中枢神经系统组织特异性启动子在基因治疗中的研究及其进展

将外源基因有效的导入神经细胞并使其靶向、高效和长期稳定的表达是目前基因治疗神经系统疾病的主要靶点。随着研究的深入,越来越多的组织特异性启动子已被用于调控目的基因在靶细胞或靶组织中的表达,成为基因治疗研究领域的热点之一。现综述目前常用的几种中枢神经系统组织特异性启动子在基因治疗中的研究及其进展。     

Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets

There is considerable interest in the viral modification of insulin-producing islets, including porcine islets, in the context of islet xenotransplantation to treat type 1 diabetes. Adenovirus (Adv) gene delivery offers the potential to modify pre-transplant islets for enhanced survival. Modifications include transfer of cytoprotective molecules to ensure islet survival immediately post-transplant, and molecules to dampen the immune system and prevent chronic islet graft rejection. In this study, we compared different promoters (three promiscuous and two tissue-specific promoters) for their efficiency in driving gene expression in neonatal pig islet tissue after Adv delivery. We also compared the efficiency of these promoters in adult islets from mouse and human pancreata. We observed that the promiscuous cytomegalovirus promoter was the most potent, eliciting high luciferase expression in neonatal pig islets, as well as in human and mouse islets. In contrast, the mammalian EF1-alpha promoter educed comparatively intermediate gene expression. The mouse major histocompatibility complex class I promoter H-2K(b) and the pancreatic-specific promoters insulin and human pdx-1 (area II) performed poorly in islets from all three species. This has important implications for the generation of modified neonatal pig islets for transplantation into humans.     

Relative promoter strengths in four human prostate cancer cell lines evaluated by particle bombardment-mediated gene transfer

BACKGROUND: The particle bombardment (gene gun) method for gene transfer provides a new and efficient means for transfection of various cell types in culture. In this study we evaluate its application to human prostate tumor cells. METHODS: Transient expression of the firefly luciferase gene driven by five viral and five cellular promoters was assessed after in vitro gene transfer using the gene gun method. The relative strengths of these promoters were quantitatively determined in four different human prostate tumor cell lines: DU145, PC-3, LNCaP, and CWR22Rv1 cells. In situ histochemical staining of cells, transfected with bacterial beta-galactosidase cDNA as a reporter gene, was also performed to evaluate the transfection efficiency. Time course of gene expression was determined using the luciferase reporter gene. RESULTS: The peak levels of transient expression of firefly luciferase are observed within 24 hr after gene transfer. Sustained but reduced luciferase levels were also detected as long as 5 days post transfection. Up to 35% of bombarded cells in vitro were found to express transgenic beta-galactosidase activity. Among tested viral promoters, cytomegalovirus early enhancer/promoter activity was observed to confer consistently the highest activity in each test cell line, whereas phosphoglycerate kinase gene promoter possessed the highest activity among the cellular promoters tested. CONCLUSIONS: The particle bombardment gene-transfer technology can be effectively employed as an efficient method for in vitro gene-transfer into prostate tumor cells. The characterization of relative promoter strength and preference may be useful for future studies of cancer gene therapy approaches.     

Development of a therapeutic adenoviral vector for cholangiocarcinoma combining tumor-restricted gene expression and infectivity enhancement

Cholangiocarcinoma is an invasive malignancy that is most often unresectable upon diagnosis and unresponsive to chemotherapy and radiation. While adenoviral gene therapy has shown promise in treating many tumors, systemic toxicity and low tumor transduction efficiency have hampered its application in many gastrointestinal cancers. To overcome these difficulties, we have constructed an adenoviral vector utilizing a tumor-specific promoter (TSP) for selective transgene expression and a vector with an RGD-motif in the fiber-knob region for infectivity enhancement. In seeking a TSP for cholangiocarcinoma, Secretory Leukoprotease Inhibitor, Midkine, Gastrin Releasing Peptide, VEGF, Cox-2M, and Cox-2L promoters were configures in adenoviral vectors, and evaluated in cholangiocarcinoma cells lines (Oz and SkChA-1). Luciferase assays demonstrated that Cox-2 promoters (M and L) showed the highest promoter activity, with Cox-2M appearing slightly stronger than Cox-2L. Infectivity enhanced vectors with RGD-motif in the fiber-knob region were also constructed with the luciferase transgene driven by a CMV control and the Cox-2M and Cox-2L promoters. Subsequent luciferase assays comparing the unmodified vectors to the RGD-modified versions demonstrated higher levels of luciferase activity than the RGD-infected cells. This paradigm was then applied to a therapeutic HSV-TK/GCV model by constructing RGD-enhanced HSV-TK vectors driven by Cox-2M and Cox-2L promoters. In vitro cytocidal effect analysis confirmed that the RGD-modified, cox-2 (M and L) driven vectors showed a stronger cytocidal effect upon gancyclovir administration than the vectors with wild-type fiber. The Cox-2 promoter demonstrates a favorable selectivity profile for cholangiocarcinoma, and RGD-modification further enhances transduction efficiency. This combination has potential to overcome the obstacles to clinical application of adenoviral gene therapy in cholangiocarcinoma. Presented at the Forty-Third Annual Meeting of The Society for Surgery of The Alimentary Tract, San Francisco, California, May 19–22, 2002 (oral presentation).     

Adenovirus-mediated gene transfer using in-situ perfusion of the liver graft

To establish an efficient technique for adenovirus-mediated gene transfer in liver transplantation, we evaluated the in situ perfusion of liver grafts. The grafts were perfused in situ with 1 × 10¹⁰ of E1-deleted, replication-defective adenoviral vectors encoding the LacZ gene driven by the human CMV promoter, either through the hepatic artery (group 1) or the portal vein (group 2). Group 3 animals served as negative controls; their liver grafts were perfused with lactated Ringer's solution through the portal vein. PCR confirmed the presence of viral DNA in every graft perfused with viral vectors. In X-gal staining, positive staining was observed almost exclusively at the portal triad in group 1, whereas in group 2 minimal staining was observed, predominantly in the parenchymal area. Protein production from the transfected gene was confirmed by a functional protein assay; the values were 0.16 % ± 0.07 % liver protein in group 1, 0.13 % ± 0.02 % in group 2, and 0.007 % ± 0.0003 % in group 3 on postoperative day 2. In conclusion, in situ perfusion of the viral vectors through the hepatic artery resulted in an effective expression of the transfected gene, predominantly at the portal triad. Received: 15 July 1996 Received after revision: 1 November 1996 Accepted: 12 November 1996     

Construction of recombinant adenoviral vector Ad—CMV—hTGFβ1 for reversion of intervertebral disc degeneration by gene transfer

Objective: To provide a highly efficient adenoviralvector Ad-CMV-hTGFβ1 for the study of gene therapy forreversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviralvector construction system was used in the study. ThecDNA of hTGFβ1 was first subcloned into a shuttle plasmidpShuttle-CMV. The resultant plasmid was linearized bydigesting with restriction endonuclease PmeI, andsubsequently transformed into E.coll. BJ5183 cells with anadenoviral backbone plasmid pAdEasy-1. Recombinantswere selected by kanamycin resistance and confirmed byrestriction endonuclease analysis. Finally, the recombinantplasmid linearized by PmeI was transfected into 293 cells.Recombinant adenoviruses were generated within 2 weeks.Results: The recombinant adenoviral plasmids werecut by BamHI and PacI respectively, and the diagnosticfragments appeared in 0.8% agarose electrophoresis. Theinfected 293 cells showed evident cytopathlc effect (CPE).The productions of PCR confirmed the presence ofrecombinant adenovirus. The expression of hTGFβ1 wasverified by immunohistochemical staining.Conclusions: The successful generation of theadenoviral vector Ad-CMV-hTGFβ1 and the confirmationof the interest gene expression make it possible for theexperimental study of the reversion of the intervertebraldisc degeneration by gene therapy.     

Specific gene expression and therapy for pancreatic cancer using the cytosine deaminase gene directed by the rat insulin promoter

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells, but cell-specific delivery of the gene is required to limit host toxicity. The objective of this study is to determine whether the rat insulin promoter (RIP) will permit cell-specific gene delivery and subsequent cell death in human pancreatic cancer cells. The RIP DNA was amplified using polymerase chain reaction (PCR), and the purified fragment was inserted into pCR-Blunt II-TOPO plasmid at the SpeI site, which contains the coding sequence of yeast cytosine deaminase (CD). Transfection assays were carried out using both RIP-lacZ and RIP-CD DNA constructs in two human pancreatic cancer cell lines, PANC-1 and MIA PaCa-2. Reporter assays using X-gal staining were performed, and the in vitro cytotoxicity was examined in RIP-CD-transfected cells treated with 5-.ucytosine for 5 days. The expression levels of CD protein in the transfected cells were determined 2 days after transfection by Western blot analysis. The expression levels of insulin promoter factor (IPF-1/PDX-1) in these human pancreatic cell lines, as well as in freshly isolated human pancreatic cancer specimens, were determined using in situ immunohistochemistry analysis. After transfection with RIP-lacZ, only PANC-1 cells, but not MIA PaCa-2 cells, were positive for RIP-lacZ expression, indicating that RIP-directed reporter gene expression occurred only in PANC-1 cells. After transfection with RIP-CD and treatment with 5-flucytosine, PANC-1 cells had a significantly increased cell death rate compared with that of MIA PaCa-2 cells, suggesting that RIP-directed suicide gene expression occurred only in PANC-1 cells. Western blot analysis demonstrated that only PANC-1 cells were able to express the CD protein and that significantly increased levels of PDX-1 were found in PANC-1 but not in Mia PaCa-2 cells. In situ immunohistochemical analysis of both cell lines showed that PDX-1 was only expressed in the nuclei of PANC-1 cells and not in MIA PaCa-2 cells. Furthermore, two freshly isolated human pancreatic cancer specimens had significantly increased levels of PDX-1. The RIP is activated in PANC-1 cells, but not in Mia PaCa-2 cells, and the mechanism of activation is via PDX-1. Pancreatic cancer-specific cytotoxicity can be achieved with the use of RIP-CD and 5-flucytosine treatment in vitro. Significantly increased levels of PDX-1 have been found in human pancreatic cancer specimens. These results suggest that RIP could be used for cell-specific suicide gene therapy to target human pancreatic tumors. Presented at the Forty-Fourth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Florida May 18–21, 2003 (oral presentation). Supported by an Advances in Technology Transfer Grant (ATP1176) from the State of Texas Department of Education and by grant NCI R01 (CA95731).     

Regulation of articular chondrocyte aggrecan and collagen gene expression by multiple growth factor gene transfer

Gene transfer is a promising approach to the delivery of chondrotrophic growth factors to promote cartilage repair. It is unlikely that a single growth factor transgene will optimally regulate these cells. The aim of this study was to identify those growth factor transgene combinations that optimally regulate aggrecan, collagen type II and collagen type I gene expression by articular chondrocytes. We delivered combinations of the transgenes encoding fibroblast growth factor-2, insulin-like growth factor I, transforming growth factor beta1, bone morphogenetic protein-2, and/or bone morphogenetic protein-7 and assessed chondrocyte responses by measuring changes in the expression of aggrecan, type II collagen and type I collagen genes. These growth factor transgenes differentially regulated the magnitude and time course of all three-matrix protein genes. In concert, the transgenes regulated matrix gene expression in an interactive fashion that ranged from synergistic to inhibitory. Maximum stimulation of aggrecan (16-fold) and type II collagen (35-fold) expression was with the combination of IGF-I, BMP-2, and BMP-7 transgenes. The results indicate that the optimal choice of growth factor genes for cell-based cartilage repair cannot be predicted from observations of individual transgenes. Rather, such gene therapy will require an empirically based selection of growth factor gene combinations.     

腺病毒驱动KDR启动子介导CD/TK融合基因系统对肝癌细胞的靶向杀伤作用

目的 研究腺病毒介导的KDR启动子驱动CD/TK双自杀基因系统（AdKDR- CDglyTK）对肝癌细胞选择性杀伤作用。方法 将质粒pAdEasy-KDR-CDglyTK在293细胞内包装、扩增后，体外感染表达KDR的BEL-7402细胞株和对照组不表达KDR的LS174T细胞株，并给予不同浓度的前药5-FC和/或GCV，观察该体系对不同细胞株的杀伤效应及其旁观者效应。结果 所得病毒滴度为2.5×10^12pfu/ml。两种细胞的感染率相似，其感染率随腺病毒滴度的递增而增加。RT-PCR方法 检测发现：感染AdKDR-CDglyTK的BEL-7402有目的 基因CDglyTK的表达，感染AdKDR- CDglyTK的LS174T细胞无目的 基因表达。表达KDR的BEL-7402细胞对前药具有较高的敏感性，不表达KDR的LS174T细胞对前药不敏感（F=750.03，P〈0.001）。融合基因的疗效优于任一单自杀基因（F=275.89，P〈0.05）。将感染腺病毒的细胞与未感染细胞以不同比例混合培养，观察到该体系明显的旁观者效应。结论 KDR基因启动子可调控双自杀基因系统选择性杀伤表达KDR的肝癌细胞。     

eNOS基因治疗对肝硬化大鼠肝内eNOS表达和门静脉压力的影响

目的 探讨eNOS基因治疗对肝硬化大鼠肝内eNOS表达和门静脉压力的影响.方法 :建立CCl4肝内型肝硬化大鼠模型.取30只肝硬化大鼠,随机分成三组:eNOS治疗组(n=10)、Tris对照组(n=10)和空质粒对照组(n=10).经门静脉分别注射脂质体-eNOS质粒、Tris缓冲液、脂质体-空质粒,分别测定注射前和注射5 d后门静脉压力,并用RT-PCR和免疫组化方法检测三组大鼠注射5 d后肝内eNOS的表达.结果 :eNOS基因治疗5 d后,eNOS mRNA和eNOS蛋白表达都显著增加.eNOS治疗组门静脉压力为(12.8±0.8)mmHg,显著低于治疗前的PVP(15.3±1.0)mmHg(P<0.01),而两对照组门静脉压力在注射前后无显著性差异.结论 :以脂质体为载体,经门静脉注射eNOS基因治疗可增加肝内eNOS表达并降低门静脉压力.     

Total vascular exclusion safely facilitates liver specific gene transfer by the HVJ (sendai virus)-liposome method in rats

BACKGROUND: Most virus mediated transfection systems are efficient; however, their highly immunogenic properties do tend to cause clinical problems. HVJ-liposome vector is a hybrid vector consisting of liposome and inactivated sendai virus (hemagglutinating virus of Japan HVJ), which has been reported to be have a low immunogenicity, while it can also be repeatedly administered. To enhance the transfection efficiency, especially in the liver, we investigated the efficacy of total vascular exclusion (TVE) during the portal vein injection (PVI) of the vectors. MATERIALS AND METHODS: beta-galactosidase and luciferase expression were used as reporter genes. Wistar rats were injected with HVJ-liposome through PVI without TVE (PVI group, n = 10) or PVI with TVE (PVI + TVE group, n = 10). The control rats were infused with equal volumes of saline through the portal vein (control group n = 12). The transfection efficiencies were assessed by beta-galactosidase staining and a luciferase assay. Biochemical and histological analyses were performed to evaluate the tissue toxicity after gene transfer. RESULTS: The reporter genes expression in the liver dramatically increased after PVI + TVE in comparison to after PVI alone (1.2 x 10(5)versus 1.5 x 10(4) RLU/mg protein, P < 0.05 according to a luciferase assay). Notably, the extrahepatic "leaky" transgene expression could be minimized by PVI + TVE, whereas the general condition remained unchanged according to both the biochemical parameters and histological findings. CONCLUSIONS: The present data indicate that PVI + TVE may thus facilitate the liver-specific gene delivery using the HVJ-liposome method and this modality might also be applicable to other gene transfer systems.     

活体转染肝细胞生长因子基因对小鼠急性肝功能衰竭的治疗作用

目的 研究肝细胞生长因子（HGF）基因治疗对小鼠急性肝衰模型的治疗作用。方法 构建人肝细胞生长因子（hHGF）表达载体，利用脂质体介导法在活体内转染肝细胞生长因子基因，利用荧光显微镜检测肝组织内绿色荧光蛋白（GFP）表达了解目的基因的表达，用酶联免疫吸附试验（ELISA）法检测血清中人肝细胞生长因子的含量，通过观察小鼠急性肝衰模型的生存率、肝功能变化、肝组织病理改变来检测肝细胞生长因子基因对急性肝衰的治疗作用，通过检测肝组织中PC—NA指数的变化了解肝脏的增殖能力的变化。结果 荧光显微镜下可见肝组织内有绿色荧光蛋白的表达，转染人肝细胞生长因子基因后在血清中可检测到hHGF的表达，而且可持续1周以上，与对照组相比。转染hHGF基因组的生存率明显提高（40．0％vs11．5％，P〈0．05），血清ALT、TBi明显降低，肝组织中PCNA指数也明显升高。结论 活体转染肝细胞生长因子基因可获得表达，而且肝细胞生长因子基因对急性肝衰小鼠有治疗作用。     

Liposomal gene transfer of keratinocyte growth factor improves wound healing by altering growth factor and collagen expression

BACKGROUND: Growth factors affect the complex cascade of wound healing; however, interaction between different growth factors during dermal and epidermal regeneration are still not entirely defined. In the present study, we thought to determine the interaction between keratinocyte growth factor (KGF) administered as liposomal cDNA with other dermal and epidermal growth factors and collagen synthesis in an acute wound. MATERIALS AND METHODS: Rats received an acute wound and were divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.22 microg, vehicle), or liposomes plus the KGF cDNA (2.2 microg) and Lac-Z gene (0.22 microg). Histological and immunohistochemical techniques were used to determine growth factor, collagen expression, and dermal and epidermal structure. RESULTS: KGF cDNA increased insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3 (IGFBP-3), and fibroblast growth factor (FGF), decreased transforming growth factor-beta (TGF-beta), while it had no effect on platelet-derived growth factor (PDGF) levels in the wound. KGF cDNA significantly increased collagen Type IV at both the wound edge as well as the wound bed, while it had no effect on collagen Type I and III. KGF cDNA increased re-epithelialization, improved dermal regeneration, and increased neovascularization. CONCLUSIONS: Exogenous administered KGF cDNA causes increases in IGF-I, IGF-BP3, FGF, and collagen IV and decreases TGF-beta concentration. KGF gene transfer accelerates wound healing without causing an increase in collagen I or III.     

Increased apoptosis and decreased proliferation of colorectal cancer cells using insulin-like growth factor binding protein-4 gene delivered locally by gene transfer

OBJECTIVE: Insulin-like growth factor (IGF)-I induces proliferation of transformed cells. Its binding proteins (IGFBP) are involved in local regulation of IGF. This study assessed the effects of overexpression of IGFBP-4 on the development of cancer in vivo. METHOD: Nude mice were subcutaneously inoculated with HT-29 colorectal cancer cells (3 x 10(6)). When the tumour became visible (1 week after inoculation), animals received either 150 microg of mammalian expression vector containing IGFBP-4 cDNA or vector alone (n = 6 each) by peritumoural injection. Tumour size was measured during the growth. After 3 weeks of IGFBP-4 induction, animals were killed and tumour tissue samples were collected for examining the level of IGFBP-4 expression. Tumour mitotic activities were determined by counting numbers of mitotic cells on the tissue section. Apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUDP nick end labelling assay. RESULTS: Following IGFBP-4 treatment, tumour showed large necrotic areas, significantly increased numbers of apoptotic cells (36.67 +/- 7.36 vs 7.07 +/- 1.91, P < 0.01 vs control), decreased cells undergoing mitosis (2.31 +/- 0.32 vs 3.61 +/- 0.27, P < 0.01 vs control) and higher expression of IGFBP-4 (P < 0.05 vs control). CONCLUSION: IGFBP-4 gene transfer increased apoptosis and decreased mitosis, but tumour volume was not significantly altered possibly due to cellular debris filling the centre of tumours.     

Systematic evaluation of distribution of transgene expression after adenovirus-mediated gene transfer to the transplanted heart

In the transplantation setting, the study and potential treatment of acute and chronic rejection by means of gene therapy will require widespread transgene expression in the donor organ. The distribution of transgene expression after adenovirus-mediated gene transfer via the coronary vasculature in a model of abdominal heterotopic heart transplantation in syngeneic rats (n = 6) was evaluated at 1 week. Reporter gene expression was evenly distributed in the base, the midventricle, and the apex of the transplanted hearts. This study demonstrates that intracoronary administration of the adenoviral vector to the donor heart results in widespread transgene expres- sion. Received: 9 December 1997 Received after revision: 24 March 1998 Accepted: 15 April 1998     

Detection of apoptotic gene expression in human osteoblast-like cells by cDNA microarrays

Global gene expression during the induction of ion pair-mediated apoptosis was evaluated by an apoptosis microarray system. Human bone marrow stromal cells were cultured in the presence of 10^–6M dexamethasone to promote osteogenesis. After 28 days, these cells expressed elevated alkaline phosphatase activity and maintained Cbfa1 expression even when challenged with an apoptogen. Apoptosis was initiated by treating cells with 3mM Ca²⁺ and 5mM Pi for 4h. ³²P-Labeled mRNA was hybridized to a human apoptosis microarray containing 205 cDNA fragments. We found that apoptosis influenced the expression of 15 genes mainly involved in cell cycle and cell signaling. These genes included IGFBPs and ERK1, known to play a role in cell survival; GST and GST mu, required for maintenance of thiol redox; TNFR1, a gene product that initiates cell death; and finally, BAD, a gene that encodes a proapoptotic protein. Real-time PCR analysis showed that the expression of ERK1, TNFR1, and GST was modulated by 1.89-, 2.66-, and 1.6 fold after 4h and by 1-, 1.91-, and 1.5 fold, respectively, after 8h treatment with the ion pair. In addition, we also measured the expression of Bcl-2 and Bax by quantitative RT-PCR. We noted that these two genes were increased 3.07 and 2.99 fold, respectively, after 8h treatment with the apoptogen. Results of this study suggest that the ion pair influenced ERK1 and TNFR1 signaling pathways and affected thiol metabolism, whereas Bcl-2 and Bax were expressed at late stages of the death process.     

In vivo targeted gene transfer in skin by the use of laser-induced stress waves

BACKGROUND AND OBJECTIVES: Much interest has been shown in the use of lasers for nonviral targeted gene transfer, since the spatial characteristics of laser light are quite well defined. The aim of this study was to demonstrate in vivo gene transfer by the use of laser-induced stress waves (LISWs). STUDY DESIGN/MATERIALS AND METHODS: After reporter genes had been intradermally injected to rat skin in vivo, a laser target was placed on the gene-injected skin. LISWs were generated by the irradiation of an elastic laser target with 532-nm nanosecond laser pulses of a Q-switched Nd:YAG laser. RESULTS: Levels of luciferase activities for the skin exposed to LISWs were two orders of magnitude higher than those for the skin injected with naked DNA. Expressions of enhanced green fluorescent protein (EGFP) and beta-galactosidase were observed only in the area that was exposed to LISWs, and in addition, epidermal cells were selectively transfected. No major side effects were observed, and luciferase activity levels as high as 10(5) RLU per mg of protein were sustained even 5 days after gene transfer. CONCLUSION: Highly efficient and site-specific gene transfer can be achieved by applying a few pulses of nanosecond pulsed LISWs to rat skin in vivo.