Analysis of Subcellular Localization and Pathogenicity of Plum Bark Necrosis Stem-Pitting Associated Virus Protein P6

Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance.However,numerous studies have indicated that they might play important roles in the pathogenesis of virus infection.The role of small hydrophobic protein P6,encoded by the open reading frame 2 of PBNSPaV,has not been well explored.In this study,we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain.Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane.To further clarify the pathogenicity of P6 proteins,we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X(PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101.Infiltration of Nicotiana benthamiana(N.benthamiana)with the PVX vector-transformed A.tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation.Meanwhile,infiltration with the PVX-P6 vector-transformed A.tumefaciens resulted in no significant symptoms.These results demonstrated that heterologous expression of P6 in N.benthamiana could not enhance the pathogenicity of PVX.Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms,and the mode of action of PBNSPaV-P6 protein remains to be further studied.

Inhibitory effect of adeno-associated virus-mediated gene transfer of human endostatin on hepatocellular carcinoma

AIM: To investigate the effect of adeno-associated virusmediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC).METHODS: HCC cell line Hep3B was infected with recombinantadeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined byELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk.RESULTS: The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P＜0.01). Intratumoral injection of rAAV2-hEndo (2×1010v.g.) led to a sustained serum endostatin level ofapproximately (86.71±5.19) ng/mL. The tumor volumeand microvessel density were less in rAAV2-hEndo group than in control groups (P＜0.01).CONCLUSION: Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.

Adeno-associated virus mediated interferon-gamma inhibits the progression of hepatic fibrosis in vitro and in vivo

AIM: To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosisin vitro and in vivo.METHODS: We constructed the recombinant AAV encoding human INF-γ (rAAV- INF-γ) and took the primary rat hepatic stellate cells and carbon tetrachloride induced rats as the experimental hepatic fibrosis model in vitro and in vivo. Immunocytochemistry analysis was used to reveal the expression of α-SMA, the marker protein expressed in hepatic stellate cells. The mRNA expression of TGF-β, TIMP-L, and MMP-13 were analyzed by RT-PCR method. In vivo study, the hydroxyproline content in liver and serum AST, ALT were also detected.RESULTS: In vitro study, AAV vector could mediated efficient expression of human INF-γ,, which inhibit the activation of hepatic stellate cells, decrease the expression of α-SMA and mRNA of TIMP-1, TGF-β, with the MMP-13unchanged. In vivo study, the histological examination revealed that rAAV- INF-γ could inhibit the progression of the hepatic fibrosis. In the rAAV-INF-γ induced group,the hydroxyproline content and serum AST, ALT level were decreased to 177±28 μg/g wet liver, 668.5±140.0,458.4±123.5 U/L, compare with the fibrosis control group 236±31 μg/g wet liver, 1 019.1±276.3, 770.5±154.3 U/L,respectively (P＜0.01). mRNA expression of TIMP-1 in the rAAV-INF-γ induced rat liver was decreased while no significant change was observed in TGF-β and MMP-13.CONCLUSION: All these results indicated that rAAV-INF-γhas potential effects for gene therapy of hepatic fibrosis,which could inhibit the progression of hepatic fibrosis.

Immunological inhibition of transplanted liver allografts by adeno-associated virus vector encoding CTLA4Ig in rats

BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno-associated virus vector pSNAV expressing CTLA4Ig was constructed, and its effects on transplanted liver allografts were investigated. METHODS: The pSNAV-CTLA4Ig construct was infused into partial liver allografts of rats via the portal vein during transplantation. CTLA4Ig expression in the transplanted livers was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Furthermore, real-time quantitative PCR was used to measure the expression of IL-2, IFN-gamma, IL-4 and IL-10 in the allografts. RESULTS: The expression of CTLA4Ig in the partial allograft was detected successfully and pSNAV-CTLA4Ig improved the survival rate of rats after liver transplantation. Agarose gel analysis of RT-PCR products indicated the presence of CTLA4Ig in the pSNAV-CTLA4Ig treatment group. Cytokines expressed in allografts on day 7 after orthotopic liver transplantation showed that IL-2, IFN-gamma, IL-4 and IL-10 mRNA levels decreased in transplant recipients treated with pSNAV-CTLA4Ig compared with those treated with pSNAV-LacZ (1.62 +/- 0.09,1.52 +/- 0.11,1.50 +/- 0.07 and 1.43 +/- 0.07 versus 1.29 +/- 0.09, 1.32 +/- 0.07, 1.34 +/- 0.06 and 1.35 +/- 0.04, respectively). CONCLUSIONS: pSNAV-CTLA4Ig effectively expressed CTLA4Ig in liver allografts. CTLA4Ig improved the pathological findings after liver transplantation. CTLA4Ig induced immune tolerance of liver transplantation, and the mechanism involved induced alteration of Th1 and Th2 cytokine transcripts. The adeno-associated virus vector encoding CTLA4Ig may be useful in the clinical study of transplantation tolerance.

Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

瞄准：测试假设这项活动的那改进他我有与周期性的肝损伤联系的纤维发生的过程的氧合酶罐头 interfere，我们调查了在表示上的治疗学的潜力他我在 CCl (4 ) 的 oxygense-1 劝诱了微榴状的肝硬化模型。方法：带老鼠 HO-1 或 GFP 基因的 Recombinant 联系 adeno 的病毒被产生。1x 10 (12 )联系 adeno 的病毒的 vg 在肝的正式就职的时候通过门注射被管理纤维变性。结果：有由 rAAV/HO-1 的 HO-1 的在表示上的老鼠肝显著地增加了的调节以一种稳定的方式的 HO 酶的活动。微榴状的肝硬化的开发显著地作为与控制相比在 rAAV/HO-1-transduced 动物被禁止。门静脉高血压显著地作为与控制相比在 rAAV/HO-1-transduced 动物被减少，而在收缩血压没有重要变化。这发现伴有激活的改进的肝生物化学，更少的渗透的巨噬细胞和更少在 rAAV/HO-1-transduced 肝的肝的星形细胞(HSC ) 。结论：改进惊讶在肝的活动压制肝硬化的发展。

Targeting adeno-associated virus and adenoviral genetherapy for hepatocellular carcinoma

Human hepatocellular carcinoma(HCC)heavily endangers human heath worldwide.HCC is one of most frequent cancers in China because patients with liver disease,such as chronic hepatitis,have the highest cancer susceptibility.Traditional therapeutic approaches have limited efficacy in advanced liver cancer,and novel strategies are urgently needed to improve the limited treatment options for HCC.This review summarizes the basic knowledge,current advances,and future challenges and prospects of adeno-associated virus(AAV)and adenoviruses as vectors for gene therapy of HCC.This paper also reviews the clinical trials of gene therapy using adenovirus vectors,immunotherapy,toxicity and immunological barriers for AAV and adenoviruses,and proposes several alternative strategies to overcome the therapeutic barriers to using AAV and adenoviruses as vectors.

Packaging and Functional Identification of Recombinant Adeno-associated Virus Encoding cdc2-siRNA

Cyclin dependent kinases （cdks） play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed recombinant adeno-associated virus （rAAV） encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA （rAAV-EGFP-U6-cdc2-siRNA） was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

ADENO-ASSOCIATED VIRUS INTRODUCED INTEGRATION AND EXPRESSION OF FOREIGN GENES IN PC12 CELLS

Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous sys- tem. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plas- mids were encapsidated as recombinant virions. PCl2 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus or AAV vectors by South- ern blot and transcript situation or foreign genes by dot blot. Results The hybridization tests showed that AAV in- troduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PCl2 cells. Conclusion AAV vectors can serve as high efficiency vectors or target genes in neuronal PC12 cells.

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

CONSTRUCTING ADENO-ASSOCIATED VIRUS-TGFβ_3 AND COMPARING ITS BIOLOGICAL EFFECT ON PROTEOGLYCAN SYNTHESIS IN DEDIFFERENTIATED NUCLEUS PULPOUS CELLS WITH ADENOVIRUS-TGFβ_1

Objective To construct adeno-associated virus (AAV) expression system for transforming growth factor β3 (TGFβ3) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFβ1. Methods TGFβ3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn Ⅰ, and its downstream contained restriction enzyme site SalⅠ. Using the restriction enzyme sites of PCR product of TGFβ3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFβ3 was subcloned into AAV. The recombinant plasmid AAV-TGFβ3 was transfected into H293 cells with LipofectamineTM 2000, and the expression of TGFβ3 gene was detected using immunofluorescent analysis. After AAV-TGFβ3 virus particle with infectious activity was packaged, TGFβ3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFβ1 in the earlier and later dedifferentiated NP cells. Results For the earlier dedifferentiated NP cells, AAV-TGFβ3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFβ1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFβ3 stably enhanced proteoglycan synthesis, but AV-TGFβ1 inhibited its synthesis. Conclusion AAV expression system can mediate TGFβ3 gene to be expressed stably, and AAV-TGFβ3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.

Anti-amyloid beta single-chain Fv ameliorates behavioral impairment in Alzheimer's disease mice via adeno-associated virus delivery

Intracranial delivery of human Fc-deleted antibody specific to amyloid-β peptide （Aβ, anti-Aβ single-chain Fv, scFv） via adeno-associated virus （AAV） inhibits amyloid deposition in transgenic mice. However, the effects of AAV-mediated Fc-deleted antibody on animal behavior remain unclear. In this study, the anti-Aβ scFv antibody gone, isolated from phage display, was fused to the 5＇ end of the scFv antibody gone for antibody secretion by 2 rounds of polymerase chain reaction amplification. The fused antibody cDNA was cloned into a pSNAV2 plasmid under the control of the cytomegalovirus promoter. The sequence verified expression vector pSNAV2/scFv was transferred to BHK-21 ceils, and stable transfected BHK-21/scFv cells were established by G418 selection and infected with the recombinant herpes simplex virus rHSV/repcap for AAV production. Recombinant AAV was injected into the left quadriceps femoris of PDAPP transgenic mice. After 3 months, Morris water-maze results confirmed significantly improved cognitive function in a mouse model of Alzheimer＇s disease. Key Words： Alzheimer＇s disease; adeno-associated virus; amyloid-β peptide; single-chain antibody; neurodegenerative diseases; neural regeneration

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.

Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis

AIM:To explore the anti-inflammatory potential of adeno-associated virus-mediated delivery of Tregitope 167 in an experimental colitis model.METHODS:The trinitrobenzene sulfonate(TNBS) model of induced colitis was used in Balb/c mice.Subsequently after intravenous adeno-associated virusmediated regulatory T-cell epitopes(Tregitope) delivery,acute colitis was initiated by intra-rectal administration of 1.5 mg TNBS in 40% ethanol followed by a second treatment with TNBS(0.75 mg in 20% ethanol) 8 d later.Control groups included mice not treated with TNBS(healthy control group) and mice treated by TNBS only(diseased group).At the time of sacrifice colon weight,the disease activity index and histology damage score were determined.Immunohistochemical staining of the colonic tissues was performed to asses the cellular infiltrate and the presence of transcription factor forkhead Box-P3(Foxp3).Thymus,mesenteric lymph nodes,liver and spleen tissue were collected and the corresponding lymphocyte populations were further assessed by flow cytometry analysis for the expression of CD4+ T cell and regulatory T cell associated markers.RESULTS:The Tregitope 167 treated mice gained an average of 4% over their initial body weight at the time of sacrifice.In contrast,the mice treated with TNBS alone(no Tregitope) developed colitis,and lost 4% of their initial body weight at the time of sacrifice(P < 0.01).The body weight increase that had been observed in the mice pre-treated with Tregitope 167 was substantiated by a lower disease activity index and a decreased colon weight as compared to the diseased control group(P < 0.01 and P < 0.001,respectively).Immunohistochemical staining of the colonic tissues for CD4+ showed that inflammatory cell infiltrates were present in TNBS treated mice with or without administration with tregitope 167 and that these cellular infiltrates consisted mainly of CD4+ cells.For both TNBS treated groups CD4+ T cell infiltrates were observed in the sub-epithelial layer and the lamina propria.CD4+ T cell infiltrates were also present in the muscularis mucosa layer of the diseased control mice,but were absent in the Tregitope 167 treated group.Numerous Foxp3 positive cells were detected in the lamina propria and sub-epithelium of the colon sections from mice treated with Tregitope 167.Furthermore,the Foxp3 and glycoprotein A repetitions predominant markers were significantly increased in the CD4+ T lymphocyte population in the thymus of the mice pre-treated with adeno-associated virus serotype 5(cytomegalovirus promoter-Tregitope 167),as cytomegalovirus promoter compared to lymphocyte populations in the thymus of diseased and the healthy control mice(P < 0.05 and P < 0.001,respectively).CONCLUSION:This study identifies adeno-associated virus-mediated delivery of regulatory T-cell epitope 167 as a novel anti-inflammatory approach with the capacity to decrease intestinal inflammation and induce longterm remission in inflammatory bowel disease.

Salvianolic acid B increases recombinant adeno-associated virus transduction through redirecting trafficking pathways and stabilizing perinuclear accumulations

OBJECTIVE To screen small molecule compounds from traditional Chinese medicine that can enhance recombinant adeno-associated virus(rAAV) transduction.METHODS Recombinant adeno-associated virus(rAAV) has been established as a powerful tool for in vivo gene transfer and achieved much promise in gene therapy applications.However,widespread clinical use has been limited by transduction efficiency.In the current study,we screened a panel of small molecule compound from traditional Chinese medicine focused on AAV intracellular trafficking process and found salvianolic acid B can significantly enhance rAAV2 transduction.RESULTS Salvianolic acid B caused a dose-depen.dent increase in rAAV2 transduction regardless of vector dose,genome architecture,and over a broad range of cell line from various cell type and species(HEK293,HeLa,HepG2,Huh-7,CHO-K1,LO-2).Salvianolic acid B treatment redirected rAAV2 particles toward large vesicles positive for late endosomal(Rab7) and lysosomal(LAMP1) markers.Furthermore,salvianolic acid B acted to increase accumulation of viral particles at the perinuclear region.CONCLUSION In summary,our results suggest that salvi.anolic acid B redirects rAAV2 toward more productive trafficking pathways and stabilizes perinuclear accumulations of vectors,facilitating productive nuclear trafficking.

Establishment of a recombinant adeno-associated virus expressing hVEGF_(165)

BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE： To construct a non-pathogenic, recombinant adeno-associated virus （AAV） simultaneously expressing human vascular endothelial growth factor 165 （hVEGF165） and green fluorescent protein （GFP）. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS： AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS： The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC （the rep/cap-gene containing plasmid）, and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES： Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter （FACS） upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS： The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene. CONCLUSION： The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.

Adeno-associated virus vectors for human gene therapy

Adeno-associated virus(AAV) is a small,non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprotein lipase deficiency. AAV made history and put human gene therapy in the forefront again. More than four decades of research on AAV vector biology and human gene therapy has generated a huge amount of valuable information. Over 100 AAV serotypes and variants have been isolated and at least partially characterized. A number of them have been used for preclinical studies in a variety of animal models. Several AAV vector production platforms,especially the baculovirus-based system have been established for commercial-scale AAV vector production. AAV purification technologies such as density gradient centrifugation,column chromatography,or a combination,have been well developed. More than 117 clinical trials have been conducted with AAV vectors. Although there are still challenges down the road,such as crossspecies variation in vector tissue tropism and gene transfer efficiency,pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients,the gene therapy community is forging ahead with cautious optimism. In this review I will focus on the properties and applications of commonly used AAV serotypes and variants,and the technologies for AAV vector production and purification. I will also discuss the advancement of several promising gene therapy clinical trials.

Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

Construction of genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus

Objective: To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing pcDNA3-Smad6/Flag and pcDNA3-Smad7/Flag were digested with BamHⅠ and XhoⅠ, respectively. Then the Smad6/Flag and Smad7/Flag gene segments obtained were cloned into plasmid pAAV-MCS respectively to construct the recombinant pAAV-Smad6/Flag and pAAV-Smad7/Flag plasmids. The resulting recombinant plasmids (pAAV-Smad6/Flag or pAAV-Smad7/Flag) or pAAV-LacZ plasmid were co-transfected into the HEK 293cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect mouse macrophages. The expressions of Smad6 and Smad7 in macrophages were detected by immunocytochemical staining and expression of b-galactosidase was evaluated by X-gal staining. Results: The recombinant AAV vector containing Smad6 or Smad7 genes was successfully constructed. More than 95% macrophage cells expressed X-gal and Smad6 and Smad7 genes at 72 h after infection. Conclusion: These results indicate that the genetically engineered macrophages can express Smad6 and Smad7 proteins effectively, laying the foundation for the studies of TGF-β-induced diseases in vivo and highlighting the feasibility of macrophage-based gene therapy.

The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus （rAAAV）, AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein （GFP） gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast （CEF） or chicken embryonic liver （CEL） cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek＇s disease virus （MDV）, avian adenovirus, chicken embryo lethal orphan （CELO） virus or infectious bursal disease virus （IBDV）. Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein （gfp） expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV- mediated transgene expression could be enhanced by super infection with the helper viruses.

Expression of Vascular Endothelial Growth Factor (VEGF) in Human Osteosarcoma Cells Transfected with Adeno-associated Virus-antisense VEGF

Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μl) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively, with the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.