Preparation of a recombinant adeno-associated viral vector with a mutation of human factor-Ⅸin large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25?012 particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitroand in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(106 cells)-1·(24h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt(2565.76?4.36) ng·(106 cells) -1·(24 h)-1 in C2C12 and453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapyresearch for hemophilia B, meanwhile, a novel packagingsystem, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation ofindustrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated withrAAV-hFⅨ.

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A,a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27%±3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeho-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.

Characteristics and advantages of adeno-associated virus vector-mediated gene therapy for neurodegenerative diseases

Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. Methods: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion: We have success- fully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.

MicroRNA-regulated viral vectors for gene therapy

Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.

A Viral Expression Vector from Foxtail mosaic virus to Express Green Fluorescent Protein

[Objective]Foxtail mosaic virus(FoMV)infects gramineous and dicotyledonous plants.In this study,we sought to construct a viral vector based on FoMV to express exogenous proteins in plants.[Method]A recombinant viral expression vector was constructed by inserting the promotor of Potato virus X(PVX)and exogenous gene sequences into the 3’non-coding region of the FoMV coat protein gene.[Results]The plasmid pCB301-FoMV-CP-PVXprom-GFP expressed green fluorescent protein in inoculated Nicotiana benthamiana leaves.[Conclusion]A recombinant viral expression vector was constructed successfully.

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.

Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo

OBJECTIVE： In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus （rAAV） serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS： Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma （NSG） mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS： We observed that treatment with pristimerin, at as low as 1 IJmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co- administration with pristimerin at 4 mg/（kg.d） for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION： These studies suggest the potential beneficial use of pristimerin and pristimerincontaining herb extract in future liver-targeted gene therapy with rAAV vectors.

非洲猪瘟病毒F334L基因编码蛋白的原核表达及多克隆抗体制备

本研究通过将非洲猪瘟病毒(African swine fever virus,ASFV)的一种非结构蛋白编码基因F334L克隆至原核表达载体pCold-Ⅰ,完成重组表达质粒pCold-Ⅰ-ASFV-F334L的构建。将pCold-Ⅰ-ASFV-F334L转化受体菌感受态细胞,经1 mmol/L的IPTG诱导原核表达。SDS-PAGE和Western blot试验检测结果表明,F334L基因得到了良好的表达,将所得的F334L基因编码蛋白纯化后免疫小鼠,获得含多克隆抗体的血清。利用该抗体检测实验室构建并拯救的表达ASFV F334L基因编码蛋白的重组猪繁殖与呼吸综合征病毒rPRRSV-F334L,试验结果显示该多克隆抗体具有良好的反应原性和特异性,证明了ASFV F334L基因可在PRRSV活载体中稳定表达,为ASFV活载体疫苗的研发提供了材料。

Increasingβ-hexosaminidase A activity using genetically modified mesenchymal stem cells

GM2 gangliosidoses are a group of autosomal-recessive lysosomal storage disorde rs.These diseases result from a deficiency of lysosomal enzymeβ-hexosaminidase A(HexA),which is responsible for GM2 ganglioside degradation.HexA deficiency causes the accumulation of GM2-gangliosides mainly in the nervous system cells,leading to severe progressive neurodegeneration and neuroinflammation.To date,there is no treatment for these diseases.Cell-mediated gene therapy is considered a promising treatment for GM2 gangliosidoses.This study aimed to evaluate the ability of genetically modified mesenchymal stem cells(MSCs-HEXA-HEXB)to restore HexA deficiency in Tay-Sachs disease patient cells,as well as to analyze the functionality and biodistribution of MSCs in vivo.The effectiveness of HexA deficiency cross-correction was shown in mutant MSCs upon intera ction with MSCs-HEXA-HEXB.The results also showed that the MSCs-HEXA-HEXB express the functionally active HexA enzyme,detectable in vivo,and intravenous injection of the cells does not cause an immune response in animals.These data suggest that genetically modified mesenchymal stem cells have the potentials to treat GM2 gangliosidoses.

鸡传染性喉气管炎疫苗研究进展

鸡传染性喉气管炎(Infectious Laryngotracheitis,ILT)是由喉气管炎病毒(Infectious Laryngotracheitis Virus,ILTV)引起的鸡的一种病毒性呼吸道传染病。ILT是一种较为严重的禽病毒病;根据ILT的临床表现分为急性型和温和型,急性型发病率和死亡率均较高,温和型死亡率低但在养殖密集地区感染逐渐增多。ILT可引起家禽死亡和蛋鸡产蛋率下降,给养禽业带来严重的经济损失。目前,通过使用疫苗接种和生物安全相结合的综合防控措施,在集约化养禽地区ILT已得到较好的控制。该文对多个国家已商品化的ILT减毒活疫苗和重组病毒载体活疫苗,以及正在研发中的ILT重组病毒载体活疫苗、基因缺失活疫苗、病毒样颗粒疫苗等新型ILT疫苗进行综述,以期为更好地防控ILT提供理论参考。

Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions

Recombinant adeno-associated virus （rAAV） serotype 2, 3 and 8 vectors are the most promising liver- tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies （Nabs） were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2 〉 AAV3 = AAVLK03 〉 AAV8. There was no difference between： 1）AAV3 and its variants; 2） male and female subjects; and 3） different age cohorts （〈 35, 36- 50, and 〉 51 years old）. People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.

HCVC基因腺病毒表达载体骨架质粒pAd.HCV-C的构建、鉴定及表达

目的构建能表达HVC基因的腺病毒表达载体的重组骨架质粒,为进一步包装能高效表达HCVC基因的腺病毒载体做准备。方法用分别含有BgⅡ及HindⅢ酶切位点的HCVC区基因上、下游引物,以含有HCV H株基因序列的质粒pBRTM/HCV1-3011为模板,通过PCR扩增获得HCVC区基因片段,基因片段回收后,以BglⅡ及HindⅢ双酶切,定向插入到腺病毒骨架质粒pAd,CMV-Link.1中CMV启动子下游BglⅡ与HindⅢ位点之间,获得重组表达质粒pAd.HCV-C通过BglⅡ/HindⅢ双酶切、PCR及插入片段序列测定对质粒进行了鉴定,以抗HCVC单克隆抗体为一抗,利用间接免疫荧光法检测了pAd.HCV-C在人肝癌细胞7721中的瞬时表达。结果酶切、PCR及测序鉴定证实,pAd,HCV-C插入片段为HCVC区基因片段,免疫荧光法检测表明其可以在7721细胞中瞬时表达。结论构建的质粒pAd.HCV-C可以在7721细胞中瞬时表达HCVC区基因,为包装表达HCVC基因的腺病毒载体奠定了基础。

丙型肝炎病毒(HCV)实验性疫苗的研究进展

丙型肝炎病毒是引起输血相关肝炎及慢性肝炎、肝硬化、肝癌的主要病原,目前尚无有效的治疗与预防手段。综述了HCV感染所引起的机体免疫应答及近年来实验性疫苗(主要是DNA疫苗、病毒载体疫苗及联合疫苗)的研究进展及面临的挑战,并提出,激发广谱、强大T细胞应答的初免-加强免疫方案是HCV实验性疫苗研制的新策略。

山羊痘病毒ORF8～ORF18缺失重组毒株的构建和鉴定

本研究旨在通过敲除山羊痘病毒（GTPV）大段基因组片段尝试构建安全性更好的基因工程减毒株和病毒表达载体。采用PCR方法,克隆了GTPV AV41株基因组ORF7-ORF8间1 242 bp的基因片段,以及ORF18~ORF19间1 355 bp的片段。接着分别以上述2片段作为左、右侧同源重组臂,构建了包含报道基因EGFP和抗性基因gpt的GTPV重组转移载体pCF-Eg。然后采用脂质体介导法,将重组转移载体pCF-Eg与GTPV AV41株共转染原代羊睾丸细胞,通过空斑纯化筛选阳性重组病毒,并鉴定重组病毒的遗传稳定性和生长特性。结果成功获得1株敲除掉ORF8~ORF18间9个ORF、长达7 kb基因组片段的重组病毒vCF-Eg。该重组病毒能稳定表达绿色荧光蛋白至少10代,并且其增殖滴度与亲本毒株基本相同。表明上述区域是GTPV的复制非必需区。

病毒载体研究进展

外源基因进入细胞通常需要“运载工具”。在漫长的自然进化过程中,病毒是以寄生形式存活下来的最小、最简单的生命体,病毒分子生物学的发展为生物工程领域研究拓展了思路,提供了有效的方法和工具。重组病毒载体又是当今病毒基因工程研究的热点之一,目前以动物病毒为基础设计的基因克隆及表达载体主要有取代型的重组病毒载体和重组的病毒-质粒载体。文章对应用较广泛的病毒载体做一综述。

EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。

重组禽α疱疹病毒研究进展

禽α疱疹病毒可以引起多种家禽重要传染病,重组病毒是研究其基因组结构功能和新型基因工程疫苗的有效手段。文章综述了禽α疱疹病毒转移载体的构建和同源重组方法、感染性细菌人工染色体系统、基因缺失疫苗和重组活载体疫苗的最新进展。

结肠灌注重组腺相关病毒2/人凝血因子Ⅸ基因载体治疗血友病B的安全性研究

目的分析结肠灌注重组腺相关病毒2/人凝血因子Ⅸ(rAAV2/CMV-hFⅨ)基因载体治疗血友病B小鼠的安全性。方法将rAAV2/CMV-hFⅨ(剂量为1×1013 v.g/kg)结肠灌注血友病B模型小鼠,分别检测抗重组腺相关病毒2(rAAV2)抗体和抗人凝血因子Ⅸ(hFⅨ)抗体,并通过组织病理学、免疫组化和PCR技术分析病毒和hFⅨ的组织分布。结果灌注后,在小鼠体内能够检测到抗rAAV2抗体和抗hFⅨ抗体;组织病理学、免疫组化和PCR技术分析结果显示灌注部位肠组织无明显病理改变,rAAV2以及hFⅨ基因仅仅在灌注的肠上皮。结论结肠灌注重组腺相关病毒基因治疗血友病B小鼠,在所观测时间内是安全的。

禽流感-火鸡疱疹病毒重组活载体疫苗的研究进展

禽流感(Avian influenza,AI)和马立克氏病(Marek′s disease,MD)是严重危害全球养禽业发展的两个主要病毒性传染病。目前,疫苗免疫是防控这两种病毒性疾病有效手段之一。在众多病毒活载体疫苗中,火鸡疱疹病毒(Herpesvirus of turkey,HVT)以其独特的优势备受青睐,而以HVT表达禽流感病毒保护性抗原是目前的研究热点之一。本文对以HVT作为疫苗载体的优势和构建载体疫苗的影响因素(外源基因、启动子、复制非必需区的选择等)进行多方面分析,并对目前AI-HVT重组疫苗的研究进展进行综述。