水上 浩明

Adeno-associated virus (AAV) vector is suitable for gene transfer to the central nervous system. However, the efficacy of gene therapy for neuroendocrine disease is still unknown. In this study, we injected AAV vector encoding arginine-vasopressin (AVP) stereotaxically into the bilateral hypothalamus of Brattleboro rats. Brattleboro rats show a central diabetes insipidus (CDI) phenotype and growth retardation due to a complete deficiency of AVP. Following injection, both urine volume and urine osmolality normalized, and these therapeutic effects persisted for more than 50 weeks. In addition to phenotypic correction, secretion of transgene-derived AVP was enhanced after 24 h water deprivation or hypertonic saline injection, and water diuresis was demonstrated after acute water loading. Also, reduced body weight and low plasma insulin-like growth factor 1 levels of Brattleboro rats were restored after AVP gene transduction, suggesting the importance of AVP in growth. These findings indicate that hypothalamic neurons of Brattleboro rats can produce and release mature AVP following AAV-mediated gene transduction, resulting in long-term phenotypic correction of CDI. Moreover, the fact that transgene-derived AVP was secreted adequately in response to stimuli, even if it was expressed constitutively, suggests advantages of gene therapy for neuroendocrine diseases and offers a basis to investigate AVP function.

Background Although adenoviral vectors primarily derived from the adenovirus serotype 5 (Ad5) are widely used for many gene transfer applications, they cannot efficiently infect hematopoietic cells, since these cells do not express the coxsackie-adenoviral receptor (CAR). Methods We have developed a soluble fusion protein that bridges adenoviral fibers and the c-Kit receptor to alter Ad5 tropism to immature hematopoietic cells. The CAR-SCF fusion protein consists of the extracellular domains of CAR and stem cell factor (SCF). The human megakaryoblastic leukemia cell lines UT-7 and M07e, human chronic myelogenous leukemia cell line K-562, and erythroleukemia cell line TF-1 were used to assess CAR-SCF-assisted Ad5-mediated gene transfer. Hematopoietic cell lines were infected with an Ad5 vector (Ad5-eGFP) or a fiber-mutant Ad5/F35 (Ad5/F35-eGFP) expressing the enhanced green fluorescent protein gene in the presence or absence of CAR-SCF. Results Twenty-four hours after infection, more than 80% of M07e cells infected in the presence of CAR-SCF were eGFP-positive, compared with very few eGFP-positive cells following AdS-eGFP infection in the absence of CAR-SCF. The enhancement of Ad5-eGFP infection by CAR-SCF was greater than that caused by Ad5/F35-eGFP (50%). The ability of CAR-SCF to enhance Ad5-eGFP infectivity was highly dependent on cellular c-Kit expression levels. Furthermore, CAR-SCF also enhanced Ad5-mediated gene transfer into human primary CD34(+) cells. Conclusions The CAR-SCF fusion protein assists Ad5-mediated transduction to c-Kit(+) CAR(-) hematopoietic cells. The use of this fusion protein would enhance a utility of Ad5-mediated hematopoietic cell transduction strategies. Copyright (C) 2003 John Wiley Sons Ltd.

Peritoneal dissemination is the major progression pathway of ovarian cancer, and its control is important for improvement of the prognosis. PTEN is a tumor suppressor gene, and is known to inhibit cancer cell growth and migration. To investigate the possibility of gene therapy using PTEN for ovarian cancer, we introduced PTEN cDNA into an ovarian cancer cell line HRA carrying wild-type PTEN, and examined the effects in vitro and in vivo. Using PTEN cDNA cloned from a human liver cDNA library, a PTEN expression vector was constructed. This vector was introduced into HRA cells by the standard calcium phosphate precipitation method, and an HRA cell line overexpressing PTEN (HRA/PTEN) was established. On the cell migration test by in vitro scratch wound healing assay, the number of migrating cells was 6.3 +/- 0.9 cells/mm(2) in HRA/PTEN, which was significantly smaller than that in the control (39.7 +/- 3.2 cells/mm(2)) (p<0.01). No significant differences were observed in the in vitro cell growth or in vivo tumor growth between HRA/PTEN and the control. The findings described above, show that enhanced expression of PTEN inhibits ovarian cancer cell migration, suggesting that gene therapy approaches using PTEN for control of peritoneal dissemination of ovarian cancer are possible.

The prognosis of cancers of various organs overexpressing thymidylate synthase (TS) has been reported to be poor. It has been suggested that the poor prognosis is partly due to a low sensitivity of TS-overexpressing tumors to TS-targeting 5-fluorouracil (5-FU). To investigate the relationship between TS expression and sensitivity to 5-FU, we used the TS-overexpressing cervical cancer cell line SKG-II/TS and SKG-I/TS that had been established by TS gene transfer. The 50% growth inhibitory concentration (IC50) of 5-FU for SKG-II/TS was 24 +/- 6.0 muM, which was 6 times as high as that for the control (4.0 +/- 1.1 muM), showing significantly decreased sensitivity to 5-FU (p < 0.01). The IC50 of 5-FU for SKG-I/TS was 90 +/- 15 muM, which was over 2 times as high as that for the control (40 +/- 0.6 muM), showing significantly decreased sensitivity to 5-FU (p < 0.05). Thus, TS-overexpressing tumors have decreased sensitivity to 5-FU, which may be one of the factors that determine the prognosis of these tumors. (C) 2003 Wiley-Liss, Inc.

Interleukin-10 (IL-10) is an immunosuppressive cytokine produced by T lymphocytes and drawing attention as an inhibitor of tumor angiogenesis. In this study, we investigated antiangiogenic and tumor suppressive effects of IL-10 in ovarian cancer cells. mIL-10-expressing phismid was transferred into two ovarian cancer cell lines, SHIN-3 [vascular endothelial growth factor (VEGF) producing] and KOC-2S (non-VEGF producing). After selection, mIL-10-expressing cells were obtained as SHIN-3/mIL-10 and KOC-2S/mIL-10. No significant differences were observed in in vitro growth properties between mIL-10-expressing cells and control (luciferase expressing) cells in either KOC-2S or SHIN-3. The angiogenic activities of mIL-10-expressing cells were measured by dorsal air sac assay, which detected the number of newly formed blood vessels within a chamber in vivo. In addition, tumor formation was evaluated by s.c. tumor transplantation, and survival was monitored after i.p. injection of ovarian cancer cells into BALB/c nude mice. Both in vivo angiogenic activity and tumor growth were significantly inhibited in SHIN-3/mIL-10 cells compared with the control. Moreover, peritoneal dissemination was inhibited, and the survival period was significantly prolonged (mean survival days >90 versus 36). In contrast, in the case of KOC-2S cells, no significant differences were observed in any of the parameters tested. These results indicate that IL-10 has suppressive effects on angiogenesis, tumor growth, and peritoneal dissemination of VEGF-producing ovarian cancer cells. Although the mechanisms of the antiangiogenic effect of IL-10 are still unclear, the potential usefulness of IL-10-mediated gene therapy of ovarian cancer was suggested.

Allogeneic bone marrow transplantation and donor lymphocyte infusion are powerful treatments for chemotherapy-resistant leukemia. Tumor eradication is attributed to a graft-versus-leukemia reaction by the donor-derived cytotoxic T lymphocytes (CTLs), but the same cell population may cause severe graft-versus-host disease. One strategy to suppress harmful CTL activity is to incorporate a suicide gene into the donor lymphocytes prior to infusion, and to destroy these cells when they aggressively attack nonmalignant host tissues. In this study, we investigated the feasibility of using a Fas-estrogen receptor fusion protein (MfasER) to control T cell-mediated cytotoxicity, based on our previous finding that the chimera transmits a Fas-mediated death signal through activation by estrogen binding. A murine CTL line CTLL-2 was transfected with a vector encoding MfasER, and the growth, viability and cytotoxic activity of the transfected cells (CTLL/MfasER) were analyzed. The expression of apoptosis-related proteins such as Fas ligand and perforin was also investigated. In the absence of estrogen, CTLL/MfasER showed similar growth to parental CTLL-2, and the killing activity was preserved. Addition of 10(-7) M estrogen induced a rapid apoptosis of CTLL/MfasER, and the cytotoxicity was severely impaired. A decrease of Fas ligand and perforin in the estrogen-treated CTLL/MfasER was seen in an immunoblot analysis. These functional and biochemical analyses showed that the estrogen-inducible apoptosis in MfasER-expressing CTLs rapidly terminated their target cell killing. The feasibility of using the MfasER-estrogen system to control graft-versus-host disease was demonstrated.

Background DNA vaccines have been used to induce both humoral and cellular immune responses against infectious microorganisms. This study explores whether DNA vaccine immunogenicity can be improved by introducing inverted terminal repeats (ITRs) from adeno-associated virus (AAV) into the regulatory region of the DNA plasmid. Methods CMV promoter-driven HIV Env expressing plasmid (pCMV-HIV) and the pCMV-HIV plasmid introduced ITRs (pITR/CMV-HIV) were transfected in HEK293 cells with LipofectAmine. The HIV Env expression was quantified with Western blot. Fifty mug of pCMV-HIV or pITR/CMV-HIV plasmid with RIBI adjuvant were immunized to BALB/c mice on days 0, 14 and 28 by intramuscular route, and HIV-specific serum IgG titer was detected 2, 6, 10, 14 and 18 weeks after the first immunization. HIV-specific tetramer assay and HIV-specific IFN-gamma ELIspot assay were performed 1 week after the last immunization. The immune mice were intravenously challenged with a vaccinia virus expressing the HIV env gene 1 week after the last immunization. Results Significantly higher level of HIV Env expression was achieved by pITR/CMV-HIV plasmid. BALB/c mice immunized with pITR/CMV-HIV plasmid generated significantly higher HIV-specific antibody, higher cellular immune responses and lower viral loading than animals immunized with pCMV-HIV plasmid. Conclusions AAV ITRs enhance CMV-dependent up-regulation of transgene expression and immunogenicity of DNA vaccine. Copyright (C) 2002 John Wiley Sons, Ltd.

Genetic modification of the gerbil hippocampal neuronal cells in vivo helps us understand the mechanisms of neuronal function under various circumstances such as ischemic insult. In this study, we examined the distinct distribution of the recombinant adeno-associated virus type 2 (rAAV2) and rAAV5 vectors for gene delivery to primary cultured cells and the gerbil hippocampus. Mixed cortical cultures containing both neurons and astrocytes from E17 rat embryos were infected with rAAVs containing the Cytomegalovirus virus (CMV) promoter. rAAV2 was preferably transduced to neurons, whereas rAAV5 was inclined to be transduced to astrocytes in vitro. rAAV2 and rAAV5 vectors, each with the CMV or Rous sarcoma virus (RSV) promoter, were injected into the gerbil hippocampus using a stereotaxic apparatus. Five days after injection, transgene expression was analyzed with X-gal staining. In the gerbil hippocampus, rAAV5 with the CMV promoter achieved, a higher overall transgene expression than rAAV2 with the CMV promoter. The transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells, while the transgene expression of rAAV5 with the RSV promoter was preferentially found in the granular cells. These findings would be valuable in optimizing rAAV-mediated gene transfer to the gerbil hippocampus. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Background Hematopoietic stem-cell-directed gene transfer has achieved limited success in transducing clinically relevant levels of target cells. The expansion of gene-modified cells is one way to circumvent the problem of inefficient transduction with current vectors. To this end, we have developed 1 selective amplifier genes' (SAGS) that encode chimeric proteins that are a fusion of granulocyte colony-stimulating factor receptor and the steroid-binding domain. Prototype SAGS conferred estrogen-responsive growth on murine hematopoietic progenitors. Methods We constructed a retroviral vector coexpressing an SAG for 4-hydroxytamoxifen (Tm)-specific proliferation and the enhanced green fluorescent protein (EGFP). Murine bone marrow cells were transduced with this vector and transplanted into myeloablated mice. Subsequently, recipients were challenged with Tm, and EGFP(+) cells were enumerated. Results The challenge induced a significant increase in EGFP(+) leukocytes (21 +/- 4% to 27 +/- 5%), while EGFP(+) cells decreased in untreated animals (21 +/- 5% to 10 +/- 3%). Three months later, bone marrow cells were transplanted from the unchallenged mice to secondary hosts. Again the administration of Tm resulted in an increase of EGFP(+) cells (16 +/- 4% to 35 +/- 3%), contrasting to a decrease in controls (22 +/- 4% to 12 +/- 4%), and the difference was significant for more than 3 months. A detailed study of lineage showed a preferential expansion of EGFP(+) cells in granulocytes and monocytes following Tm administration. Conclusions Long-term repopulating cells were transduced with the SAG, and the transduced granulocyte/monocyte precursors were most likely to be expandable in vivo upon Tm stimulation. Copyright (C) 2002 John Wiley Sons, Ltd.

Adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for amyotrophic lateral sclerosis (ALS) and other motor neuron diseases. The purpose of this report was to investigate transgenic GDNF expression at different time points post AAV mediated GDNF intramuscular delivery. An AAV vector was constructed to encode a recombinant fusion of GDNF tagged with a FLAG sequence at the C-terminal (AAV-GDNF) to distinguish it from its endogenous counterpart. A single intramuscular injection of AAV-GDNF led to substantial expression of transgenic GDNF which remained for at least 10 months in transduced gastrocnemius muscle. This transgenic GDNF was distributed in a large number of myofibers, mainly in the vicinity of the sarcolemma and predominantly concentrated at the sites of neuromuscular junctions (NMJs). Furthermore, transgenic GDNF, but not P-galactosidase expressed as a control, was detected in the motoneurons that projected axons to the injected muscles, thus, indicating retrograde axonal transportation of the transgenic GDNF. This study provides a basis for a strategy of intramuscular AAV-GDNF delivery to protect motoneurons as a possible means of ALS treatment. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Although gene transfer into hematopoietic stem cells holds a considerable therapeutic potential, clinical trials targeting this cell compartment have achieved limited success. Poor transduction efficiency with gene transfer vectors used in human studies has hindered delivering therapeutic genes to clinically relevant numbers of target cells. One way to overcome the low-efficiency problem is by selecting or expanding the number of genetically modified cells to a suprathreshold level to achieve clinical efficacy. This approach may be further classified into 2 categories: one is to transfer a drug resistance gene and eliminate unmodified cells with cytotoxic drugs, and the other is to confer a direct growth advantage on target cells. This review aims at an overview of recent advances involving these strategies, with some details of "selective amplifier genes," a novel system that we have developed for specific expansion of genetically modified hematopoietic cells.

Objective. To evaluate both the potential for transferring genes to periosteal cells using an adeno-associated virus (AAV) vector and the potential for gene expression after transplantation of those cells to a cartilage defect in vivo. Methods. Periosteum was obtained from the tibia of 6-week-old rabbits and enzymatically digested. The isolated periosteum derived cells were cultured and the subconfluence cells were infected with a recombinant AAV expressing the LacZ gene (AAV-LacZ). Collagen gel containing the LacZ transferred, periosteum derived cells was transplanted into a full thickness articular cartilage defect in 10 rabbits. Results. Infected cells still growing on the plate continued to express LacZ at least 12 weeks after AAV infection, with the highest percentage of LacZ positive cells reaching 74.4%. The LacZ positive cells were recognized at the transplant sites in 8 out of 10 knees. Conclusion. Gene expression in periosteum derived cells was sustained in vitro for at least 12 weeks using the AAV vector, and for 2 weeks ex vivo after transplantation into a cartilage defect.

Oral vaccines can induce both systemic and mucosal immunity. Mucosal immunity, especially regional cell-mediated immunity, plays an important role in protecting individuals from infectious diseases such as acquired immunodeficiency syndrome. In this study, a recombinant adeno-associated virus vector expressing human immunodeficiency virus type 1 env gene (AAV-HIV) was orally administered to BALB/c mice. Systemic and regional immunity was induced in the mice. Furthermore, the immunization significantly reduced viral load after an intrarectal challenge with a recombinant vaccinia virus expressing HIV env gene. Moreover, we also show that dendritic cells might contribute to the AAV-HIV vector- induced immune responses.

The cytokine receptor common gamma chain (gammac) plays a pivotal role in transmitting multiple interleukin signals. Its gene mutations cause profound lymphoid maldevelopment, a disorder known as X-linked severe combined immunodeficiency (X-SCID). Without successful bone marrow transplantation, affected patients would die in infancy or early childhood due to severe and recurrent infections. Therefore, development of gene therapy strategies has been awaited for those without suitable marrow donors. We constructed a retrovirus vector carrying the gammac and the enhanced green fluorescent protein (EGFP) genes for a preclinical study with a murine X-SCID model. After transduction of X-SCID bone marrow and infusion into myeloablated X-SCID recipients, robust lymphoid reconstitution was observed in all the examined lymphocyte subsets such as CD4(+)-T, CD8(+)-T, B and NK cells. A long-term EGFP expression was demonstrated in both lymphoid and myeloid cells, suggesting that this type of vector is useful in evaluating regimens of stem cell-directed gene therapy.

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for ALS and other motor neuron diseases. Because adeno-associated virus (AAV) has been developed as an attractive gene delivery system with proven safety, we explored the therapeutic efficacy of intramuscular delivery of the GDNF gene mediated by an AAV vector (AAV-GDNF) in the G93A mouse model of ALS. We show here that AAV-GDNF leads to substantial and long-lasting expression of transgenic GDNF in a large number of myofibers with its accumulation at the sites of neuromuscular junctions. Detection of GDNF labeled with FLAG in the anterior horn neurons, but not beta-galactosidase expressed as a control, indicates that most of the transgenic GDNF observed there is retrogradely transported GDNF protein from the transduced muscles. This transgenic GDNF prevents motoneurons from their degeneration, preserves their axons innervating the muscle, and inhibits the treated-muscle atrophy. Furthermore, four-limb injection of AAV-GDNF postpones the disease onset, delays the progression of the motor dysfunction, and prolongs the life span in the treated ALS mice. Our finding thus indicates that AAV-mediated GDNF delivery to the muscle is a promising means of gene therapy for ALS.

The cytokine receptor common gamma chain (gammac) plays a pivotal role in multiple interleukin signaling, and gammac gene mutations cause an X-linked form of SCID (X-SCID). Recently, gammac gene transfer into the autologous X-SCID BM achieved appreciable lymphocyte reconstitution, contrasting with the limited success in previous gene therapy trials targeting hematopoietic stem cells. To understand the mechanisms underlying this success, we examined the repopulating potential of the wild-type (WT) BM cells using an X-SCID mouse model. Limited numbers of WT cells were infused into non-ablated WT and X-SCID hosts. Whereas no appreciable engraftment was observed in WT recipients, donor-derived lymphocytes repopulated well in X-SCID, reaching 37% (10(6) cells given) and 53% (10(7) cells given) of the normal control value 5 months post BMT. A lineage analysis showed a predominance of the donor-derived lymphocytes (CD4(+) T, CD8(+) T, B and NK cells) in X-SCID while the donor-derived granulocytes and monocytes engrafted poorly. These results showed a selective advantage of WT cells in X-SCID, and that the advantage was restricted to lymphocytes. In human gene therapy for X-SCID, an analogous growth advantage would greatly enhance the repopulation of lymphocytes derived from a very small number of gammac gene-supplemented precursors.

Purpose: PTEN is a tumor suppressor gene that was identified on chromosome 40q23. In addition to its original function as a tumor suppressor, this gene product was recently reported to enhance the sensitivity of cancer cells to anticancer agents. It is for the purpose of this study to investigate its function and the mechanisms by which PTEN enhances the sensitivity of ovarian cancer to antitumor agents. Experimental Design: PTEN cDNA was introduced into the ovarian cancer cell line SHIN-3 and a high-expression cell line (SHIN-3/PTEN) was established. This cell line and a control were further analyzed. Results: SHIN-3 cells did not carry any mutations in its genome after sequencing. In vitro examination of sensitivity to anticancer agents showed that the 50% growth -inhibitory concentration value for irinotecan metabolite (SN-38) in SHIN-3/PTEN was 800 nM, a 6.6-fold higher sensitivity compared with that of the control (5300 nM). There were no differences in sensitivity to cisplatin, paclitaxel, or gemcitabine between SHIN-3/PTEN and the controls. The percentage of apoptotic cells in SHIN-3/PTEN was 16.6 +/- 0.7% 24 h after addition of SN-38, a significant increase over controls (8.6 +/- 0.9%; P < 0.01). Lower topoisomerase I activity was observed in SHIN-3/PTEN, compared with controls. Conclusions: These results indicate that high PTEN expression enhances the sensitivity, of ovarian cancer cells to irinotecan and the induction of apoptosis and the suppression of topoisomerase I activity in cancer cells are suggested as possible mechanisms attributable to high PTEN expression.

Vascular endothelial growth factor (VEGF), a bifunctional protein enhancing vascular permeability and stimulating endothelial growth, is thought to be responsible for fluid accumulation and angiogenesis in ascites tumors. To investigate the effects of stable expression of the soluble form of Fit-1 VEGF receptor (sFit-1), a known endogenous inhibitor of VEGF, on the malignant ascites tumors, we cotransduced RMG-1 human ovarian cancer cells with adeno-associated virus vectors carrying the sFlt-1 cDNA and Neo gene or Neo gene alone and isolated both the sFlt-1-expressing clone and the Neo-expressing clone. In vitro growth characteristics were essentially the same. As expected, conditioned medium collected from the sFlt-1-expressing cells significantly inhibited the human umbilical vein endothelial cell proliferation in the presence of recombinant VEGF. Expression of sFlt-1 significantly suppressed RMG-1 cell-induced angiogenesis in vivo in the mouse dorsal air sac assay model. We then inoculated sFlt-1- or Neo alone-expressing cells i.p. into female BALB/c nude mice. The average volume of ascites fluid, number of leaked RBCs, and number of cancer cells were significantly lower in mice injected with sFlt-1-expressing cells than in the controls. Survival time was significantly prolonged in mice injected with sFlt-1-expressing cells. These results suggest that inhibition of VEGF activity by sFlt-1 expression may provide a means to control carcinomatous ascites and angiogenesis of malignant ascites tumors.

Objectives: Clinical trials on therapeutic angiogenesis using vascular endothelial growth factor (VEGF) are ongoing, however the benefits of these therapies are still controversial. To establish a more efficient gene transfer method for ischemic diseases, we investigated the therapeutic potential of adeno-associated virus (AAV)-mediated VEGF gene transfer. Methods: We produced VEGF(165)-express in AAV vectors (AAV-VEGF). HEK-293 cells were transduced with AAV-VEGF in vitro and VEGF expression and secretion were examined. We used a rat ischemic hindlimb model and AAV-VEGF was administered intramuscularly into the ischemic limb. Gene expression was evaluated by RT-PCR and ELISA. Six weeks after gene transfer, we measured the blood flow of limb vessels and the skin temperature of limbs. Histochemical examination was performed to illustrate capillary growth. Results: Western blotting and ELISA revealed VEGF protein expression and secretion from AAV-VEGF-transduced HEK-293 cells. VEGF mRNA and protein expression was consistently observed in the injected muscle at least 10 weeks after the injection, while no VEGF mRNA could be detected at remote organs. The mean blood flow in AAV-VEGF-transduced ischemic limbs was significantly higher than in AAV-LacZ-transduced limbs. Capillary density was significantly higher in AAV-VEGF-injected tissues than in AAV-LacZ-injected tissues. Conclusions: This study demonstrates that (1) AAV-mediated VEGF gene transfer into rat skeletal muscles is efficient and stable without ectopic expression, and (2) AAV-mediated VEGF gene transfer stimulates angiogenesis and thereby improves blood flow in a rat hindlimb ischemia model. These findings suggest that AAV-mediated VEGF gene transfer may be useful for treatment of ischemic diseases. (C) 2002 Elsevier Science BY All rights reserved.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AA V) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AA V vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ), into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AA V-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AA V vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.

It has been shown that there is an inverse relationship between the level of thymidylate synthase (TS) and therapeutic outcomes in patients with malignancies including cervical cancer. To clarify the mechanism of the poor prognosis of cervical cancer with high TS expression, we introduced TS cDNA to the human uterine cervical cancer cell line SKG-II and evaluated the effect of TS expression on its radiosensitivity. After selection, stable transformants of SKG-II cells expressing high level of TS (SKG-II/TS) and control cells (SKG-II/luciferase) were obtained. The level of TS measured by the FdUMP-TS binding assay was significantly higher (p < 0.05) in SKG-II/TS than in control (2.0 +/- 0.1 and 1.3 +/- 0.1 pmol/mg, respectively). No difference was observed in in vitro cell growth or in vivo tumor growth. On evaluation of in vitro radiosensitivity, the 50% growth inhibitory dose (ID50) was 3.1 +/- 0.1 Gy in SKG-II/TS and was significantly higher (p < 0.01) than that in control (2.2 +/- 0.1 Gy). From these results, it is suggested that one of the reasons of poor outcome of cervical cancer to radiation is high TS expression.

The ability of an adeno-associated virus vector expressing the env gene from HIV-1 (AAV-HIV) to induce a protective mucosal immune response when administered orally was studied. A single oral administration of AAV-HIV stimulated strong HIV-specific mucosal, humoral immunity and generated MHC class I restricted CTL in BALB/c mice. This response significantly reduced the viral load after intra-rectal challenge with a recombinant vaccinia virus expressing the HIV env gene. These results demonstrate that oral administration of an AAV-based HIV vaccine elicits a strong immune response that protects against mucosal infection.

Since hematopoietic stem cells (HSCs) differentiate readily ex vivo resulting in the loss of self-renewal and engraftment abilities, the transient block of differentiation is essential to maintain those abilities during their ex vivo expansion culture. To this end, we developed a method of reversible integration of the dominant negative retinoic acid receptor (DN-RAR) gene, a differentiation-blocking gene, into cells utilizing the Cre/loxP-dependent gene recombination system. The murine immature hematopoietic 32D cells differentiate into mature neutrophils upon G-CSF treatment. However, 32D cells transduced with a retroviral vector expressing the DN-RAR gene put between two loxP sites continued to proliferate without showing differentiation even in the presence of G-CSF. After the cells were fully amplified, the cells were transduced with the Cre recombinase gene. The cells then restored the ability to differentiate into mature neutrophils upon G-CSF treatment. PCR analysis showed that the DN-RAR gene was efficiently removed from the genome by introduction of the Cre gene. This system may eventually be applicable to the ex vivo expansion of HSCs. (C) 2001 Academic Press.

Adeno-associated virus (AAV) vector has several unique properties suited for gene therapy applications. However, relatively low efficiency of transgene expression, which is mainly due to a limited second-strand synthesis from the single-stranded AAV genome, can be a problem in some applications that require potent gene expression such as antitumor applications. Recently, gamma -ray irradiation has been reported to enhance the second-strand synthesis of the AAV genome, and consequently transgene expression. We demonstrate here that an AAV vector harboring the herpes simplex virus type-1 thymidine kinase (HSVtk) is able to kill cancer cells more efficiently when used in combination with gamma -ray irradiation. A human maxillary sinus cancer cell line, NKO-1, was efficiently killed in combination with HSVtk transduction and ganciclovir (GCV), as expected. More importantly, gamma -ray irradiation of practical dosages augmented the cytocidal effect of the HSVtk/GCV system. Southern analysis indicated that gamma -rays enhanced the double-strand synthesis of the rAAV genome in NKO-1 cells. These findings suggest that the combination of rAAVtk/GCV suicide gene therapy with radiotherapy has synergistic effects in the treatment of cancers and may lead to a reduction of the potential toxicity of both rAAVtk/GCV and gamma -ray irradiation.

Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related dependovirus AAV-3, We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2, The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID50 of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.

A 65-year-old man with marked leukocytosis was admitted for diagnosis and treatment. His peripheral blood leukocyte count was 37,500/mu-l and the leukocytes consisted of mature neutrophil-like cells. A high neutrophil alkaline phosphatase score and a normal bone marrow cell karyotype suggested that the patient had chronic neutrophilic leukemia rather than chronic myeloid leukemia. Several neutrophil functions, such as superoxide production, nitroblue tetrazolium reduction activity, and phagocytosis, were elevated. These data and the morphological features (toxic granules and Dohle bodies) indicated that the patient's neutrophils were in an activated stage.

Granulocyte colony-stimulating factor (G-CSF) is known to act on the neutrophilic granulocytes from chronic myelogenous leukemia (CML) patients to induce neutrophil alkaline phosphatase (NAP) activity. Gamma-interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been reported to suppress NAP induction with G-CSF. We confirmed that this inhibitory effect of GM-CSF is accompanied by the decrease of the NAP mRNA level. Moreover, we found that the simultaneous addition of retinoic acid completely neutralized this inhibitory effect of GM-CSF. Recovery of the NAP activity brought about by the retinoic acid was also accompanied by the increase of NAP mRNA level. These results indicate that retinoic acid neutralizes the inhibitory effect of GM-CSF on the induction of NAP activity through the change of the NAP mRNA level.

To investigate the physiologic role of macrophage colony-stimulating factor (M-CSF) in hematological recovery from bone marrow hypoplasia, we used an enzyme-linked immunosorbent assay to measure serial changes of the serum M-CSF level during 25 intensification chemotherapy courses given to seven patients with acute non-lymphocytic leukemia who were in complete remission. Three M-CSF peaks were observed during therapy: the first peak was during or just after chemotherapy, the second peak was around the leukocyte nadir, and the third peak coincided with a rapid increase in the monocyte count. We could find no significant correlation between the height of the second peak and the time from the initiation of therapy to hematological recovery. On the other hand, there was a significant positive correlation between the height of the second peak and the interval from the last day of chemotherapy to the peak (r = 0.62, p = 0.001), and there was a significant negative correlation between the peak height and the time from the peak until hematological recovery (defined as a neutrophil count of over 500/mu-l (r = -0.63, p = 0.001) and a leukocyte count of over 1,000/mu-l (r = 0.55, p = 0.008). However, we found only a weak correlation between the peak height and monocyte recovery. These data suggest that increased M-CSF levels lead to the stimulation of granulocyte progenitors, and that we can predict the time of neutrophil recovery by monitoring the serum M-CSF level and finding its peak.

A 71-year-old woman with leukocytosis was admitted for treatment of malignant lymphoma. During the clinical course, neutrophilia of unknown origin occurred in parallel with the progression of the malignant lymphoma. The supernatant of lymphoma tissue culture contained a high titer of granulocyte colony-stimulating factor (G-CSF), and lymphoma cells were positive when immunohistochemically stained by anti-G-CSF antibody. Western blot analysis and mouse colony assay of the supernatant also confirmed that the lymphoma produced G-CSF.

We examined steady-state levels of mRNA for alkaline phosphatase in neutrophils (NAP) treated with granulocyte colony-stimulating factor (G-CSF). The amount of mRNA for NAP was shown to increase after 6 hours of culture with G-CSF when no increase in NAP activity was yet observed, and the transcript was the greatest after 20-24 h of culture with G-CSF. Treatment of neutrophils with both G-CSF and retinoic acid augmented the amount of mRNA for NAP over the amount obtained by G-CSF alone, which was most marked at 24 h. These results show that both G-CSF-mediated NAP induction and its potentiation by retinoic acid are due to the increased levels of mRNA for NAP.