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.

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.

重组腺相关病毒(rAAV)介导基因SMCKA3999对Duchenne肌营养不良病理和功能的影响

目的 研究重组腺相关病毒 (rAAV)载体介导的dystrophin小基因SMCKA3999治疗DMD模型鼠mdx ,从病理和功能观察rAAVSMCKA3999治疗对DMD模型小鼠mdx的疗效。方法 以dystrophin小基因SMCK A3999为目的基因 ,将SMCKA3999克隆至rAAV并包装成rAAVSMCKA3999,以 5× 10 10 病毒颗粒单点注射于DMD模型鼠mdx腓肠肌 ,基因治疗后 4个月及 7个月 ,采用免疫荧光、光镜组织病理、肌电图等方法 ,从形态和功能观察rAAVSMCKA3999治疗对DMD模型小鼠mdx的疗效。 结果 rAAVSMCKA3999使肌膜缺失的dys trophin恢复并稳定表达持续 7个月以上 ,肌肉组织病理改变好转 ,肌病肌电图改变明显改善 ,疗效持续 4个月以上。 结论 rAAVSMCKA3999能改善mdx小鼠骨骼肌的病理及功能 ,采用rAAV介导的dystrophin小基因SMC KA3999对Duchenne肌营养不良基因治疗是有希望的治疗方法。

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

Objective:Plant-derived cytotoxic transgene expression,such as trichosanthin(tcs),regulated by recombinant adeno-associated virus(r AAV)vector is a promising cancer gene therapy.However,the cytotoxic transgene can hamper the vector production in the r AAV producer cell line,human embryonic kidney(HEK293)cells.Here,we explored micro RNA-122(miR122)and its target sequence to limit the expression of the cytotoxic gene in the r AAV producer cells.Methods:A miR122 target(122 T)sequence was incorporated into the 30 untranslated region of the tcs c DNA sequence.The firefly luciferase(fluc)transgene was used as an appropriate control.Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells.The effects of miR122 overexpression on cell growth,transgene expression,and r AAV production were determined.Results:The presence of 122 T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line(in vitro),fresh human hepatocytes(ex vivo),and mouse liver(in vivo).Also,the normal liver physiology was unaffected by delivery of 122 T sequence by r AAV vectors.Compared with the parental cells,the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122 T,as well as the ability to produce liver-targeting r AAV vectors.Fascinatingly,the yield of r AAV vectors carrying the tcs-122 T gene was increased by 77.7-fold in HEK293-mir122 cells.Moreover,the tcs-122 T-containing r AAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.Conclusion:HEK293-mir122 cells along with the 122 T sequence provide a potential tool to attenuate the cytotoxic transgene expression,such as tcs,during r AAV vector production.

重组腺相关病毒载体的研究进展

重组腺相关病毒(rAAV)载体作为基因治疗的工具,具有无致病性、长期表达、低免疫原性和感染多种细胞等特点。因此,rAAV载体越来越受到重视。就rAAV载体在生物学特性、构建、安全性和应用方面作一介绍。

重组腺辅助病毒载体的制备、纯化及其介导的基因转移

目的 :探讨一种制备、纯化重组腺辅助病毒 (r AAV)载体的有效方法。方法 :以质粒 PDG和含有人 apo AIc DNA的AAV载体共转染 2 93T细胞 ,继以 Iodixanol密度梯度离心结合肝素亲和层析法分离、纯化 ,斑点杂交鉴定 r AAV病毒颗粒数 ;并以含人 apo AIc DNA的 r AAV感染 C2 C12细胞。结果 :r AAV颗粒数为 7× 10 1 0 / ml;r AAV成功介导了人 apo AIc DNA在 C2 C12细胞中的表达并持续了 30天 ,在分化为肌管细胞后仍有此表达能力。结论 :本实验采取的制备、纯化 r AAV的方法简便、高效 ,r AAV成功介导 apo AI基因在肌源性细胞 C2 C12中的表达。

HIF-1α siRNA Leads to Apoptosis of Pancreatic Cancer Cells under Hypoxic Conditions

OBJECTIVE To explore the role （HIF-1α） in the proliferation and cells under hypoxic conditions. of hypoxic inducible factor-1α apoptosis of pancreatic cancer METHODS A cassette encoding small interference RNA （siRNA） targeting HIF-1α mediated by recombinant adeno-associated virus （rAAV） was constructed, giving rAAV-siHIE rAAV-siHIF or rAAV- hrGFP was transfected into exponentially growing MiaPaCa2 cells under hypoxic conditions. Then, the expression of HIF-1α mRNA and protein, the proliferation and apoptosis of MiaPaCa2 cells were examined, using real-time PCR, Western Blot, MTT and TUNEL, respectively. RESULTS Under hypoxic conditions, rAAV-siHIF inhibited the expression of HIF-1α mRNA and protein in MiaPaCa2 cells. At the same time, rAAV-siHIF decreased MiaPaCa2 cell proliferation and induced apoptosis. However, rAAV-hrGFP had no effect on the expression of HIF-1α as well as the proliferation and apoptosis of MiaPaCa2 cells under hypoxic conditions. CONCLUSION Under hypoxic conditions, HIF-1α plays a key role in the proliferation of MiaPaCa2 cells, and inhibition of HIF- 1α expression can lead to MiaPaCa2 cell apoptosis.

Gene editing for corneal disease management

Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.

表达HCV包膜蛋白的两种重组腺相关病毒疫苗的制备及免疫原性分析

重组腺相关病毒载体(rAAV)可在动物体内高水平地持久表达外源基因,本研究采用两种rAAV载体(rAAV1与rAAV2)构建了表达丙型肝炎病毒中国分离株包膜糖蛋白(E1E2)的载体疫苗并以之免疫小鼠,分别采用免疫荧光证实其表达与总抗体,用HCV假病毒系统检测其中和抗体水平,用ELISpot分析其细胞免疫应答,结果表明:rAAV1-E1E2重组载体疫苗单针免疫激发的体液应答明显高于rAAV2-E1E2,rAAV1-E1E2单针注射后3个月可在肌肉组织中检出E2蛋白表达及特异性T细胞应答。上述结果提示HCV重组腺相关病毒载体疫苗单针免疫可引起明显持久的体液与细胞免疫应答。

重组腺相关病毒载体在基因治疗中的应用

重组腺相关病毒(rAAV)载体具有安全性好、免疫原性低、能感染分裂细胞和非分裂细胞、能介导基因长期稳定表达等优点。因此,作为一种基因导入系统,rAAV载体在基因治疗的研究和开发中越来越受到关注。本文就rAAV载体在基因治疗中的应用作一综述。

降低重组腺相关病毒载体免疫反应的策略

重组腺相关病毒（Recombinant adeno-associated virus,rAAV）载体在基因治疗研究中应用广泛,但可能引发机体产生免疫反应,限制了其在基因治疗领域的临床应用,如何降低rAAV载体的免疫反应已经成为基因治疗领域的研究重点之一。本文从对rAAV载体的选择和对患者免疫抑制调控两个方面介绍降低rAAV载体免疫反应的策略。

化学修饰的重组腺相关病毒载体

重组腺相关病毒(Recombinant adenovirus-associated virus,rAAV)载体源于腺相关病毒(Adeno-associated virus,AAV),以其无致病性、低免疫原性、可定点整合及在宿主细胞内长期表达等优势,广泛应用于肿瘤和遗传疾病等基因治疗研究,被视为最有前途的基因治疗载体之一。但是人体内广泛存在AAV中和抗体,以及rAAV对体内特定组织或细胞的靶向性欠理想,限制了其在临床上的应用。经化学修饰的rAAV可以抵御血清中和抗体,提高转导靶向性,还可实现rAAV体内动态监测,这为解决当前rAAV载体的问题提供了新的思路和方法。本文对化学修饰的rAAV展开系统阐述,并对其未来发展趋势作出展望。

Recent development and gene therapy for glycogen storage disease type Ia

Glycogen storage disease type Ia(GSD-Ia)is an autosomal recessive metabolic disorder caused by a deficiency in glucose-6-phosphatase-α(G6Pase-αor G6PC)that is expressed primarily in the liver,kidney,and intestine.G6Pase-αcatalyzes the hydrolysis of glucose-6-phosphate(G6P)to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis,and is a key enzyme for endogenous glucose production.The active site of G6Pase-αis inside the endoplasmic reticulum(ER)lumen.For catalysis,the substrate G6P must be translocated from the cytoplasm into the ER lumen by a G6P transporter(G6PT).The functional coupling of G6Pase-αand G6PT maintains interprandial glucose homeostasis.Dietary therapies for GSD-Ia are available,but cannot prevent the long-term complication of hepatocellular adenoma that may undergo malignant transformation to hepatocellular carcinoma.Animal models of GSD-Ia are now available and are being exploited to both delineate the disease more precisely and develop new treatment approaches,including gene therapy.