辅助病毒依赖型腺病毒载体研究进展

摘要：辅助病毒依赖型腺病毒载体（helper-dependent adenoviral vector,HDAd）缺乏所有腺病毒的编码序列,与非复制型的第一代腺病毒载体（first-generation adenovirus vector,FGAd）相比,具有载体免疫原性低、安全、转移容量大和持续表达等特点,现广泛用于遗传性疾病、神经退行性疾病和肿瘤等的基因治疗和特异性靶向治疗研究。本文综述了HDAd构建和应用等方面的研究进展及未来的发展方向。     

Generation of GGTA1 Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors

This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS₁-Cas9-NLS₂ plasmid can both generate KO pigs by direct pronuclear microinjection. In total, 41 and 53 fertilized eggs were microinjected on 1-site and 4-site strategies, respectively. The 1-site construction generated a litter of 8 piglets, and 2 were mono-allelic mutant (mMt). The injection of 4-site constructions resulted in one biallelic mutant (bMt) and one mMt piglet in a litter of 7. Those 3 mMt pigs had a 4?bp deletion, 5?bp insertion, or 7?bp insertion at site I, and the bMt pig had 5 types of mutations at cleavage sites I and III. The expression of alpha-Gal on the bMt peripheral blood mononuclear cells (PBMCs) was reduced, and survival rate of bMt PBMCs was maintained as indicated by results of cultivation with sera of humans or Formosan Macaques. We concluded that mutant pigs could be generated by direct pronuclear microinjection of ppU6-GGTA1-gRNA CRISPR vectors with the pCX-Flag2-NLS₁-Cas9-NLS₂ plasmid and that the 4-site strategy has a better mutant efficiency. Porcine U6 promoter was firstly used to express KO vectors and effectively generate mutant pigs, worthily to adopt for future KO studies.     

一种辅助病毒依赖型腺病毒载体的构建及其在小鼠体内的应用

腺病毒载体具有在离体细胞和动物体内高效转移和表达外源基因的优点,但由于第一代腺病毒载体能在靶细胞内表达病毒结构蛋白,并诱导机体的细胞和体液免疫反应,影响了目的基因在体内的稳定表达。为了克服这一缺点,构建了一种辅助病毒依赖型腺病毒载体ＨＡｄＩ－ｈＦＶＩＩ。该载体去除了病毒基因组的ｌ３、Ｌ１、Ｌ２、ＶＡＩ－ＶＡＩ、ｐＴＰ等基因序列。在第一代重组病毒ＡｄＩ－ｈＦＶＩＩ辅助下,能在２９３细胞包装和扩增。经氯化铯梯度超速离心后,能与辅助病毒有效分离。小鼠体内研究表明,该载体能在体内高效转移和表达ｈＦＶＩＩ基因。与笫一代腺病毒载体比较,外源基因表达稳定性明显提高,提示该载体在体内具有较低的免疫原性。     

反义c-myc重组腺病毒载体的构建及其对大鼠胸腺淋巴细胞的增殖抑制作用

目的:观察反义c-myc重组腺病毒载体对大鼠胸腺淋巴细胞的增殖抑制作用.方法:构建大鼠反义及正义c-myc细菌质粒,并将所得细菌质粒与E1缺失腺病毒质粒导入293细胞系,经共转染得到正义及反义重组腺病毒载体.MTS法检测重组腺病毒载体对大鼠淋巴细胞增殖的抑制作用,RT-PCR检测重组腺病毒载体对c-myc mR-NA表达的影响.结果:反义c-myc重组腺病毒载体可抑制大鼠淋巴细胞增殖,降低淋巴细胞c-myc mRNA的表达.结论:反义c-myc重组腺病毒载体可抑制大鼠淋巴细胞增殖.     

AZI基因被捕获的鉴定实验

目的 确立基因捕获细胞中被捕获的基因名称. 方法 Southern印迹确定合适的限制性内切酶,用质粒拯救（plasmid rescue）获得含有细胞染色体DNA的质粒,测序.结果 本次实验中,被捕获载体整合的基因是AZI基因. 结论质粒拯救方法能确立质粒整合细胞染色体上准确的位置.     

Expression of human IL-1alpha after intramarrow gene transfer into healthy non-human primate by adenoviral vector

Interleukin-1alpha (IL-1alpha) is a multifunctional cytokine that stimulates myelopoiesis in macaque. However, daily systemic injections of IL-1alpha are associated with severe side effects. We therefore investigated the feasibility of a gene therapy strategy aimed at increasing the IL-1alpha local production in bone marrow with limited release of the vector into the blood circulation. Intra-medullar administration of adenoviral vector containing human IL-1alpha (huIL-1alpha) gene resulted in enhanced neutrophil, monocyte and platelet counts during the two first weeks after injection. The DNA vector, the transgene expression and the huIL-1alpha production was detected in treated bone marrow without significant detection of huIL-1alpha in the peripheral blood. Associated with huIL-1alpha production, we observed concomitant plasma C reactive protein and IL-1Ra peaks in the acellular fraction of treated bone marrow at days 3 and 7. No abnormal clinical side effects were observed in any of the animals following the adenoviral vector injection.     

Viral Transduction of Cocaine Hydrolase in Brain Reward Centers

1. Site-directed mutagenesis of human plasma butyrylcholinesterase has led to novel hydrolases that rapidly destroy cocaine. We are investigating whether viral gene transfer of such enzymes might reduce addiction liability by blocking cocaine from its sites of action.2. As groundwork for a possible gene therapy, we previously studied adenoviral transduction of cocaine hydrolases in the rat. Systemically injected vectors raised plasma cocaine hydrolase activity greatly, reduced pressor responses to cocaine, and lowered cocaine's tissue burden.3. In the present study, to reduce cocaine's brain access still further, vectors were injected directly into the nucleus accumbens. Six days later, medium sized neurons gained dramatic butyrylcholinesterase activity. Species-selective immunohistochemistry proved that the transgene accounted for this activity.4. Since the transgene product is so efficient with cocaine as a substrate, it is now reasonable to begin testing gene therapy in rodent models of cocaine addiction.     

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRalpha1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRalpha1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.     

利用PiggyBac系统在家蝇中表达蜜蜂王浆蛋白MRJP1

家蝇Musca domestica是一种重要的资源昆虫,作为饲料蛋白已广泛应用于动物养殖产业。MRJP1蛋白（Major Royal Jelly Proteins 1, MRJP1）是蜂王浆的主要蛋白成分,具有营养作用和跨物种促细胞增殖作用。构建表达意大利蜜蜂Apis mellifera王浆蛋白基因Ammrjp1的家蝇可望提高家蝇幼虫的应用价值。本研究构建了携带Ammrjp1基因的重组转座质粒pBac[fa PUb-mrjp1-DsRed],显微注射家蝇胚胎,成功建立Ammrjp1转基因家蝇品系,RT-PCR证明Ammrjp1基因在转基因家蝇中正常转录;SouthernBlot证实Ammrjp1基因是以单拷贝的形式插入到家蝇基因组内;利用Inverse-PCR技术获得Ammrjp1基因在家蝇基因组上插入位点侧翼序列。与野生型家蝇比较,G8代Ammrjp1转基因家蝇4龄幼虫的百头重增加8.6%。家蝇遗传转化体系的成功构建,为建立新型转基因生物反应器和开发高值动物蛋白饲料提供技术支持。     

腺病毒载体衣壳蛋白质的遗传修饰

腺病毒载体是最早用于基因治疗研究的病毒载体之一,也是目前肿瘤基因治疗中最为常见的病毒载体之一,其主要通过靶细胞表面的天然柯萨奇腺病毒受体(coxsackie and adenovirus receptor,CAR)感染宿主细胞。由于大多数肿瘤细胞表面该受体表达水平较低,降低了腺病毒载体对靶细胞的感染效率,从而制约了腺病毒载体在肿瘤基因治疗中的应用。因此,如何提高腺病毒载体对靶细胞的感染效率是腺病毒载体应用于肿瘤基因治疗的关键。目前对腺病毒载体衣壳蛋白质(capsid protein)的遗传修饰是提高其对宿主细胞感染效率的主要途径。本文将对这一领域的主要研究进展作一综述,为该方面的研究提供有用的信息。     

Adenoviral vector-directed expression of neurotrophin-3 in rat dorsal root ganglion explants results in a robust neurite outgrowth response

The neurotrophins are a family of proteins that promote neuronal survival and neurite outgrowth during development and can also enhance the regeneration of injured adult neurons. The local and continuous delivery of these proteins at the site of injury is problematic, since this requires repeated intraparenchymal injections or the use of invasive canula-micropump devices. In the present study we report the generation and characterization of an adenoviral vector for a member of the neurotrophins, neurotrophin-3 (Ad-NT-3). Using Ad-NT-3, we examined the expression and biological activity of NT-3 in dorsal root ganglia (DRG) explant cultures. Gene transfer with Ad-NT-3 results in the synthesis of genuine NT-3 and in a dosage-dependent neurite outgrowth response in DRG explants. Transduction of DRG explants with a viral vector dosage of 5 × 10⁵ to 5 × 10⁶ plaque-forming units induced the formation of a dense halo of neurites comparable to outgrowth observed following the addition of 100 ng/mL exogenous NT-3. In addition, a single infection with Ad-NT-3 produced biologically active NT-3 for at least 20 days in culture, as evidenced by continued neurite extension. This indicates that adenoviral vector-mediated expression of NT-3 results in high-level production of biologically active NT-3 and could therefore be used as a strategy to promote the regeneration of injured peripheral and central nerve projections. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 172–184, 1997     

P27特异性siRNA重组腺病毒载体的构建及功能鉴定

目的构建针对P27的siRNA腺病毒载体及相应的对照病毒载体,并鉴定重组腺病毒在小鼠胰岛中对内源性皿7基因表达的影响。方法合成针对p27的siRNA的靶DNA序列及相应的阴性对照序列,连接至pShuttle-H1质粒中,然后用NotI和HindHI限制性内切酶将H1-siRNA片段从pShuttle—H1-siRNA质粒上双酶切下来,克隆至空载pAdTrack穿梭质粒上,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染QBI-293A细胞,包装得到pAd—P27-siRNA和pAd—NC重组腺病毒。用病毒体外感染小鼠胰岛,Western印迹法检测P27蛋白表达水平。结果重组腺病毒载体经测序鉴定正确,制备的病毒感染效率高,能有效抑制小鼠胰岛中P27的表达。结论成功构建了针对P27的siRNA重组腺病毒载体,为进一步研究P27在胰岛β细胞生长中的作用提供了基础。