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 ...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.展开更多
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 v...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.展开更多
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 lipoprote...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.展开更多
Objective: To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165(hVEGF165) and bone morphogenetic protein 7(hBMPT), measure the virus titer and verify the recom...Objective: To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165(hVEGF165) and bone morphogenetic protein 7(hBMPT), measure the virus titer and verify the recombination. Methods:The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid plRES was cut down and subcloned into ITR/ MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then, recombinant plasmid pAAV- hVEGF165-IRES-hBMP7, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells, and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results:Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5 ×10^11vp/ml, and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion:Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer, which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.展开更多
BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provid...BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.展开更多
H7N9 subtype avian influenza virus poses a great challenge for poultry industry.Newcastle disease virus(NDV)-vectored H7N9 avian influenza vaccines(NDV_(vec)H7N9)are effective in disease control because they are prote...H7N9 subtype avian influenza virus poses a great challenge for poultry industry.Newcastle disease virus(NDV)-vectored H7N9 avian influenza vaccines(NDV_(vec)H7N9)are effective in disease control because they are protective and allow mass administration.Of note,these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition(HI)but high IgG antibodies in chickens.However,the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.Herein,immunization with an NDV_(vec)H7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin(HA)-binding IgG antibodies in chickens.Three residues(S150,G151 and S152)in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDV_(vec)H7N9 immune serum.Passively transferred NDV_(vec)H7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.The NDV_(vec)H7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor,suggesting the engagement of the complement system.Moreover,mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDV_(vec)H7N9 immune serum,indicating the requirement of antibody-antigen binding for complement activity.Therefore,antibodies induced by the NDV_(vec)H7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.Our findings unveiled a novel role of the complement in protection conferred by the NDV_(vec)H7N9,highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.展开更多
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 v...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.展开更多
A novel method for recombinant adeno-associ-ated virus (rAAV) purification on large scale is described. The method involves three steps, including chloroform treatment, PEG/NaCl precipitation and chloroform extraction...A novel method for recombinant adeno-associ-ated virus (rAAV) purification on large scale is described. The method involves three steps, including chloroform treatment, PEG/NaCl precipitation and chloroform extraction. The whole procedure can be performed in four hours. Using this purification method, we can reproducibly obtain, from 4 × 109 of proviral cells cultured in roller bottles, purified rAAV-GFP stocks with titers of around 5×1013 particles/mL and purity greater than 95%. The infectious titers of the vector stocks were up to 2×1012 TU/mL, thus particle-to-infectivity rate was about 25. Under an electronic microscope, most rAAV particles appeared full and a few were in intermediate form. Empty particles were rarely seen. The purified rAAV-GFP stocks have been successfully used in in vitro and in vivo transfection experiments. Therefore, this new method offers a simple, rapid and cost-effective way for large-scale rAAV purification.展开更多
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) s...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.展开更多
Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells ...Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.展开更多
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 susceptibil...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.展开更多
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...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.展开更多
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...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展开更多
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 ...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.展开更多
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 r...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.展开更多
In recent years,significant breakthroughs have been made in the field of gene ther-apy.Adeno-associated virus(AAV)is one of the most promising gene therapy vectors and a powerful tool for delivering the gene of intere...In recent years,significant breakthroughs have been made in the field of gene ther-apy.Adeno-associated virus(AAV)is one of the most promising gene therapy vectors and a powerful tool for delivering the gene of interest.Among the AAV vectors,AAV serotype 8(AAv8)has attracted much attention for its efficient and stable gene transfection into specific tissues.Currently,recombinant AAv8 has been widely used in gene therapy research on a va-riety of diseases,including genetic diseases,cancers,autoimmune diseases,and viral diseases.展开更多
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...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.展开更多
Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constr...Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.展开更多
Numerous studies have confirmed that vascular endothelial growth factor (VEGF) improves the function of neural cells following spinal cord injury (SCI). However, some studies have also verified that VEGF cannot si...Numerous studies have confirmed that vascular endothelial growth factor (VEGF) improves the function of neural cells following spinal cord injury (SCI). However, some studies have also verified that VEGF cannot significantly induce the increase in vascular density at or surrounding the lesion, and that VEGF therapy exacerbated secondary damage following SCI. Based on the dual effects of VEGF on SCI, we constructed the recombinant adeno-associated viruses (rAAV)-hVEGF165-IRES-human recombinant green fluorescent protein (hrGFP) (AAV-VEGF) and rAAV-IRES-hrGFP (AAV-GFP). Our results suggested that rAAV expressed hVEGFles, and a low dose of VEGF relieved increased vascular permeability, improved microcirculation in the local spinal cord, lessened spinal cord edema, and decreased neuronal apoptosis. These results verified that the releasing effects of the rAAV virus vector had protective effects on the spinal cord.展开更多
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 tr...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.展开更多
文摘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.
文摘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.
文摘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.
基金National Natural Science Foundation of China(No.30600624)
文摘Objective: To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165(hVEGF165) and bone morphogenetic protein 7(hBMPT), measure the virus titer and verify the recombination. Methods:The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid plRES was cut down and subcloned into ITR/ MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then, recombinant plasmid pAAV- hVEGF165-IRES-hBMP7, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells, and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results:Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5 ×10^11vp/ml, and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion:Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer, which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.
基金Supported by the National Natural Science Foundation of China,No.82172297Natural Science Foundation of Jiangsu Province of China,No.BK20211346 and No.BK20201011+1 种基金Natural Science Foundation of Jiangsu Higher Education Institutions of China,No.22KJA310007Xuzhou Science and Technology Project,No.KC22055.
文摘BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.
基金supported by the earmarked fund for China Agriculture Research System(CARS-40)the Key Research and Development Project of Yangzhou(Modern Agriculture),China(YZ2022052)the‘‘High-end Talent Support Program’’of Yangzhou University,China。
文摘H7N9 subtype avian influenza virus poses a great challenge for poultry industry.Newcastle disease virus(NDV)-vectored H7N9 avian influenza vaccines(NDV_(vec)H7N9)are effective in disease control because they are protective and allow mass administration.Of note,these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition(HI)but high IgG antibodies in chickens.However,the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.Herein,immunization with an NDV_(vec)H7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin(HA)-binding IgG antibodies in chickens.Three residues(S150,G151 and S152)in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDV_(vec)H7N9 immune serum.Passively transferred NDV_(vec)H7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.The NDV_(vec)H7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor,suggesting the engagement of the complement system.Moreover,mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDV_(vec)H7N9 immune serum,indicating the requirement of antibody-antigen binding for complement activity.Therefore,antibodies induced by the NDV_(vec)H7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.Our findings unveiled a novel role of the complement in protection conferred by the NDV_(vec)H7N9,highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.
基金This work was supported by the National High Technology Development Program.
文摘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.
基金the National High-technology Development Program (Grant No. Z20-05-02).
文摘A novel method for recombinant adeno-associ-ated virus (rAAV) purification on large scale is described. The method involves three steps, including chloroform treatment, PEG/NaCl precipitation and chloroform extraction. The whole procedure can be performed in four hours. Using this purification method, we can reproducibly obtain, from 4 × 109 of proviral cells cultured in roller bottles, purified rAAV-GFP stocks with titers of around 5×1013 particles/mL and purity greater than 95%. The infectious titers of the vector stocks were up to 2×1012 TU/mL, thus particle-to-infectivity rate was about 25. Under an electronic microscope, most rAAV particles appeared full and a few were in intermediate form. Empty particles were rarely seen. The purified rAAV-GFP stocks have been successfully used in in vitro and in vivo transfection experiments. Therefore, this new method offers a simple, rapid and cost-effective way for large-scale rAAV purification.
基金supported in part by the Alex's Lemonade Foundation,and Bankhead-Coley Cancer Research Program,Florida Department of Health(to CL), Public Health Service grants R01 HL-097088 and R21 EB-015684 from the National Institutes of Health,and an institutional grant from the Children's Miracle Network (to AS,CL and GA)supported in part by the state-sponsored program for Graduate Students from China Scholarship Council,Government of China, and the National Natural Science Foundation of China (No.30730114)
文摘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.
文摘Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.
基金Supported by National Natural Science Foundation of China.No.81272687Zhejiang Provincial Public Welfare Technology Application Research Projects,No.2014C33275+1 种基金Zhejiang Provincial Natural Science Foundation of China,No.LZ13H160004the Grant for 521 Talent Project of Zhejiang Sci-Tech University,Hangzhou,China
文摘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.
文摘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.
基金the National Natural Science Foundation of China,No. 30670741
文摘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
基金a grant from National Natural Sciences Foundation of China (No. 30572065/ C03031103)
文摘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.
基金supported by grants from the National Natural Sciences Foundation of China (No. 30400141, No. 30670737).
文摘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.
文摘In recent years,significant breakthroughs have been made in the field of gene ther-apy.Adeno-associated virus(AAV)is one of the most promising gene therapy vectors and a powerful tool for delivering the gene of interest.Among the AAV vectors,AAV serotype 8(AAv8)has attracted much attention for its efficient and stable gene transfection into specific tissues.Currently,recombinant AAv8 has been widely used in gene therapy research on a va-riety of diseases,including genetic diseases,cancers,autoimmune diseases,and viral diseases.
基金the National Natural Science Foundation of China, No. 30600624
文摘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.
基金sponsored by the National Natural Science Foundation of China,No.30970992
文摘Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.
基金the National Natural Science Foundation of China, No. 30772189
文摘Numerous studies have confirmed that vascular endothelial growth factor (VEGF) improves the function of neural cells following spinal cord injury (SCI). However, some studies have also verified that VEGF cannot significantly induce the increase in vascular density at or surrounding the lesion, and that VEGF therapy exacerbated secondary damage following SCI. Based on the dual effects of VEGF on SCI, we constructed the recombinant adeno-associated viruses (rAAV)-hVEGF165-IRES-human recombinant green fluorescent protein (hrGFP) (AAV-VEGF) and rAAV-IRES-hrGFP (AAV-GFP). Our results suggested that rAAV expressed hVEGFles, and a low dose of VEGF relieved increased vascular permeability, improved microcirculation in the local spinal cord, lessened spinal cord edema, and decreased neuronal apoptosis. These results verified that the releasing effects of the rAAV virus vector had protective effects on the spinal cord.
文摘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.