The development of clinical candidates that modify the natural progression of sporadic Parkinson's disease and related synucleinopathies is a praiseworthy endeavor,but extremely challenging.Therapeutic candidates ...The development of clinical candidates that modify the natural progression of sporadic Parkinson's disease and related synucleinopathies is a praiseworthy endeavor,but extremely challenging.Therapeutic candidates that were successful in preclinical Parkinson's disease animal models have repeatedly failed when tested in clinical trials.While these failures have many possible explanations,it is perhaps time to recognize that the problem lies with the animal models rather than the putative candidate.In other words,the lack of adequate animal models of Parkinson's disease currently represents the main barrier to preclinical identification of potential disease-modifying therapies likely to succeed in clinical trials.However,this barrier may be overcome by the recent introduction of novel generations of viral vectors coding for different forms of alpha-synuclein species and related genes.Although still facing several limitations,these models have managed to mimic the known neuropathological hallmarks of Parkinson's disease with unprecedented accuracy,delineating a more optimistic scenario for the near future.展开更多
Researchers commonly use cyclization recombination enzyme/locus of X-over P1(Cre/loxP)technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and L...Researchers commonly use cyclization recombination enzyme/locus of X-over P1(Cre/loxP)technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis.However,the shortcomings of these genetic tools include high costs,lengthy experimental periods,and limited accessibility for researchers.Therefore,exploring alternative gene silencing techniques is of great practical value.In this study,we employed adeno-associated virus(AAV)as a vector for gene silencing in Sertoli and Leydig cells.Our findings demonstrated that AAV serotypes 1,8,and 9 exhibited high infection efficiency in both types of testis cells.Importantly,we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection.We achieved cell-specific knockouts of the steroidogenic acute regulatory(Star)and luteinizing hormone/human chorionic gonadotropin receptor(Lhcgr)genes in Leydig cells,but not in Sertoli cells,using AAV9-single guide RNA(sgRNA)-mediated gene editing in Rosa26-LSL-Cas9mice.Knockdown of androgen receptor(Ar)gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpinRNA(shRNA)-mediated targeting.Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.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
AIM:To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosis in vitro and in vivo. METHODS: We constructed the recombinant...AIM:To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosis in 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-1, 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-13 unchanged. 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-γ has potential effects for gene therapy of hepatic fibrosis, which could inhibit the progression of hepatic fibrosis.展开更多
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-associ...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.展开更多
AIM： To test the hypothesis that enhancement of the activity of heine oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-e...AIM： To test the hypothesis that enhancement of the activity of heine oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heine oxygense-1 in a CCl4-induced micronodular cirrhosis model. METHODS： Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated, 1×10^12 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis. RESULTS： Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controls, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells （HSCs） in rAAV/ HO-1-transduced livers. CONCLUSIONS： Enhancement of HO activity in the livers suppresses the development of cirrhosis.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
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
Whether direct manipulation of Parkinson’s disease(PD)risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue.Here,we used an adeno-associated virus serotype 9(AAV9)-deliver...Whether direct manipulation of Parkinson’s disease(PD)risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue.Here,we used an adeno-associated virus serotype 9(AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras(SNs)of two monkey groups:an old group and a middle-aged group.After the operation,the old group exhibited all the classic PD symptoms,including bradykinesia,tremor,and postural instability,accompanied by key pathological hallmarks of PD,such as severe nigral dopaminergic neuron loss(>64%)and evidentα-synuclein pathology in the gene-edited SN.In contrast,the phenotype of their middle-aged counterparts,which also showed clear PD symptoms and pathological hallmarks,were less severe.In addition to the higher final total PD scores and more severe pathological changes,the old group were also more susceptible to gene editing by showing a faster process of PD progression.These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys.Taken together,this system can effectively develop a large number of genetically-edited PD monkeys in a short time(6–10 months),and thus provides a practical transgenic monkey model for future PD studies.展开更多
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
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 ham...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.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
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 ra...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.展开更多
Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheles...Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.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
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
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
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...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.展开更多
基金supported by grants PID2020-120308RB-I00 and PID2023-147802OB-I00 funded by MICIU/AEI/10.13039/501100011033FEDER,UE,by Aligning Science Across Parkinson’s(ref.ASAP-020505)through the Michael J.Fox Foundation for Parkinson’s Research+1 种基金by CiberNed Intramural Collaborative Projects(ref.PI2020/09)by the Spanish Fundación Mutua Madrile?a de Investigación Médica(to JLL)。
文摘The development of clinical candidates that modify the natural progression of sporadic Parkinson's disease and related synucleinopathies is a praiseworthy endeavor,but extremely challenging.Therapeutic candidates that were successful in preclinical Parkinson's disease animal models have repeatedly failed when tested in clinical trials.While these failures have many possible explanations,it is perhaps time to recognize that the problem lies with the animal models rather than the putative candidate.In other words,the lack of adequate animal models of Parkinson's disease currently represents the main barrier to preclinical identification of potential disease-modifying therapies likely to succeed in clinical trials.However,this barrier may be overcome by the recent introduction of novel generations of viral vectors coding for different forms of alpha-synuclein species and related genes.Although still facing several limitations,these models have managed to mimic the known neuropathological hallmarks of Parkinson's disease with unprecedented accuracy,delineating a more optimistic scenario for the near future.
基金supported by the National Natural Science Foundation of China(No.82070872 and No.82370854 to JXL)Innovative and Entrepreneurial Team of Jiangsu Province(No.JSSCTD2021 to JXL)+1 种基金China Postdoctoral Science Foundation(2023M741790 to JP)Jiangsu Funding Program for Excellent Postdoctoral Talent(2023ZB558 to JP).
文摘Researchers commonly use cyclization recombination enzyme/locus of X-over P1(Cre/loxP)technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis.However,the shortcomings of these genetic tools include high costs,lengthy experimental periods,and limited accessibility for researchers.Therefore,exploring alternative gene silencing techniques is of great practical value.In this study,we employed adeno-associated virus(AAV)as a vector for gene silencing in Sertoli and Leydig cells.Our findings demonstrated that AAV serotypes 1,8,and 9 exhibited high infection efficiency in both types of testis cells.Importantly,we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection.We achieved cell-specific knockouts of the steroidogenic acute regulatory(Star)and luteinizing hormone/human chorionic gonadotropin receptor(Lhcgr)genes in Leydig cells,but not in Sertoli cells,using AAV9-single guide RNA(sgRNA)-mediated gene editing in Rosa26-LSL-Cas9mice.Knockdown of androgen receptor(Ar)gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpinRNA(shRNA)-mediated targeting.Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.
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Supported by the National High Technology Research and Development Program of China, 863 Program, No. 2003AA2Z347A
文摘AIM:To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosis in 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-1, 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-13 unchanged. 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-γ has potential effects for gene therapy of hepatic fibrosis, which could inhibit the progression of hepatic fibrosis.
基金Supported by the National Natural Science Foundation of China, No. 20074031
文摘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.
文摘AIM： To test the hypothesis that enhancement of the activity of heine oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heine oxygense-1 in a CCl4-induced micronodular cirrhosis model. METHODS： Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated, 1×10^12 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis. RESULTS： Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controls, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells （HSCs） in rAAV/ HO-1-transduced livers. CONCLUSIONS： Enhancement of HO activity in the livers suppresses the development of cirrhosis.
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.
基金This work was supported by the National Key R&D Program of China(2018YFA0801403)the Key-Area Research and Development Program of Guangdong Province(2019B030335001)+6 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB32060200)the National Program for Key Basic Research Projects(973 Program:2015CB755605)the National Natural Science Foundation of China(81471312,81771387,81460352,81500983,31700897,31700910,31800901,31625013,and 91732302)the Applied Basic Research Programs of Science and Technology Commission Foundation of Yunnan Province(2017FB109,2018FB052,2018FB053,2019FA007,and 202001AT070130)Chinese Academy of Sciences"Light of West China"Program,Shanghai Brain-Intelligence Project from Science and Technology Commission of Shanghai Municipality(16JC1420501)Shanghai Municipal Science and Technology Major Project(2018SHZDZX05)Open Large Infrastructure Research of Chinese Academy of Sciences,and China Postdoctoral Science Foundation(2018M631105).
文摘Whether direct manipulation of Parkinson’s disease(PD)risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue.Here,we used an adeno-associated virus serotype 9(AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras(SNs)of two monkey groups:an old group and a middle-aged group.After the operation,the old group exhibited all the classic PD symptoms,including bradykinesia,tremor,and postural instability,accompanied by key pathological hallmarks of PD,such as severe nigral dopaminergic neuron loss(>64%)and evidentα-synuclein pathology in the gene-edited SN.In contrast,the phenotype of their middle-aged counterparts,which also showed clear PD symptoms and pathological hallmarks,were less severe.In addition to the higher final total PD scores and more severe pathological changes,the old group were also more susceptible to gene editing by showing a faster process of PD progression.These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys.Taken together,this system can effectively develop a large number of genetically-edited PD monkeys in a short time(6–10 months),and thus provides a practical transgenic monkey model for future PD studies.
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.
基金supported by the China Postdoctoral Science Foundation(No.2019M651381)the Shanghai Talent Development Funding(No.2019115)to Chen Zhong。
文摘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.
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Supported by the National Natural Sciences Foundation of China(30271318).
文摘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.
基金sponsored by grants from the National Natural Science Foundation of China(No.82030117)the Wenzhou major scientific and technological innovation project(No.ZY2022001).
文摘Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.
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.
