Efficient gene transduction in pigs and macaques with the engineered AAV vector AAV.GT5 for hemophilia B gene therapy.

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. We examined the potential of a novel engineered liver-tropic AAV3B-based vector, AAV.GT5, for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination.

The seroprevalence of neutralizing antibodies against the adeno-associated virus capsids in Japanese hemophiliacs.

Adeno-associated virus (AAV) vectors are promising modalities of gene therapy to address unmet medical needs. However, anti-AAV neutralizing antibodies (NAbs) hamper the vector-mediated therapeutic effect. Therefore, NAb prevalence in the target population is vital in designing clinical trials with AAV vectors. Hence, updating the seroprevalence of anti-AAV NAbs, herein we analyzed sera from 100 healthy individuals and 216 hemophiliacs in Japan. In both groups, the overall seroprevalence against various AAV serotypes was 20%-30%, and the ratio of the NAb-positive population increased with age. The seroprevalence did not differ between healthy participants and hemophiliacs and was not biased by the concomitant blood-borne viral infections. The high neutralizing activity, which strongly inhibits the transduction with all serotypes in vitro, was mostly found in people in their 60s or of older age. The multivariate analysis suggested that "60s or older age" was the only independent factor related to the high titer of NAbs. Conversely, a large proportion of younger hemophiliacs was seronegative, rendering them eligible for AAV-mediated gene therapy in Japan. Compared with our previous study, the peak of seroprevalences has shifted to older populations, indicating that natural AAV exposure in the elderly occurred in their youth but not during the last decade.

A phase III, 52-week, open-label study to evaluate the safety and efficacy of 5% sofpironium bromide (BBI-4000) gel in Japanese patients with primary axillary hyperhidrosis.

A long-term study was conducted in Japanese patients with primary axillary hyperhidrosis who completed the preceding 6-week phase III, confirmatory study of 5% sofpironium bromide gel (hereinafter referred to as sofpironium) to evaluate the safety and efficacy of 52-week treatment with sofpironium. In the long-term study, 185 patients who completed the confirmatory study (94 and 91 patients in the vehicle and sofpironium groups, respectively) started to receive sofpironium (switching and extension groups, respectively), and all these patients were included in both the full analysis set (FAS) and the safety analysis set (SAF). In the FAS, there were more females than males (73.0% vs. 27.0%), and median age was 38.0 years. A total of 161 patients (86 and 75 patients in the switching and extension groups, respectively) completed the study at week 52. The proportions of patients with hyperhidrosis disease severity score of 1 or 2 and a 50% or more reduction in total gravimetric weight of sweat were 57.4% in the switching group and 58.2% in the extension group at week 52. The proportions of patients who achieved this efficacy end-point in the long-term study were similar to that (53.9%) in the sofpironium group in the confirmatory study. In the SAF, the incidences of adverse events (AEs) were 80.9% in the switching group and 83.5% in the extension group, and the incidences of adverse drug reactions were 39.4% and 45.1%, respectively. AEs that occurred in at least 20% of patients in both treatment groups were application site dermatitis (25.5% and 33.0%, respectively) and nasopharyngitis (31.9% and 23.1%, respectively). Reported AEs were generally mild, and there were no deaths. Serious AEs occurred in three patients, but none were considered related to the study drug. In this study, the efficacy of sofpironium was maintained during 52-week treatment, and no new safety risk was observed.

A phase 3, multicenter, randomized, double-blind, vehicle-controlled, parallel-group study of 5% sofpironium bromide (BBI-4000) gel in Japanese patients with primary axillary hyperhidrosis.

A phase 3 study was conducted to verify the efficacy and safety of 5% sofpironium bromide (BBI-4000) gel (hereinafter referred to as sofpironium) administrated for 6 weeks in Japanese patients with primary axillary hyperhidrosis. The primary efficacy end-point was the proportion of patients who satisfied both criteria of a Hyperhidrosis Disease Severity Score (HDSS) of 1 or 2 at the end of 6-week treatment and a 50% or more reduction in total gravimetric weight of sweat at the end of treatment relative to baseline. A total of 281 patients were randomized to receive 5% sofpironium (141 patients) or vehicle (140 patients), and all patients were included in the full analysis set (FAS). In the FAS, 70.1% of patients were female, and the median age was 35.0 years. The proportion of patients who achieved the primary efficacy end-point was 53.9% in the sofpironium group and 36.4% in the vehicle group, with a statistically significant difference of 17.5% (95% confidence interval, 6.02-28.93) between these two groups (P = 0.003). The incidence of adverse events was 44.0% in the sofpironium group and 30.7% in the vehicle group, and the incidence of adverse drug reactions was 16.3% in the sofpironium group and 5.0% in the vehicle group. Reported adverse events were generally mild or moderate in severity. In the sofpironium group, common events (incidence, ≥5%) were nasopharyngitis (14.2%) and dermatitis/erythema at the application site (8.5%/5.7%), with no serious adverse events reported. This study demonstrated the efficacy and safety of 5% sofpironium.

Aspects of Gene Therapy Products Using Current Genome-Editing Technology in Japan.

The development of genome-editing technology could lead to breakthrough gene therapy. Genome editing has made it possible to easily knock out or modify a target gene, while current gene therapy using a virus vector or plasmid hampering modification with respect to gene replacement therapies. Clinical development using these genome-editing tools is progressing rapidly. However, it is also becoming clear that there is a possibility of unintended gene sequence modification or deletion, or the insertion of undesired genes, or the selection of cells with abnormalities in the cancer suppressor gene p53; these unwanted actions are not possible with current gene therapy. The Science Board of the Pharmaceuticals and Medical Devices Agency of Japan has compiled a report on the expected aspects of such genome-editing technology and the risks associated with it. This article summarizes the history of that discussion and compares the key concepts with information provided by other regulatory authorities.

Comprehensive Metabolomic Analysis of IDH1R132H Clinical Glioma Samples Reveals Suppression of ß-oxidation Due to Carnitine Deficiency.

Gliomas with Isocitrate dehydrogenase 1 (IDH1) mutation have alterations in several enzyme activities, resulting in various metabolic changes. The aim of this study was to determine a mechanism for the better prognosis of gliomas with IDH mutation by performing metabolomic analysis. To understand the metabolic state of human gliomas, we analyzed clinical samples obtained from surgical resection of glioma patients (grades II-IV) with or without the IDH1 mutation, and compared the results with U87 glioblastoma cells overexpressing IDH1 or IDH1R132H. In clinical samples of gliomas with IDH1 mutation, levels of D-2-hydroxyglutarate (D-2HG) were increased significantly compared with gliomas without IDH mutation. Gliomas with IDH mutation also showed decreased intermediates in the tricarboxylic acid cycle and pathways involved in the production of energy, amino acids, and nucleic acids. The marked difference in the metabolic profile in IDH mutant clinical glioma samples compared with that of mutant IDH expressing cells includes a decrease in ß-oxidation due to acyl-carnitine and carnitine deficiencies. These metabolic changes may explain the lower cell division rate observed in IDH mutant gliomas and may provide a better prognosis in IDH mutant gliomas.

AAV6-Mediated IL-10 Expression in the Lung Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative disorder with limited therapeutic options. An aberrant wound healing process in response to repetitive lung injury has been suggested for its pathogenesis, and a number of cytokines including transforming growth factor ß1 play pivotal roles in the induction and progression of fibrosis. Thus, the regulation of these pro-inflammatory conditions may reduce the progression of IPF and ameliorate its symptoms in patients. Interleukin-10 (IL-10), a pleiotropic cytokine, exerts anti-inflammatory and anti-fibrotic effects in numerous biological settings. In the present study, we investigated the preventive effects of IL-10 on bleomycin-induced pulmonary fibrosis in mice with the continuous expression of this cytokine via an adeno-associated virus serotype 6 vector. Mice were administered the adeno-associated virus serotype 6 vector encoding mouse IL-10 by intratracheal injection, and osmotic minipumps containing bleomycin were subcutaneously implanted seven days later. Lung histology and the expression levels of pro-inflammatory cytokines and fibrogenic cytokines were then analyzed. In mice exhibiting persistent IL-10 expression on day 35, the number of infiltrated inflammatory cells and the development of fibrosis in lung tissues were significantly reduced. Increases in transforming growth factor ß1 and decreases in IFN-γ were also suppressed in treated animals, with changes in these cytokines playing important roles in the pathogenesis of pulmonary fibrosis. Furthermore, IL-10 significantly improved survival in bleomycin-induced mice. Our results provide insights into the potential benefit of the anti-fibrotic effects of IL-10 as a novel therapeutic approach for IPF.

Eradication of cervical cancer in vivo by an AAV vector that encodes shRNA targeting human papillomavirus type 16 E6/E7.

The major causative agent of cervical cancer is human papilloma virus (HPV); the viral proteins E6 and E7 induce carcinogenesis through the inactivation of the host tumor-suppressor gene. Therefore, the stable expression of specific inhibitors of E6 and E7 in cancer cells is expected to provide effective treatment for cervical cancer without affecting normal tissue. In this study, we propose a novel therapeutic approach using an adeno-associated virus (AAV) vector encoding short hairpin RNA (shRNA) against the oncoproteins E6 and E7 (shE6E7) of HPV type 16 (HPV­16), termed AAV­shE6E7. Three different HPV­16-positive cervical cancer cell lines (BOKU, SiHa and SKG-IIIa cells) were tested for gene transfer efficiency using serotypes of AAV vectors. For in vitro analysis, the cells were transduced AAV­shE6E7; alternatively, in vivo studies were performed via the administration of a direct injection of AAV­shE6E7 into cervical cancer cell-derived tumors in mice. The high gene transfer efficiency was observed using AAV2 in all three cervical cancer cell lines. Following transduction, we observed apoptosis, G1 phase arrest and cell growth inhibition. Additionally, in the transduced cells, the E6, E7 and p16 expression levels decreased, whereas the expression levels of p53, p21 and pRb levels were enhanced. The growth of subcutaneously transplanted tumors was markedly inhibited by the single administration of AAV2­shE6E7, and the tumors were almost completely eradicated without any adverse effects. These results provided evidence of the utility of AAV2­shE6E7 as a novel treatment approach for cervical cancer.

Novel anti-tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cells.

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

An R132H mutation in isocitrate dehydrogenase 1 enhances p21 expression and inhibits phosphorylation of retinoblastoma protein in glioma cells.

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to α-ketoglutarate (α-KG) and acquires new activity whereby it converts α-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1(R132H)-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1(R132H)-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progression of the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation.

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models.

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19(+) tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19(+) human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.

Suppression of lymph node and lung metastases of endometrial cancer by muscle-mediated expression of soluble vascular endothelial growth factor receptor-3.

Lymph node metastasis is the most important prognostic factor of endometrial cancer. However, effective therapy has not been established against lymph node metastasis. In this study, we explored the efficacy of gene therapy targeting lymph node metastasis of endometrial cancer by suppressing the action of vascular endothelial growth factor (VEGF)-C through soluble VEGF receptor-3 (sVEGFR-3) expression. For this purpose, we first conducted a model experiment by introducing sVEGFR-3 cDNA into an endometrial cancer cell line HEC1A and established HEC1A/sVEGFR-3 cell line with high sVEGFR-3 expression. The conditioned medium of HEC1A/sVEGFR-3 cells inhibited lymphatic endothelial cell growth in vitro, and sVEGFR-3 expression in HEC1A cells suppressed in vivo lymph node and lung metastases without inhibiting the growth of a subcutaneously inoculated tumor. To validate the therapeutic efficacy, adeno-associated virus vectors encoding sVEGFR-3 were injected into the skeletal muscle of mice with lymph node metastasis. Lymph node and lung metastases of HEC1A cells were completely suppressed by the muscle-mediated expression of sVEGFR-3 using adeno-associated virus vectors. These results suggest the possibility of gene therapy against lymph node and lung metastases of endometrial cancer by using muscle-mediated expression of sVEGFR-3.

NF-κB activity regulates mesenchymal stem cell accumulation at tumor sites.

Mesenchymal stem cells (MSC) accumulate at tumor sites when injected into tumor-bearing mice, perhaps offering cellular vectors for cancer-targeted gene therapy. However, the molecular mechanisms involved in MSC targeting the tumors are presently little understood. We focused on MSC-endothelial cell (EC) adhesion following TNF-α stimulation in an attempt to elucidate these mechanisms. Interestingly, stimulation of MSCs with TNF-α enhanced the adhesion of MSCs to endothelial cells in vitro. This adhesion was partially inhibited by blocking antibodies against vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4). It is well known that TNF-α induces VCAM-1 expression via the NF-κB signaling pathway. Parthenolide has an anti-inflammatory activity and suppressed NF-κB activity by inhibition of IκBα phosphorylation after TNF-α stimulation and strongly inhibited TNF-α-induced VCAM-1 expression on MSCs. In vivo imaging using luciferase-expressing MSCs revealed that the bioluminescent signal gradually increased at tumor sites in mice injected with untreated MSCs. In contrast, we observed very weak signals at tumor sites in mice injected with parthenolide-treated MSCs. Our results suggest that NF-κB activity regulates MSC accumulation at tumors, by inducing VCAM-1 and thereby its interaction with tumor vessel endothelial cells. These findings have implications for the ongoing development of efficient MSC-based gene therapies for cancer treatment.

Recovery of neurogenic amines in phenylketonuria mice after liver-targeted gene therapy.

Phenylketonuria (PKU) is a common genetic disorder arising from a deficiency of phenylalanine hydroxylase. If left untreated, the accumulation of phenylalanine leads to brain damage and neuropsychological dysfunction. One of the abnormalities found in hyperphenylalaninemic patients and a mouse model of PKU is an aminergic deficit in the brain. We previously showed correction of hyperphenylalaninemia and concomitant behavioral recovery in PKU mice after liver-targeted gene transfer with a viral vector. Here, we addressed whether such a functional recovery was substantiated by an improved amine metabolism in the brain. After gene transfer, brain dopamine, norepinephrine, and serotonin levels in the PKU mice were significantly elevated to normal or near-normal levels, along with systemic improvement of phenylalanine catabolism. The results of biochemical analyses validated the efficacy of PKU gene therapy in the central nervous system.

Development of a mouse model for lymph node metastasis with endometrial cancer.

Controlling lymph node metastasis is currently a key issue in cancer therapy. Lymph node metastasis is one of the most important prognostic factors in various types of cancers, including endometrial cancer. Vascular endothelial growth factor-C (VEGF-C) plays a crucial role in lymphangiogenesis, and is implicated to play an important role in lymph node metastasis. To evaluate the role of VEGF-C in lymph node metastasis, we developed an animal model by using an endometrial cancer cell line, HEC1A. This cell line is not invasive by nature and secretes moderate amounts of VEGF-C; intrauterine injection of HEC1A cells into Balb/c nude mice resulted in uterine cancer with lymph node metastasis after 8 weeks. To analyze the contribution of VEGF-C to lymph node metastasis, its corresponding gene was stably introduced into HEC1A cells (HEC1A/VEGF-C), which then produced more than 10 times the amount of VEGF-C. The number of lymph node metastases was significantly higher in HEC1A/VEGF-C cells than in HEC1A cells (3.2 vs 1.1 nodes/animal, respectively). Augmented lymphangiogenesis was observed within tumors when HEC1A/VEGF-C cells were inoculated. These results indicate that VEGF-C plays a critical role in lymph node metastasis, in addition to serving as a platform to test the efficacy of various therapeutic modalities against lymph node metastasis.

Complete restoration of phenylalanine oxidation in phenylketonuria mouse by a self-complementary adeno-associated virus vector.

BACKGROUND: Classical phenylketonuria (PKU) arises from a deficiency of phenylalanine hydroxylase (PAH) that catalyses phenylalanine oxidation in the liver. Lack of PAH activity causes massive hyperphenylalaninemia and consequently severe brain damage. Preclinical studies showed that conventional adeno-associated virus (AAV) vectors could correct hyperphenylalaninemia in a mouse model of PKU, although limitations such as very large dose requirement and relative inefficiency in female animals were recognized. METHOD: An AAV8-pseudotyped vector was constructed with a self-complementary AAV (scAAV) genome for efficient liver transduction and expression. Following vector injection to PKU mice, blood Phe was periodically measured by an enzymatic fluorometric assay. In vivo Phe oxidation was evaluated by a non-invasive breath test using [1-(13) C]Phe. Vector copy number in the host tissues was determined by quantitative polymerase chain reaction. RESULTS: A single injection of 1 × 10(11) -1 × 10(12) particles of the scAAV8 vector resulted in a reduction of blood Phe to normal or near-normal levels for more than 1 year in both genders. The treated animals showed normal level of in vivo Phe oxidation. The presence of > 1 copy of vector DNA per diploid genome in the liver was associated with normal blood Phe in the AAV-treated PKU mice. CONCLUSIONS: Complete phenotypic correction of PKU mice was achieved by the scAAV8 vector for the longest duration reported to date. The vector overcame the female-specific disadvantage in AAV-mediated liver transduction; thus, it offers a promising platform of long-lasting gene therapy for PKU.

A phase I study of aromatic L-amino acid decarboxylase gene therapy for Parkinson's disease.

Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinson's disease (PD). We evaluated the safety, tolerability, and potential efficacy of adeno-associated virus (AAV) vector-mediated gene delivery of aromatic L-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinson's Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[(18)F]fluoro-L-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.

Kinetics and effect of integrin expression on human CD34(+) cells during murine leukemia virus-derived retroviral transduction with recombinant fibronectin for stem cell gene therapy.

The CH-296 recombinant fragment of human fibronectin is essential for murine leukemia virus (MLV)-derived retroviral transduction of CD34(+) cells for the purpose of stem cell gene therapy. Although the major effect of CH-296 is colocalization of the MLV-derived retrovirus and target cells at specific adhesion domains of CH-296 mediated by integrins expressed on CD34(+) cells, the precise roles of the integrins are unclear. We examined the kinetics of integrin expression on CD34(+) cells during the course of MLV-derived retrovirus-mediated gene transduction with CH-296. Flow cytometry revealed that the levels of both very late activation protein (VLA)-4 and VLA-5 on CD34(+) cells freshly isolated from cord blood were insufficient for effective MLV-derived retroviral transduction. However, increases were achieved during culture for preinduction and MLV-derived retrovirus-mediated gene transduction in the presence of a cocktail of cytokines. In addition, we confirmed by using specific antibodies that inhibition of the cell adhesion mediated by the integrins significantly reduced transduction efficiency, indicating that integrin expression is indeed important for CH-296-based MLV-derived retroviral transduction. Only a few cytokines are capable of inducing integrin expression, and stem cell factor plus thrombopoietin was found to be the minimal combination that was sufficient for effective transduction of an MLV-derived retrovirus based on CH-296. Our findings should be useful for improving the culture conditions for CH-296-based MLV-derived retroviral transduction in stem cell gene therapy.

Retroviral vector-producing mesenchymal stem cells for targeted suicide cancer gene therapy.

BACKGROUND: Mesenchymal stem cells (MSCs) are a promising vehicle for targeted cancer gene therapy because of their potential of tumor tropism. For efficient therapeutic application, we developed retroviral vector-producing MSCs that enhance tumor transduction via progeny vector production. METHODS: Rat bone marrow-derived MSCs were nucleofected with the proviral plasmids (vesicular stomatitis virus-G protein-pseudotyped retroviral vector components) (VP-MSCs) or pLTR plasmid alone (non-VP-MSCs). The luciferase-based in vivo imaging system was used to assess gene expression periodically. To evaluate the anticancer effects, we administered MSCs expressing herpes simplex virus-thymidine kinase (HSV-tk) into the left ventricular cavity of nude mice engrafted with 9L glioma cells subcutaneously. RESULTS: In vivo imaging revealed that administration of luciferase-expressing non-VP-MSCs enhanced the bioluminescence signal at the inoculation sites of 9L cells, whereas no accumulation was observed in mice at the site of the control Rat-1 fibroblasts. Compared to non-VP-MSCs, the administration of VP-MSCs resulted in significant augmentation of the signal with an increase in transgene copy number. Immunohistochemical analysis showed marked luciferase expression at the tumor periphery in mice injected with VP-MSCs, whereas little expression was detected in those injected with non-VP-MSCs. Under the continuous infusion of ganciclovir, systemic administration of VP-MSCs expressing HSV-tk suppressed tumor growth more effectively than non-VP-MSC administration, whereas no anticancer effect was observed without ganciclovir treatment. Furthermore, VP-MSC administration caused no transgene transduction in the normal tissues and organs. CONCLUSIONS: VP-MSCs accumulated at the site of tumors after intravascular injection in tumor-bearing mice, followed by in situ gene transfer to tumors without transduction of normal organs. When applied to the HSV-tk/ganciclovir suicide gene therapy, more efficient tumor growth suppression was observed using VP-MSCs compared to non-VP-MSCs. This VP-MSC-based system has great potential for improved cancer gene therapy.

Genomic organization of regions that regulate chicken glycine decarboxylase gene transcription: physiological and pathological implications.

Regions required for chicken glycine decarboxylase gene transcription were examined. A region between -82 and +22 (-82/+22) with motifs similar to binding sites for Sp1, NF-Y and CP2 was assigned to the proximal promoter active in both chicken hepatoma cell line, LMH, and hepatocytes in primary culture. In LMH cells, a genomic region, KX, between KpnI (-4155) and XbaI (-2113) sites changed promoter activity with the aid of four additional genomic regions termed upstream regulator regions for suppression (UpRS) and activation (UpRA) of transcription. Those precise segments are UpR1S (-376/-346), UpR1A (-345/-291), UpR2S (-137/-108) and UpR2A (-107/-83). Within KX, -4155/-3605 activates and -3604/-3367 suppresses the promoter. -3366/-3024 activates or suppresses the promoter, probably with different UpR counterparts. -2197/-2113 restores the actions of -3366/-3024. While in LMH cells, the upstream UpRs abrogate the functions of immediately downstream UpRs, UpR1S or UpR2S or both may be at least less active in hepatocytes than in LMH cells. Nuclear extracts from various chicken tissues and LMH cells had UpR2A binding proteins in different populations, suggesting that together with the UpRs, the segments in KX are involved in the regulation of cell type-specific transcription of this gene.