Integration of active human beta-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal beta-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of beta-galactosidase (beta-gal) could be detected in human beta-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of beta-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated beta-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed beta-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and beta-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.     

Neuronal precursor-restricted transduction via in utero CNS gene delivery of a novel bipartite HSV amplicon/transposase hybrid vector.

The ability to modify genetically in utero the precursors of neuronal lineage contributing to multiple postmitotic cell types in the adult central nervous system would provide a means to evaluate strategies to ameliorate conditions affecting cellular patterning, metabolism, or survival. The herpes simplex virus (HSV)-derived amplicon, a vector devoid of viral genes and with the largest known payload capacity, normally exists episomally within nuclei of transduced cells, thus precluding conveyance during mitosis. Herein, we modify the Tc1-like Sleeping Beauty (SB) transposon system to create an integrating amplicon vector platform wherein provision of transposase in trans effectively catalyzes integration of a transgenomic segment. Cotransduction with a Rous sarcoma virus promoter-driven beta-galactosidase-neomycin (betageo) fusion flanked by SB terminal repeats (HSVT-betageo) and a second expressing the SB transposase gene under HSV immediate-early 4/5 gene promoter control (HSVsb) resulted in integration and extension of expression duration. Most notably, in utero intraventricular application led to extensive transgene expression within neuronal precursors and their derivatives without attendant adverse consequences, suggesting this new platform could be used to evaluate prenatally the function of gene products in neuronal lineages and evaluate therapeutic strategies for correction of genetic abnormalities affecting the developing CNS.     

Functional recovery in a Friedreich's ataxia mouse model by frataxin gene transfer using an HSV-1 amplicon vector.

There is currently no effective treatment for Friedreich's ataxia (FA), the most common of the hereditary ataxias. The disease is caused by mutations in FRDA that drastically reduce expression levels of the mitochondrial protein frataxin. In FA animal models, a key difficulty is obtaining the precise levels of frataxin expression in the appropriate tissues to provoke pathology without early lethality. To develop strategies to circumvent these problems, conditional frataxin transgenic mice have been generated. We now show that frataxin expression can be eliminated in neurons from these loxP[frda] mice by infection with CRE-expressing herpes simplex virus type 1 (HSV-1) amplicon vectors. We have also achieved in vivo delivery by stereotaxic injection of these CRE-expressing vectors into the brainstem of loxP[frda] mice to generate a localized gene knockout model. These mice develop a behavioral deficit in the rotarod assay detectable after 4 weeks, and when re-injected with HSV-1 amplicon vectors expressing human frataxin complementary DNA (cDNA) exhibit behavioral recovery as early as 4 weeks after the second injection. To the best of our knowledge, this is the first proof of principle of recovery of neurological function by a therapeutic agent aimed at correcting frataxin deficiency.     

Regulation of glucose-transporter gene expression in vitro and in vivo

Understanding of the fundamental mechanisms underlying the complex regulation of glucose homeostasis has been dramatically transformed recently by the realization that glucose transport in mammalian tissues is mediated by a family of structurally related but genetically distinct glucose-transporter proteins. The regulatory factors and intracellular signaling pathways that influence expression of the genes encoding these proteins are just being identified. Factors that regulate glucose-transporter gene expression in vitro include oncogenes, growth factors, insulin, oral hypoglycemic agents, vanadate, glucocorticoids, ambient glucose levels, and the state of cellular differentiation. In vivo, glucose-transporter gene expression in adipose cells, skeletal muscle, and liver is markedly affected by various altered nutritional and metabolic states. Recent studies have demonstrated that two glucose transporters expressed in the same tissue may be regulated differently in response to the same metabolic perturbation. Furthermore, transporter regulation appears to be tissue specific. These observations lay the groundwork for future studies aimed at unraveling the functional roles of the individual transporter species in different tissues, the molecular processes involved in regulating the expression of these genes, and the impact of dysregulated glucose-transporter gene expression in the pathogenesis of insulin-resistant states such as diabetes.     

HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. Previous research has shown that it is possible to correct the hereditary deficiency A-T by DNA transfection in cell culture, but the large size of the ATM cDNA (9 kb) limits the use of many vector types for gene replacement. HSV-1 amplicon vectors provide a means to deliver large genes to cells efficiently and without toxicity. In this study, the FLAG-tagged cDNA for human ATM was inserted into an HSV-1 amplicon under control of the CMV promoter (designated as HGC-ATM). FLAG-ATM expression was confirmed in 293T/17 cells and human A-T fibroblasts (GM9607) after transduction, by immunoprecipitation, Western analysis, and immunocytochemistry. Functional recovery was assessed by two independent assays. First, in vitro kinase assay showed that vector-derived ATM in GM9607 cells could successfully phosphorylate wt p53 using recombinant GST-p53(1-101). Second, in A-T cells infected with the HGC-ATM vector, the extent of accumulation in G2/M phase at 24 h postirradiation was similar to that observed in cells with wild-type endogenous ATM and lower than that observed in A-T cells infected with a control vector. Thus, these vectors provide a tool to test the feasibility of HSV-amplicons as gene therapy vectors for A-T.     

Dextran-spermine polycation: an efficient nonviral vector for in vitro and in vivo gene transfection

Dextran-spermine cationic polysaccharide was prepared by means of reductive amination between oxidized dextran and the natural oligoamine spermine. The formed Schiff-base imine-based conjugate was reduced with borohydride to obtain the stable amine-based conjugate. The transfection efficiency of the synthetic dextran-spermine was assessed in vitro on HEK293 and NIH3T3 cell lines and found to be as high as the DOTAP/Chol 1/1 lipid-based transfection reagent. Modification of the dextran-spermine polycation with polyethylene glycol resulted in high transfection yield in serum-rich medium. Intramuscular injection in mice of dextran-spermine-pSV-LacZ complex induced high local gene expression compared to low expression of the naked DNA. Intravenous injection of a dispersion of the dextran-spermine-pSV-LacZ complex resulted with no expression in all examined organs. When the partially PEGylated dextran-spermine-pSV-LacZ complex was intravenously applied, a high gene expression was detected mainly in the liver. Preliminary targeting studies indicated that the PEGylated dextran-spermine-pSV-LacZ complex bound to galactose receptor of liver parenchymal cells rather than the mannose receptor of liver nonparenchymal cells. This work offers a new biodegradable polycation based on natural components, which is capable of transfecting cells and tissues in vitro and in vivo.     

Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.     

In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer

Increasing the level and duration of transgene expression and restricting expression to vascular cells are important goals for clinically useful gene therapy vectors. We evaluated several promoters, enhancers and introns in endothelial, smooth muscle and liver cells in tissue culture and in vivo, comparing local delivery to the carotid artery with intravenous delivery to the liver. A 1800-bp fragment of the oxidized LDL receptor (LOX-1) promoter showed highest in vivo activity in the carotid artery, achieving 39% the activity of the reference cytomegalovirus promoter, with 188-fold greater specificity for carotid artery over liver. An enhancer from the Tie2 gene in combination with the intracellular adhesion molecule-2 promoter improved endothelial specificity of plasmid vectors, increased the expression from adenoviral vectors in cultured endothelial cells and doubled the specificity for carotid artery over liver in vivo. Adding a short intron to expression cassettes increased expression in both endothelial and smooth muscle cells in vitro; however, the eNOS enhancer failed to consistently increase the expression or endothelial specificity of the vector. In conclusion, elements from the LOX-1 promoter and Tie2 enhancer together with an intron can be used to improve vectors for vascular gene transfer.     

Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression in vivo

There is currently an urgent need to develop efficient gene-delivery systems for the lung that are free of inflammatory effects. The LID vector is a synthetic gene delivery system, comprised of lipofectin (L), an integrin-targeting peptide (I) and DNA (D) that has previously been shown to have high transfection efficiency in the lung. We have assessed the effect of alternative methods of complex preparation on structural features of the complex, levels and duration of reporter gene expression and the host response to the LID vector. We have demonstrated that making the complex in water affects the structure of the LID complexes making them smaller and more stable with a more cationic surface charge than complexes prepared in phosphate-buffered saline (PBS). When the LID vector was constituted in water and instilled intratracheally into the lungs of mice there was a 10-fold increase in luciferase activity compared with preparation in PBS. Furthermore, luciferase activity was still evident 1 week following vector instillation. This enhancement may be because of altered complex structure, although effects of the hypotonic vector solution on the lung cannot be excluded. The inflammatory effects of instilling the LID vector in water were minimal, even after three administrations of the LID vector, with only mild alterations in cytokine and broncho-alveolar lavage fluid (BALF) cell profiles. These results demonstrate that the LID vector can generate high, and prolonged, levels of gene expression in the lung from small quantities of DNA and that careful attention to synthetic polyplex structure may be important to optimize efficiency of gene expression in vivo.     

表达降钙素基因相关肽的单纯疱疹病毒Ⅰ型扩增子载体的构建、包装和扩增

目的对单纯疱疹病毒Ⅰ型扩增子质料装载降钙素基因相关肽,并进行包装和扩增。方法分别钓取pac基因和γ134.5基因,克隆入pMD18-T载体中。pSV载体经HindⅢ和BamHⅠ双酶切,获得lacZ片段,自身环化形成pUPO-γ134.5-lacZ。合成CGRP基因序列,将此基因替换pUPO-γ134.5-lacZ质粒中的lacZ基因,插入γ134.5基因位点中,即pUPO-Δγ134.5-CGRP。再用单纯疱疹病毒温敏株辅助在BHK细胞中包装、扩增,通过检测lacZ的表达反映扩增子病毒滴度。结果酶切鉴定证实重组子构建成功。重组扩增子质粒的包装、扩增由BHK细胞的形态变化和X-gal原位检测载体lacZ的表达证实。扩增子假型病毒滴度达3.5×105TU/ml。结论本研究构建、包装、扩增的携带降钙素基因相关肽的假型病毒有较高感染性,这种扩增子病毒可作为实验研究的有力工具。     

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

Bicistronic vectors are essential to achieve efficient expression of multiple genes in gene therapy protocols and biomedical applications. Internal ribosome entry site (IRES) elements have been utilized to initiate expression of an additional protein from a bicistronic vector. The IRES element commonly used in current bicistronic vectors originates from the encephalomyocarditis virus (EMCV). As IRES-mediated translation is dependent on availability of IRES trans-acting factors, which vary between cell types and species, adequate gene expression from the EMCV IRES element is not always achieved. To identify a novel IRES element that mediates gene expression consistently with a higher efficiency than the EMCV IRES, we tested 13 bicistronic reporter constructs containing different viral and cellular IRES elements. The in vitro screening in human and mouse fibroblast and hepatocarcinoma cells revealed that the vascular endothelial growth factor and type 1 collagen-inducible protein (VCIP) IRES was the only IRES element that directed translation more efficiently than the EMCV IRES in all cell lines. Furthermore, the VCIP IRES initiated greater reporter expression levels than the EMCV IRES in transfected mouse livers. These results suggest that VCIP-IRES containing vectors improve gene expression compared with those harboring an EMCV-IRES. This could increase the potential benefits of bicistronic vectors for experimental and therapeutic purposes.     

Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo

Regulated expression of therapeutic genes is required for long-term gene therapy applications for many disorders. Here we describe a doxycycline (dox)-regulated lentiviral vector system consisting of two HIV-1-based self-inactivating viruses. One of the vectors is constitutively expressing a novel improved version of the tetracycline reverse transactivator rtTA2(S)-M2 and the other has a rtTA responsive promoter driving the expression of beta-galactosidase gene (lacZ). The rtTA2(S)-M2 has highly improved properties with respect to specificity, stability and inducibility. Functionality of the system by dox was confirmed after in vitro cotransduction of Chinese hamster ovary and human endothelial hybridoma (EAhy926) cells. Regulation of the system showed tight control of the gene expression. Dose dependence for dox was seen with concentrations that can be obtained in vivo with doses normally used in clinical practice. LacZ expression could be switched on/off during long-term (3 months) culturing of cotransduced cells. The system was next tested in vivo after cotransduction into rat brain and studying expression of the lacZ gene in dox-treated and control rats. Nested RT-PCR confirmed that the tight control of the gene expression was achieved in vivo. Also, X-gal staining showed positive cells in the dox-treated rats, but not in the controls 10 days after cotransduction with 4 days preceding treatment with dox. It is concluded that our doxycycline-regulated vector system shows significant potential for long-term gene therapy treatments.     

表达降钙素基因相关肽的单纯疱疹病毒I型扩增子载体免疫效果观察

目的 探讨重组pUPO-△γ134.5-CGRP扩增子对免疫系统的作用.方法 用重组扩增子感染BALB/C小鼠,分离血清及淋巴细胞,流式细胞仪法、免疫组化法、LDH法、分别检测外周血CD4、CD8分类、TCR、NK细胞、CTL细胞以及LAK细胞活性杀伤活性.结果 重组扩增子明显增强了机体的免疫力.结论 该重组扩增子具有增强机体免疫力的作用.     

Functional long-term thymidine kinase suicide gene expression in human T cells using a herpesvirus saimiri vector

Herpesvirus saimiri transforms human T lymphocytes to stable growth and persists episomally without genomic integration and without virus production. The transformed T cells retain essential features of their parental cells including the MHC-restricted antigen specificity which may be useful for applications in adoptive immunotherapy. In order to improve the biological safety of such vectors, the prodrug activating gene thymidine kinase of herpes simplex virus was inserted into the genome of herpesvirus saimiri by homologous recombination. After infection with wild-type or cloned recombinant viruses, T cells from tamarin monkeys and from humans were transformed to stable growth. Thymidine kinase-expressing transformed T cells were efficiently eliminated in the presence of low concentrations of ganciclovir. This elimination mechanism remained fully functional over an observation period of 12 months. The potentially immunogenic neomycin resistance gene expression cassette was deleted from the genome of established mutant viruses by using the prokaryotic Cre/LoxP recombination system. At any time during the course of a therapeutic application, thymidine kinase-expressing transformed human T cells might be eliminated after administration of ganciclovir. In principle, this function could be useful for the T cell-dependent immunotherapy of resistant blood cancer while avoiding the risk of uncontrolled graft-versus-host disease.     

真核表达载体pcDNA3-CTLA41g的构建及体内表达

背景:CTLA-41g的制备过程比较复杂,单克隆抗体的价格比较昂贵,而且应用CTLA-41g单克隆抗体注射本身存在着血浆药物浓度不稳定的问题.目的:探讨Ctla41g表达质粒构建的可行性.设计、时间及地点:基因水平观察实验,于2005/2006在中国医科大学中心实验室完成.材料:雄性Wistar大鼠10只,近交系昆明小鼠10只.方法:提取Wistar大鼠总RNA,反转录成cDNA第1链,PCR扩增CTLA4基因,将其和真核表达载体pcDNA3酶切、连接.转化感受态菌DH5α,培养后挑取刚性克隆、提取质粒,酶切鉴定重组子、测序,最后转染近交系昆明小鼠,应用Westen Blot法检测血清CTLA41g水平.主要观察指标:构建的Ctla41g表达质粒基因序是否正确以及能否在小鼠体内进行蛋白表达.结果:对含有pcDNA3-CTLA4质粒的LB菌液进行鉴定测序,目的基因片断大小为288 bp,与Genebank上公布的CTLA4序列完全一致,质粒构建正确.利用阳性脂质体载体包被pcDNA3一CTLA41g转染小鼠,有3只在第7天血清检测CTLA41g阳性,显示pcDNA3-CTLA41g能够在鼠肌细胞内表达.结论:利用基因合成和重组技术成功构建了真核表达载体pcDNA3-CTLA41g,利用脂质体法成功的将pcDNA3-CTLA41g转染到鼠肌细胞内并进行表达.