Syusuke Moriuchi

Double suicide gene therapy using a replication defective herpes simplex virus vector reveals reciprocal interference in a malignant glioma model

Herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli cytosine deaminase (CD) are non-mammalian enzymes capable of converting innocuous prodrugs into cytotoxic metabolites. Both enzymes have been utilized independently, as well as together in ‘suicide’ gene therapy protocols to eliminate tumor cells in vitro and in vivo. We have used a set of replication defective HSV vectors expressing either or both enzymes to compare the efficacies of single and double suicide gene therapies in the 9L gliosarcoma model in vitro and in vivo. In cell culture experiments at high and low multiplicities of infection, combined expression of the two genes by vector TOCD/TK along with exposure to the matching prodrugs (ganciclovir and 5-fluorocytosine) showed increased cytotoxicity compared with exposure to either prodrug alone. However, the two gene combination was inferior to single gene treatments, suggesting that HSVtk and CD are mutually counteractive in the prodrug-dependent killing of glioma cells. In animal experiments, survival was not significantly prolonged by administration of both prodrugs to TOCD/TK-treated animals, while each single gene/prodrug pair resulted in increased survival. These results indicate that single suicide gene systems employing HSVtk or CD may be preferable over combinations of the two.

HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma.

Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (tk) gene to tumor cells in combination with ganciclovir (GCV) administration may provide an effective suicide gene therapy for destruction of malignant glioblastomas. However, because HSV is a highly cytotoxic agent, gene expression from the virus is short-lived which may limit the effectiveness of HSVtk/GCV therapy. Using different replication-defective HSVtk gene vectors, we compared HSV vector backgrounds for their cytotoxic activity on infection of 9L gliosarcoma cells in culture and brain tumors in rats and evaluated the impact of vector toxicity on the effectiveness of tk/GCV-mediated suicide gene therapy. As reported previously for other cell lines, a vector deleted for both copies of the immediate–early (IE) gene ICP4 (SOZ.1) was highly toxic for 9L cells in culture while a vector deleted in addition for the ICP22 and ICP27 IE genes (T.1) reduced or arrested 9L cell proliferation with more limited cell killing. Nevertheless, both vectors supported widespread killing of uninfected cells in the presence of GCV following low multiplicity infections, indicating that vector cytotoxicity did not preempt the production of vector-encoded TK enzyme necessary for the killing of uninfected cells by the HSV-tk/GCV bystander effect. Although an SOZ.1-related vector (SHZ.2) caused tumor cell necrosis in vivo, injection of SHZ.2 at multiple coordinates thoughout the tumor followed by GCV administration failed to prolong markedly the survival of tumor- bearing rats. In contrast, a single injection of T.1 produced a life-extending response to GCV. These results indicate that vector cytotoxicity can limit the efficacy of HSV-tk/GCV treatment in vivo, which may be due to premature termination of tk gene expression with attendant abortion of the bystander effect.

Humanization of mouse ONS-M21 antibody with the aid of hybrid variable regions.

Mouse monoclonal antibody, ONS-M21, directed against human medulloblastoma cells, has been humanized by complementarity determining region (CDR) grafting. A humanized ONS-M21 VH region, comparable to the original mouse ONS-M21 VH region, was easily constructed based on framework regions (FRs) 1, 2 and 3 from human EU antibody and on FR4 from human ND antibody. Five alterations in the FRs were made at amino acids 27, 28, 29, 30 and 94 which are all part of the canonical structure for CDR1 (H1). The humanized ONS-M21 VL regions were constructed based on the FRs from human REI antibody. We first identified five amino acid residues in the FRs at positions 20, 21, 71, 73 and 87 as having a possible adverse influences on antigen binding. None of the versions with a variety of combinations at these five positions showed any bindings to antigen. In order to identify the mouse residues that must be retained in the human FRs, hybrid VL regions were constructed by joining the mouse ONS-M21 VL region and the first humanized version within CDR2. The hybrid VL regions revealed that residues in FR1 and/or FR2 were critical in creating a functional antigen binding site. Redesigning several versions with alterations in FR1 and FR2 revealed that the Pro-46 residue was the only critical residue for creating an antigen binding site. This approach should be helpful in identifying key residues in difficult cases of antibody humanization.

Sustained release of low-dose ganciclovir from a silicone formulation prolonged the survival of rats with gliosarcomas under herpes simplex virus thymidine kinase suicide gene therapy

A silicone formulation of ganciclovir (GCV-pellet) was developed to enhance the cytotoxic effects of herpes simplex virus thymidine kinase suicide gene therapy. The effectiveness of this drug delivery system was assessed in a rat 9L gliosarcoma model. The GCV-pellets (1 mm in length and in diameter) used in this experiment contained a total amount of 0.15 mg of GCV. In vitro experiments demonstrated that GCV was gradually released over a period of 7 days. Five days after stereotactic tumor inoculation into the right caudate nucleus, a herpes simplex virus type 1 (HSV-1) vector expressing herpes simplex virus thymidine kinase (HSV-tk) (T1, 2×106 pfu) was administered at the same location. The survival rate of the group treated with the GCV-pellet was compared with that of the T1 group injected intraperitoneally (IP) with GCV (30 mg/kg/day for 7 days). The GCV-pellet-treated group had a significantly prolonged survival (a median of more than 80 days) compared with the GCV IP group (a median of 65 days) and with control groups (P<0.05). The control groups (untreated or receiving only the virus vector) had a survival of 35–38 days. The survival rate of the GCV-pellet group over 80 days was 75%, and all the rats that survived more than 80 days and did not show tumors upon histological examination of the brain were deemed cured. No toxic effects or immunological reactions were observed histologically around the pellet in brain sections from the rats treated with the GCV-pellet. After GCV-pellet inoculation into the tumor, drug concentrations were kept at 1–10 μg/g tissue for 3–4 days. When the same dose of GCV (0.15 mg) in aqueous solution was injected into the tumor, GCV concentrations reached a peak of 0.5 mg/g tissue after 30 min and decreased below measurable level within 12 h. After IP injections of 3 mg GCV, GCV concentrations in the tumor reached a peak of 5.7 μg/g tissue after 30 min and also decreased below measurable level within 12 h. This sustained release of a low and effective GCV dose with the silicone formulation significantly prolonged survival in combinations with HSV-tk expression if compared to IP administration of GCV. Histological examination suggests that the treatment appears to be safe.

Characterisation of a new mouse monoclonal antibody (ONS-M21) reactive with both medulloblastomas and gliomas

We developed an IgG1 mouse monoclonal antibody (ONS-M21) directed against a cell surface antigen of medulloblastomas and gliomas in immunisation of mice with the ONS-76 medulloblastoma cell line. The antibody specifically reacted with medulloblastomas, supratentorial primitive neuroectodermal tumours (SPNETs) and gliomas, but not with other neuroectodermally derived tumours (neuroblastoma and melanoma) or with other kinds of tumours (meningioma, neurinoma, leukaemia, and small cell lung cancer). No reactivity was identified with normal body tissues, including peripheral blood cells. Characterisation of the ONS-M21 antigen showed that it was a trypsin-sensitive glycoprotein with a molecular weight of 80 kDa on SDS-PAGE. The pattern of reactivity and the biochemical properties of this antigen were different from those of other markers of medulloblastoma. These results indicate that ONS-M21 detects a new tumour-associated cell surface antigen specifically expressed by medulloblastomas, SPNETs, and gliomas. This is the first report that medulloblastomas may share common cell surface antigens with gliomas, although most studies have concluded that medulloblastoma has a predominantly neuronal phenotype. The lack of reactivity with normal tissue implies that ONS-M21 has potential applications as both a diagnostic tool and a therapeutic agent.

Neural transplantation in mouse Parkinson's disease.

A complete recovery from the methamphetamine-induced rotational response was shown in C57BL/6 (H-2b) mice which had had unilateral 6-OHDA lesions in the nigrostriatal pathway about 60 days after transplantation of approximately 1 x 10(6) dopamine-rich cells from syngeneic or allogeneic (C3H/HeN, H-2k) mouse embryos (ED 15), without immunosuppressive agents. Morphological examination showed tyrosine-hydroxylase-immunoreactive cell clusters around the needle tract in the mice which were transplanted not only with syngeneic cells but also with allogeneic cells. This might indicate that so-called immunosuppressive agents are not necessary for grafted embryonic cells to survive in an allogeneic mouse brain.

Biological Characterization of Human Medulloblastoma (ONS-76 and ONS-81) Cell Lines

Medulloblastomas are the most common malignant brain tumors in children. The origins of medulloblastoma cells are controversial. Most neuropathologists believe them to have originated from immature precursor cells which have the potentiality to differentiate into either neuronal or glial cells [1]. Although it is very difficult to establish a cell line of human medulloblastoma [2,3], we have established 2 cell lines with neuronal characteristics.

Murine Models with Leptomeningeal Dissemination of Human Medulloblastoma Cells

The 5-year survival rate of patients with medulloblastoma has improved as much as 70% in the past 15 years. This dramatic improvement is mainly due to advances in radiotherapy [1–3]. Although medulloblastomas are certainly radiosensitive, they are not always radiocurable because of limited irradiation dosages and adverse effects on the whole neural axis. Sooner or later, tumor regrowth takes place, and the meningeal dissemination of the tumor cells then results in the demise of most patients with medulloblastoma [4].

Correlation between graft survival and host immunological responses after intracerebral transplantation of fetal brain cells in mouse Parkinson's models

SURVIVE AND GROW RAPIDLY WITHOUT IMMUNOSUPPRESSION Joseph Yanai 1, Thomas C. Doetschman 5, Neri Laufer 3, Julia Maslaton t Shlomo Mor-Yosef ~, Anat Safran~, Moshe Shani 4, Dov Softer ~and Emanuel A. Winston 1. Ross Lab for Neural Birth Defects Lab Dept. Anatomy & Embryol. t , Pathoi. 2, & Dept. Obst. Gynec 3. Hebrew Univ. -Hadassah Med. Schl,, Jerusalem, Dept. Animal Sci. Volcani Ct~. Israel & Univ Cinc. Ohio, USA 5.

Investigation of MHC Antigens on Neural Graft in Mouse Parkinson’s Models

Parkinson’s disease is one of the most common disorders that affect the nigrostriatal system in human. Loss of neurons in the substantia nigra and severe depletion of dopamine (DA) in the corpus striatum are the hallmarks of this condition. The most effective treatment of Parkinson’s disease, particularly in its early phase, is the administration of both the L-isomer of 3,4-dihydroxyphenylalanine (L-DOPA) and dopamine-receptor agonists. However, it is clear that L-DOPA and the receptor agonists provide only limited ameliorable treatment and that most patients inexorably become progressively debilitated regardless of the type of therapy. On the other hand, an alternative approach in the therapeutic management of Parkinson’s disease is surgical implantation of either DA neurons or adrenal chromaffin cells that have the ability to release DA.1