMMARY 2001-2004   Molecular Medicine   Our research interests are directed at the elucidation of the molecular mechanisms underlying disease and their applications for the better treatment of patients.  Various novel techniques of gene therapy and regenerative medicine are developed by us and are applied for preclinical studies.  In collaboration with clinical groups, several clinical studies in gene thrapy and regenerative medicine are ongoing. Professor Hirofumi Hamada, M.D., Ph.D. Interests: Gene therapy for cancer, Regenerative medicine for hematopoietic, cardiovascular, and neural diseases.   Associate Professor Kazunori Kato, Ph.D. Interests: Cancer immunology and immunogene therapy.   Instructor Yoshinori Ito, M.D. Kiminori Nakamura, D.D., Ph.D. sachie Hirai     1. Reduction of natural adenovirus tropism. The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR). This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression level of CAR and alphav integrin, both CAR binding and fiber shaft played important roles in efficient transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no significant differences in transduction efficiency were observed among adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue types with specific receptors.   2. Gene therapy for neovascular formation.  Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) play important roles in vascular formation and maturation, suggesting that the combination of these two would be a promising therapy for ischemia. However, it remains unclear what the best schedule of administration of these cytokines might be.  Six experimental groups were used to prepare the rabbit ischemic hindlimb model following naked plasmid intramuscular administration as follows: empty vector (C), single gene (Ang1, A; VEGF, V), Ang-1 followed by VEGF (A - V), co-administration of Ang1 and VEGF (A + V), and VEGF followed by Ang1 (V - A).   Thirty days after gene administration, A - V showed a significantly increased blood pressure and blood-flow recovery in the ischemic limb compared with the control group. Histological findings by alpha-smooth muscle-actin (alpha-SMA) staining revealed that the two combination groups had more mature vessels as compared with the control group. Significantly, A - V revealed the highest density of alpha-SMA-positive vessels compared with VEGF alone or Ang1 alone. Angiographic assessment revealed that A - V had a greater increased arterial diameter compared with VEGF alone. Edema, one of the major adverse effects induced by VEGF, was not found in A - V throughout the experiments, while VEGF alone and V - A showed severe edema induced by VEGF.   The pre-administration of Ang1 followed by VEGF resulted in an improvement of hemodynamic status, an increased number of vessels covered with alpha-actin-positive mural cells, and prevention of VEGF-mediated edema. Thus, priming by Ang1 gene administration would be beneficial for therapeutic angiogenesis in VEGF gene therapy.   3. Hematopoietic support of human stromal cells. Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However, the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34(+) cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells, and its receptor molecules were detected in CD34(+) cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential, which was elevated compared with control stromal cells, as indicated by the colony-forming units in culture (CFU-Cs) (26-fold +/- 2-fold versus 59-fold +/- 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's], 63-fold +/- 37-fold versus 349-fold +/- 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency-ss(2)m(-/-) (NOD/SCID-ss(2)m(-/-)) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results, and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID-ss(2)m(-/-) RCs proliferated efficiently on Ihh stromal cells, compared with control stromal cells. These results indicate that Ihh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells.   4. BDNF gene-modified mesenchymal stem cells (MSC) for cerebral infarction. 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.   5. Antitumor effect of genetically engineered MSC for malignant glioma. The prognosis of patients with malignant glioma is extremely poor, despite the extensive surgical treatment that they receive and recent improvements in adjuvant radio- and chemotherapy. In the present study, we propose the use of gene-modified mesenchymal stem cells (MSCs) as a new tool for gene therapy of malignant brain neoplasms. Primary MSCs isolated from Fischer 344 rats possessed excellent migratory ability and exerted inhibitory effects on the proliferation of 9L glioma cell in vitro. We also confirmed the migratory capacity of MSCs in vivo and showed that when they were inoculated into the contralateral hemisphere, they migrated towards 9L glioma cells through the corpus callosum. MSCs implanted directly into the tumor localized mainly at the border between the 9L tumor cells and normal brain parenchyma, and also infiltrated into the tumor bed. Intratumoral injection of MSCs caused significant inhibition of 9L tumor growth and increased the survival of 9L glioma-bearing rats. Gene-modification of MSCs by infection with an adenoviral vector encoding human interleukin-2 (IL-2) clearly augmented the antitumor effect and further prolonged the survival of tumor-bearing rats. Thus, gene therapy employing MSCs as a targeting vehicle would be promising as a new therapeutic approach for refractory brain tumor.   List of Main Publications from 2001 to 2004 Technology development for gene therapy: Nakamura T, Sato K. and Hamada H.  Effective Gene Transfer to Human Melanomas via Integrin-Targeted Adenoviral Vectors.  Hum. Gene Ther.,13(5): 613-626 (2002). Nakamura T, Sato K and Hamada H.  Reduction of natural adenovirus tropism to the liver by both ablation of fiber-Coxsackievirus and adenovirus receptor interaction and use of replaceable short fiber.  J. Virol., 77(4): 2512-2521 (2003). Regenerative medicine and gene therapy for cardiovascular diseases: Yamauchi, A, Ito, Y, Morikawa, M, Kobune, M, Huang, J, Sasaki, K, Takahashi, K, Nakamura, K, Dehari, H, Niitsu, Y, Abe, T and Hamada, H. 　Pre-administration of angiopoietin-1 followed by VEGF induces functional and mature vascular formation in a rabbit ischemic model.  J. Gene Med., 5(11):994-1004(2003). Takahashi K, Ito Y, Morikawa M, Kobune M, Huang J, Tsukamoto M, Sasaki K, Nakamura K, Dehari H, Ikeda K, Uchida H, Hirai S, Abe T and Hamada H  Adenoviral delivered angiopoietin-1 reduces the infarction and attenuates the progression of cardiac dysfunction in the rat model of acute myocardial infarction.  Mol. Ther. 8(4):584-592(2003). Regenerative medicine using bone marrow stem cells: Tsuda H., Wada T., Ito Y., Uchida H., Dehari H., Nakamura K., Sasaki K., Kobune M., Yamashita T. and Hamada H.  Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber-mutant adenoviral vector.  Mol. Ther., 7(3): 354-365(2003). Kawano Y, Kobune M, Yamaguchi M, Nakamura K, Ito Y, Sasaki K, Takahashi S, Nakamura T, Chiba H, Sato T, Matsunaga T, Azuma H, Ikebuchi K, Ikeda H, Kato J, Niitsu Y and Hamada H.  Ex vivo　expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subnit (hTERT)-transfected human stromal cells. Blood, 101(2): 532-540 (2003). Kobune M, Ito Y, Kawano Y, Sasaki K, Uchida H, Nakamura K, Dehari H, Chiba H, Takimoto R, Matsunaga T, Terui T, Kato J, Niitsu Y, Hamada H.  Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-{beta}2m-/- repopulating cells.  Blood. 104(4):1002-1009 (2004). Kurozumi, K., Nakamura, K., Tamiya, T., Kawano, Y., Kobune, M., Hirai, S., Uchida, H., Sasaki, K., Ito, Y., Kato, K., Honmou, O., Houkin, H., Date, I., and Hamada, H.   BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model.  Mol. Ther.  9(2):189-97 (2004). Cancer Gene Therapy and Immunothrapy Nakamura K, Ito Y, Kawano Y, Kurozumi K, Kobune M, Tsuda H, Bizen A, Honmou O, Niitsu Y, and Hamada, H.   Anti-tumor effect of genetically engineered mesenchymal stem cells in a rat glioma model.  Gene Ther. 11(14):1155-64 (2004). Uchida H, Tanaka T, Sasaki K, Kato K, Dehari H, Ito Y, Kobune M, Miyagishi M, Taira K, Tahara H, Hamada H.  Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo.  Mol Ther. 10(1):162-71 (2004). Key words: Gene therapy, Regenerative medicine, Cancer, Immunotherapy, Mesenchymal stem cell, Adenoviral vector

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