久米 晃啓

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.

Peritoneal dissemination is the most frequent progression pathway of ovarian cancer and is therefore a key step, to improve the prognosis. NK4, a large part of the a-chain of hepatocyte growth factor, is known to inhibit cancer cell migration. To characterize the function of NK4 and investigate its potential role in gene therapy of ovarian cancer, we introduced NK4 cDNA to an ovarian cancer cell line HRA and investigated its effects both in vitro and in vivo. HRA cells were transfected with either NK4 or luciferase-expression plasmids. After selection, NK4-expressing HRA cells (HRA/NK4) and the control cells (HRA/LUC) were obtained. NK4 was detected in the culture supernatant of HRA/NK4 by Western analysis. Migration capabilities of the cells were evaluated in vitro by scratch wound healing assay. The number of migrated cells was significantly smaller in the HRA/NK4 cultures than that in the control cultures (HRA or HRA/LUC). Also, the, culture supernatant of HRA/NK4 significantly suppressed migration of control cells. This suppressive effect was observed when NK4-expressing cells were mixed with control cells at the ratio of 25% or more. in the in vivo experiments, HRA transfectants were injected intraperitoneally. The number of intraperitoneal tumors of HRA/NK4 was much smaller than that of control. In mice injected with HRA/NK4, ascites formation was suppressed and the survival was significantly prolonged. These findings suggest that NK4-mediated gene therapy can improve the prognosis of ovarian cancer by suppressing peritoneal dissemination.

We investigated whether nitric oxide (NO) synthase gene transfer could attenuate growth of cultured cardiac myocytes. First, we investigated the effects of exogenous NO and cGMP analog on protein synthesis of cultured neonatal rat cardiac myocytes. The NO donor 3-morpholino-sydnonimine-hydrochloride (SIN-1) and 8-bromo-cGMP caused concentration-dependent decreases in phenylephrine-stimulated incorporation of H-3-leucine into cardiac myocytes. We then transferred endothelial constitutive NO synthase (ecNOS) gene into cultured neonatal rat cardiac myocytes using adeno-associated virus (AAV) vectors. ecNOS gene transfer into cardiac myocytes induced 140 kD ecNOS protein expression and significantly increased cGMP contents of myocytes compared with control cells. ecNOS gene transfer inhibited H-3-leucine incorporation into cardiac myocytes in response to phenylephrine, which was significantly recovered in the presence of the NOS inhibitor N-G-monomethyl-L-arginine acetate. These results indicate that endogenously generated NO by ecNOS gene transfer using AAV vectors inhibits the alpha -adrenergic agonist-induced cardiac protein synthesis at least partially via cGMP production.

A recently reported system for recombinant adenoassociated virus (rAAV) production does not require infection of a helper virus and depends on the transfection with a huge amount of three plasmids: AAV-vector, AAV-helper, and adenovirus-helper plasmids. Toward simplifying rAAV production, as a first step, we tested the use of the rAAV itself instead of the AAV-vector plasmid as a source of rAAV DNA and determined the optimal timing of infection and dose of the input rAAV. When 293 cells were infected just after transfection with 100 particles/cell of rAAV, irrespective of the purity, CsCl-purified or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fold self-amplification), a yield comparable to that obtained by an adenovirus-free transfection. These results indicate that infection of rAAV can greatly reduce the amount of plasmid DNA for a large-scale transfection. This strategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins. (C) 2000 Academic Press.

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy, but current gene transfer methodologies are inadequate for efficient HSC transduction and perpetual transgene expression. To improve gene transfer vectors and transduction protocols, it is vital to establish a system to evaluate transgene expression and the long-term behavior of transduced cells in vivo. For this purpose, we constructed a bicistronic retrovirus encoding the human CD24 (as the first cistron) and the enhanced green fluorescent protein (EGFP; as the second cistron). Murine bone marrow cells were transduced with this vector and the transgene expression was monitored along with hematopoietic reconstitution. Stable expression of CD24 and EGFP was demonstrated in the long-term repopulating cells for at least 6 months, and multi-parameter flow cytometry illustrated expression of both markers in all the lymphohematopoietic lineages examined (B and T lymphoid, erythroid and myeloid). Sustained expression was also shown in the secondary transplants for 6 months, suggesting that self-renewing HSCs were transduced by this vector. Overall, EGFP-tagged bicistronic retroviruses would provide powerful tools for detailed in vivo analysis of transduced hematopoietic cells, such as transgene expression in conjunction with lineage differentiation.

Adeno-associated virus (AAV) vectors have a number of attractive features, including lack of cytotoxicity, ability to transduce nondividing cells, and long-term transgene expression. We investigated whether rat renal cells could be efficiently transduced with AAV vectors. Rat glomerular mesangial cells were transduced with AAV-lacZ vector containing beta-galactosidase gene in vitro, and the expression of beta-galactosidase was evaluated by X-gal staining and ELISA, For ex vivo experiments, sections of rat kidneys were incubated with AAV-lacZ, and then evaluated by X-gal histochemical staining, The level of beta-galactosidase expression in cultured rat mesangial cells increased in a dose-dependent manner (ranging from 1 x 10(5) to 5 x 10(6) particles/cell). When transduced with 5 x 10(6) vector particles/cell of AAV-lacZ, about 50% of mesangial cells were stained positively with X-gal, and the level of beta-galactosidase expression reached 9.9 +/- 1.5 ng/mg protein. Expression was detectable during the culture period for at least 7 days. X-gal histochemical examination of the ex vivo transduced renal tissue revealed tubular cell and interstitial tissue staining. However, gene transfer was not clearly observed in glomeruli. These findings suggest that AAV vectors have the potential for gene therapy of renal diseases. Copyright (C) 2000 S. Karger AG, Basel.

When NIH 3T3 fibroblasts were transduced with a retroviral vector containing a cDNA for porcine pancreatic elastase 1 and cultured in the presence of affinity-purified human plasminogen, the exogenously added plasminogen was digested to generate the kringle 1-3 segment known as angiostatin, a potent angiogenesis inhibitor. This was evidenced by immunoblot analysis of the plasminogen digests using a monoclonal antibody specifically reacting with the kringle 1-3 segment, acid by efficient inhibition of proliferation of human umbilical vein endothelial cells by the plasminogen digests isolated from the culture medium of 3T3 fibroblasts. However, when Lewis lung carcinoma cells were transduced with the same vector and injected subcutaneously into mice in their back or via the tail vein, their growth at the injection sites or in the lungs was markedly suppressed compared with the growth of similarly treated nontransduced Lewis lung carcinoma cells. Nevertheless, the transduced cells were able to grow as avidly as the control cells in vitro. Assuming that the elastase 1 secreted from the transduced cells is likely to be exempt from rapid inhibition by its physiological inhibitor, alpha(1)-protease inhibitor, as shown in the inflammatory tissues, the elastase 1 secreted from the tumor cells may effectively digest the plasminogen that is abundantly present in the extravascular spaces and generate the kringle 1-3 segment in the vicinity of implanted tumor cell clusters. Although the selection of more profitable virus vectors and cells to be transduced awaits further studies, such a protease gene transfer strategy may provide us with a new approach to anti-angiogenesis gene therapy for malignant tumors and their metastasis in vivo.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)]. The most appropriate gene-delivery vehicles for neurons are adeno-associated virus (AAV) vectors, which are derived from non-pathogenic virus. Therefore, TH and AADC genes were introduced into the striatum in the lesioned side using separate AAV vectors in parkinsonian rats, and the coexpression of TH and AADC resulted in better behavioral recovery compared with TH alone. Another strategy for gene therapy of PD is the protection of dopaminergic neurons in the substantia nigra using an AAV vector containing a glial cell line-derived; neurotrophic factor (GDNF) gene. Combination of dopamine-supplement gene therapy and GDNF gene therapy would be a logical approach to the treatment of PD.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)]. The most appropriate gene-delivery vehicles for neurons are adeno-associated virus (AAV) vectors, which are derived from non-pathogenic virus. Therefore, TH and AADC genes were introduced into the striatum in the lesioned side using separate AAV vectors in parkinsonian rats, and the coexpression of TH and AADC resulted in better behavioral recovery compared with TH alone. Another strategy for gene therapy of PD is the protection of dopaminergic neurons in the substantia nigra using an AAV vector containing a glial cell line-derived neurotrophic factor (GDNF) gene. Combination of dopamine-supplement gene therapy and GDNF gene therapy would be a logical approach to the treatment of PD.

Hematopoietic stem cells (HSCs), because they have a self-renewal ability and can generate progeny of all kinds of blood cells throughout one's life, are an ideal target for gene therapy. Retroviral vectors are predominantly used for transduction of HSCs, but the gene transfer efficiency is extremely low. Several efforts have been made at achieving clinically relevant gene transfer efficiencies. First, new cytokines such as Flt-3 ligand and thrombopoietin, and coculture with stromal elements such as fibronectin fragments, have been successfully tried during ex vivo culture of HSCs with retroviral vectors. Second, new vectors that meet the host requirements have been developed: pseudotyped retroviral vectors and lentiviral vectors. Finally, positive selection of transduced cells has been designed in vitro before reinfusion or in vivo after engraftment to compensate for the low transduction efficiency of HSCs. A novel method of in vivo expansion of transduced hematopoietic cells using the selective amplifier gene may also help overcome the low transduction efficiency of HSCs. It has recently been reported that immunological tolerance against xenogeneic gene products can be induced by introduction of their genes into HSCs. This distinctive feature further enhances the value of HSCs as a target of gene therapy.

We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, beta-galactosidase with the nuclear localization signal of SV40 (np-gal), within the cytoplasm, n beta-gal was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor (G-CSFR), a membrane protein. To liberate the n beta-gal portion hom the fusion protein, we used a protease derived from a plant virus, whose recognition sequence was inserted between the G-CSFR and n beta-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescence microscopy. In contrast, when expressed together with the protease, beta-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the protease, indicating that this protease is not cytotoxic. These results suggest that the viral protease-mediated alteration of subcellular localization can potentially regulate the function of nuclear proteins. (C) 1999 Academic Press.

Since the Rep proteins of adeno-associated virus (AAV) are harmful to cells, it is difficult to obtain stable cell lines that express them constitutively. In this study, stable 293 cell lines were obtained in which large Rep expression was inducible by using the Cre/loxP switching system. To determine the function of the induced Rep proteins, the packaging capacity was examined after supplementation with a plasmid expressing small Rep and Cap proteins. A significant amount of recombinant AAV (5.5 x 10(8) vector particles per 10 cm dish) was produced by transfection with a vector plasmid and infection with Cre-expressing recombinant adenovirus, indicating that the large Rep proteins retained the function required for packaging. These findings indicate that large Rep protein expression can be strictly regulated by the Cre/loxP system and will also serve as a basis for the development of an efficient AAV-packaging cell line.

Adeno-associated virus (AAV) vectors are potentially useful vehicles for the delivery of therapeutic genes into human cells. To determine the optimal expression pattern of AAV proteins (Rep78, Rep68, Rep52, Rep40, and Cap proteins) for packaging the recombinant AAV genome, helper plasmids were split into two portions. In this study, two sets of split-type helper plasmids were prepared: i.e., 1) a Rep expression plasmid (pR78/68) and Cap expression plasmid (pCap), and 2) a large Rep expression plasmid (pR78/68 and small Rep plus Cap expression plasmid (pR52/40Cap). When AAV vectors were produced using these sets of split-type helper plasmids at various ratios, the optimal ratio of (large) Rep expression plasmid and Cap expression plasmid was 1 to 9 for both sets. More importantly, the titers were comparable to or even higher than that of a conventional helper plasmid (pIM45) (4.9+/-2.1x10(11) vector particles/10 cm dish for pRep and pCap; 2.9+/-1.6x10(11) vector particles/10 cm dish for pR78/68 and pR52/40Cap: and 1.8+/-0.16x10(11) particles/10 cm dish for pIM45). Western analysis of AAV proteins suggests that the expression of a relatively small amount of large rep and a large amount of Cap is important for optimal vector production. The present study shows that the AAV helper plasmid can be split without losing the ability to package the recombinant AAV genome, and provides us with valuable basic information for the development of efficient AAV packaging cell lines.

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication, Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed far their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A and R138A mutations completely abolished the Binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing β-galactosidase (β-gal) gene in vitro and the expression of β-gal in CM was evaluated by X-gal staining and β-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the β-gal expression increased to 31.1 ± 4.6 ng/mg protein in a MOI-dependent manner (MOI: 104 to 106 particles/cell). The β-gal expression was also increased in an incubation period-dependent manner (1-24 h). β-gal expression was maximal at day 3 and then gradually decreased with time. However, β-gal expression at day 14 was almost the same level as that at day 1 (45.5 ± 5.9 v 55.2 ± 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the β-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.