腺相关病毒介导的体内睾丸组织基因特异性敲除系统的建立

目的建立一套可用于体内睾丸组织特异性敲除基因的系统。方法利用同源重组技术将CRISPR/Cas9系统中sgRNA功能元件插入到AAV表达载体,将人源Wee1 2#sgRNA构建入改造的AAV-sgRNA(新版)载体中进行病毒包装,并感染稳定表达Cas9的HeLa-spCas9细胞验证该载体的基因敲除效率。筛选睾丸组织特异表达基因Sycp3的sgRNA靶点,构建入AAV-sgRNA(新版)载体中,进行病毒包装,利用显微注射技术将病毒注射入小鼠睾丸组织曲细精管内,通过T7E1分析体内细胞的基因敲除效果。结果构建成功的AAV-sgRNA载体能够进行病毒包装并在体外细胞水平上对人源Wee1进行基因编辑,与慢病毒介导的CRISPR/Cas9系统中的载体编辑效率相比无明显差异。同时,该系统能在体内水平对Sycp3进行基因编辑。结论成功建立一套可用于在体内睾丸组织中进行特异性敲除目标基因的系统,为生殖体内功能研究提供一个新的思路和方法。     

miR-210通过MMP-2下调M2/M1巨噬细胞比例抑制肺癌细胞生长

目的研究miR-210通过MMP-2调控M2/M1巨噬细胞比例对肺癌细胞生长的影响及对相关机制进行初步探讨。方法将小鼠Lewis lung carcinoma (LLC)-1细胞皮下植入C57BL/6小鼠构建小鼠肺癌模型,荧光定量PCR检测miR-210表达。进一步取40只肺癌建模成功的小鼠,并随机分为4组:对照组小鼠尾静脉注射AAV9-Fugw腺相关病毒;AAV9-OE-miR-210组小鼠尾静脉注射AAV9-OE-miR-210腺相关病毒; AAV9-OE-MMP-2组小鼠尾静脉注射AAV9-OE-MMP-2腺相关病毒;AAV9-OE-miR-210+AAV9-OE-MMP-2组小鼠尾静脉注射AAV9-OE-miR-210和AAV9-OE-MMP-2腺相关病毒。所有小鼠注射腺相关病毒滴度为108TU,病毒注射时间为4周。检测4组小鼠肿瘤大小变化,WGA染色检测4组小鼠肺癌细胞大小变化。另外,取生长融合至80%的LLC1细胞,并随机分为2组:WT组转染包含野生型MMP-2 3’-UTR的构建物,Mut组转染突变型MMP-2 3’-UTR的构建物。细胞转染48 h后,荧光素酶实验检测m...     

不同载体转基因效率及AAV2介导bFGF基因在肌腱中表达的研究

目的:比较腺病毒、腺相关病毒和脂质体-质粒三种载体在肌腱中的基因转染效率并观测腺相关病毒载体介导bFGF基因在肌腱愈合过程中表达.方法:用微量注射器将携带增强绿色荧光蛋白报告基因的三种载体注射入正常肌腱中,用荧光显微镜观测术后不同时间点肌腱内EGFP的表达.将AAV2-bFGF病毒颗粒注入损伤肌腱,使用免疫组化染色观测bFGF在肌腱内表达.结果三种不同的载体转染肌腱3天后有EGFP表达、7天最明显、14天时下降;21天时少有表达.在同期的时间点,注射腺病毒、腺相关病毒组肌腱内EGFP表达明显强于质粒载体,而腺病毒、腺相关病毒两组之间无较大区别.免疫组化显示注射AAV2-bFGF后的第2、3和4周的肌腱表达很强的bFGF.结论:腺病毒、腺相关病毒载体的基因转染效率高于脂质体-质粒载体,AAV2携带的外源性bFGF基因在肌腱细胞内较强表达,提示我们将来在体内转基因到肌腱的研究中能够选择腺病毒和腺相关病毒载体作为运载基因的工具.     

共表达2个独立的抗关节炎分子TNFR-Fc和CTLA4-FasL重组表达系统的建立与鉴定

目的构建能同时独立表达双抗关节炎分子TNFR-Fc和CTLA4-FasL的重组腺相关病毒(AAV)载体,并对蛋白表达进行鉴定。方法应用furin-2A新型剪切策略,构建TNFR-Fc和CTLA4-FasL双融合基因重组AAV载体,体外转染293T细胞,收集转染上清,以ELISA和Western blot等方法检测目的蛋白的表达。结果成功构建双抗炎分子重组表达载体TFCF,在转染细胞上清中检测到目的蛋白TNFR-Fc和CTLA4-FasL的独立共表达。结论获得了可同时拮抗TNF和T细胞的新型双抗关节炎分子共表达系统,为今后动物体内研究奠定了基础。     

Complexin-1/2过表达重组载体对阿尔茨海默病小鼠记忆损伤的实验研究

目的　构建Complexin-1/2（CPLX-1/2）基因的重组腺相关病毒过表达载体pAAV- CPLX-1/2并研究其在阿尔茨海默病记忆损伤中的作用。 方法　将用Oligo 7软件对Genbank上小鼠CPLX-1/2 mRNA的CDS两端序列进行分析,合成CPLX-1/2基因,构建过表达载体并转化至大肠杆菌Top10;选取正确的克隆进行PCR和测序鉴定。重组腺相关病毒载体测序正确后,转染AAV293细胞,包装腺相关病毒。将重组腺相关病毒经脑立体定位注射到三转基因阿尔茨海默病(3XTg-AD)小鼠海马CA3区,经免疫组化和Western-Blot检测CPLX-1/2蛋白表达情况,水迷宫检测CPLX-1/2过表达对3XTg-AD小鼠记忆的影响。 结果　PCR及测序结果表明成功构建了含小鼠CPLX-1/2基因的重组腺相关病毒表达载体。CPLX-1/2在海马CA3表达显著增加。CPLX-1/2过表达显著改善3XTg-AD小鼠的记忆损伤。 结论　本研究成功构建了CPLX-1/2重组腺相关病毒过表达载体并在整体动物水平成功高效表达; CPLX-1/2过表达可显著改善3XTg-AD小鼠空间记忆的损伤。为在整体动物水平深入研究CPLX-1/2的功能提供了新的手段;研究结果提示CPLX-1/2可能作为AD记忆损伤治疗的关键靶点。     

人肿瘤坏死因子受体-Fc融合蛋白的表达及生物活性分析

目的构建携带人肿瘤坏死因子受体(TNFR)胞外区融合人IgG Fc片段的重组腺相关病毒载体(pAAV/hTNFR-Fc),并对融合蛋白hTNFR-Fc的表达和生物学活性进行鉴定。方法构建hTNFR-Fc融合基因重组腺相关病毒(AAV)载体,体外转染人胚肾HEK293T细胞,收集转染上清,以Western blot法检测目的蛋白的表达,应用L929细胞测定重组表达产物对人和大鼠TNF-α细胞毒中和活性,采用ELISA比较重组表达产物对人和大鼠TNF-α的结合活性。结果成功构建重组表达载体pAAV/hTNFR-Fc,在转染细胞上清中检测到目的蛋白hTNFR-Fc的表达,重组表达产物可有效结合人TNF-α并中和其L929细胞毒活性,并对大鼠TNF-α具有一定程度的结合和中和活性。结论成功构建了hTNFR-Fc融合基因,验证了其在AAV载体上的分泌性表达,并对其生物活性进行了鉴定,为进一步病毒包装和大鼠关节炎动物实验研究奠定了基础。     

含HIV-1 gp120基因的重组腺相关病毒和重组腺病毒疫苗联合免疫的研究

目的 探讨含HIV-1 gp120基因的重组腺相关病毒(rAAV)和重组腺病毒(rAdV)疫苗在BALB／c小鼠中联合免疫的效果。方法 将密码子优化的HIV-1 gp120基因分别插入腺相关病毒(AAV)和腺病毒(AdV)载体质粒，构建含该基因的rAVV和rAdV载体疫苗。将两种疫苗以不同的联合方式免疫BALB／c小鼠，ELISA检测小鼠血清中的gp120特异性抗体，细胞内细胞因子染色法检测小鼠的特异性细胞毒性T淋巴细胞(CTL)应答。结果 两种重组病毒均可表达目的基因gp120；在小鼠体内两种重组病毒联合免疫可诱导特异性的CTL应答和血清1gG抗体反应，但用rAAV初免2次，再用rAdV加强3次所诱发的CTL和血清1gG反应最强。结论 rAAV和rAdV疫苗联合免疫可在小鼠体内诱导特异性的CTL应答和血清1gG抗体反应。     

玻璃体注射腺相关病毒2和慢病毒转染大鼠视网膜的比较

目的通过玻璃体注射重组腺相关病毒2载体(r AAV2)和慢病毒载体(LV),比较两者转染成年大鼠视网膜的异同。方法实验组大鼠60只,每组12只玻璃体内分别注射重组腺相关病毒2载体-增强绿色荧光蛋白(r AAV2-EGFP)、重组腺相关病毒2载体-神经突起素-增强绿色荧光蛋白(r AAV2-neuritin-EGFP)、慢病毒载体-红色荧光蛋白(LV-RFP)和慢病毒载体-神经突起素-红色荧光蛋白(LV-neuritin-RFP),对照组注射等量的生理盐水,4周后取材,采用免疫荧光和CTB-FITC检测2种病毒载体在视网膜中转染的细胞及转染率,然后分别采用Real-time PCR和Western blotting检测神经突起素(neuritin)在视网膜中的表达变化。结果 r AAV2能够转染约70%视网膜节细胞(RGCs),LV主要转染色素上皮细胞,RGCs转染率仅为30%;r AAV2-neuritin-EGFP组神经突起素mRNA表达约是r AAV2-EGFP组和对照组的16倍,蛋白表达约为3倍;LV-neuritin-RFP组神经突起素mRNA表达约是LVRFP组和对照组的5.5倍,蛋白表达约为1.7倍。结论 r AAV2玻璃体注射后,转染大部分RGCs且神经突起素表达量高于LV,LV主要转染色素上皮细胞,提示基因治疗眼科疾病和损伤时,涉及到转染RGCs时应采用r AAV2载体,转染色素上皮时宜采用LV载体。     

抗阿尔茨海默病重组腺相关病毒的制备与活性研究北大核心CSCD

目的:制备可表达抗阿尔茨海默病单链抗体AT8-scFv的重组腺相关病毒AAV8-AT8-scFv,并探究其体内外活性。方法:利用293T细胞对重组病毒进行包装与纯化;SDS-PAGE、RT-qPCR对重组病毒的理化性质进行表征;细胞感染、Western blot法检测重组病毒的体外感染活性;以BALB/c小鼠为动物模型进行免疫,ELISA法、活体成像检测重组病毒的体内表达情况。结果:成功制备重组腺相关病毒AAV8-AT8-scFv,该病毒具有较好的感染能力,并可在小鼠体内长期稳定表达。结论:成功获得具有良好生物学活性的重组腺相关病毒,为阿尔茨海默病的免疫治疗提供新思路。     

HBV靶向腺相关病毒载体的构建与鉴定

目的：构建和鉴定HBsAg靶向性的重组2型腺相关病毒（ adeno?associated virus, AAV2）载体。方法运用噬菌体展示技术筛选特异性结合HBsAg的多肽,通过重组PCR技术将特异性多肽插入到2型腺相关病毒（AAV2）核衣壳蛋白的587位氨基酸处,构建靶向结合HBsAg的重组AAV2病毒。流式细胞术检测重组病毒感染HepG2?215细胞的特异性。结果筛选得到HBsAg特异性多肽,序列为SSYAPYVWQ?PIA。成功构建了携带靶向 HBsAg 多肽的重组 AAV2,命名为 rAAVssyU6?hrGFP。 rAAVssyU6?hrGFP 对HepG2、 HepG2?215细胞的感染率较AAV2明显升高,对HepG2?215细胞的感染率最高, HBsAb封闭实验明显降低rAAVssyU6?hrGFP病毒感染HepG2?215细胞的效率。结论 rAAVssyU6?hrGFP对HepG2?215细胞的感染具有特异性,有望成为介导siRNA分子有效治疗HBV的靶向载体。     

Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units

Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology.     

Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector

The ability to manipulate the expression of genes within neurons provides unique opportunities to study the role of individual gene products in nervous system function. Virus vectors are a potentially rapid tool for the experimental manipulation of gene expression in the mammalian nervous system. However, a block to the use of virus vector systems in neurobiology is often the lack of cell-specific expression of the gene within the nervous system, and the immune and inflammatory responses to both the virus vector and the delivered gene. We have generated an adeno-associated virus vector that exploits the restricted expression pattern of the rat preprotachykinin-A promoter to support reporter gene expression. We demonstrate that this virus has a neuronal-specific expression pattern. Moreover, it is shown for the first time that the proximal rat preprotachykinin-A promoter is nerve growth factor inducible. This virus will be a useful tool to (i) modify neuronal phenotype by expressing therapeutic molecules or antisense nucleic acid and (ii) dissect the signal transduction pathways that regulate promoter function in vivo.     

Muscle-specific expression of hepatitis B surface antigen: no effect on DNA-raised immune responses.

The injection of plasmid DNA encoding hepatitis B virus (HBV) envelope proteins in mouse muscle leads to the induction of specific humoral and cellular immune responses. Most studies on DNA-based immunization have used viral promoters to drive antigen expression. In this study, we compared the efficiency of a muscle-specific promoter, the human desmin gene promoter, with the commonly used cytomegalovirus (CMV) early gene promoter. We showed that increased in vitro expression of HBV envelope proteins from the human desmin gene promoter has no effect on the in vivo immune response even after the injection of as little as 10 micrograms of DNA. The injection of vectors encoding HBV envelope proteins under the control of either the human desmin gene promoter or the CMV promoter induced humoral and cytotoxic immune responses at comparable levels and of the same duration. The recruitment of antigen-presenting cells to the DNA injection site by pretreatment of muscle with a necrotizing agent increases the precocity and the intensity of the responses, particularly when the nonspecific CMV vector was used.     

Effects of the fruit essential oil of Cuminum cyminum Linn. (Apiaceae) on acquisition and expression of morphine tolerance and dependence in mice

Congenital deafness, affecting 1 in 1000 neonates, can lead to major problems in speech, cognitive and psychosocial development. Congenital deafness is mainly caused by mutations in connexins, hemi-channel proteins forming gap-junctions between supporting cells in the sensory epithelia. We describe a high tropism of AAV5 serotype for the supporting cells of the cochlea, both in vitro in postnatal day 4 mouse explants, and in vivo in the adult guinea-pig inner ear, through scala media perfusion. AAV5 transduction correlates with PDGFRalpha expression, previously reported as AAV5 receptor. This vector could be of major interest in addressing gene therapy approaches to deafness as well as for studying basic aspects of inner-ear development and hearing mechanisms.     

Adeno-associated virus (AAV) capsid genes isolated from rat and mouse liver genomic DNA define two new AAV species distantly related to AAV-5

Using polymerase chain reactions and genome walking strategies, adeno-associated virus (AAV)-like capsid genes were isolated from rat and mouse liver genomic DNA, where they are present at <5 copies per cell. These genes define two new species of AAVs since their amino acid sequences are <60% identical to each other or to any other AAV capsid. They are most similar to the AAV-5 and goat AAV capsids. A recombinant vector with the mouse AAV capsid and a lacZ transgene (rAAV-mo.1 lacZ) was able to transduce rodent cell lines in vitro. However, it was not able to transduce eight human cell lines or primary human fibroblasts in vitro. It did not bind heparin and its ability to transduce cells in vitro was not inhibited by heparin, mucin, or sialic acid suggesting it uses a novel entry receptor. rAAV-mo.1 lacZ was 29 times more resistant to in vitro neutralization by pooled, purified human IgG than AAV-2. In vivo, rAAV-mo.1 lacZ efficiently transduced murine ocular cells after a subretinal injection. Intramuscular injection of a rAAV-mo.1 human factor IX (hFIX) vector into mice resulted in no detectable hFIX in plasma, but intravenous injection resulted in high plasma levels of hFIX, equivalent to that obtained from a rAAV-8 hFIX vector. Biodistribution analysis showed that rAAV-mo.1 primarily transduced liver after an intravenous injection. These AAV capsids may be useful for gene transfer in rodents.     

Progress in the use of adeno-associated viral vectors for gene therapy

The development of safe and efficient gene transfer vectors is crucial for the success of gene therapy trials. A viral vector system promising to meet these requirements is based on the apathogenic adeno-associated virus (AAV-2), a member of the parvovirus family. The advantages of this vector system is the stability of the viral capsid, the low immunogenicity, the ability to transduce both dividing and non-dividing cells, the potential to integrate site specifically and to achieve long-term gene expression even in vivo, and its broad tropism allowing the efficient transduction of diverse organs including the skin. All this makes AAV-2 attractive and efficient for in vitro gene transfer and local injection in vivo. This review covers the progress made in AAV vector technology including the development of AAV vectors based on other serotypes, summarizes the results obtained by AAV targeting vectors and outlines potential applications in the field of cutaneous gene therapy.