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1.
Site-specific integration of adeno-associated virus-based plasmid vectors in lipofected HeLa cells 总被引:7,自引:0,他引:7
Adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on human chromosome 19q13.3-qter. Similarly, there is accumulating evidence that this site-specific integration occurs by transfection of AAV-based plasmid vectors. In order to further define the process of plasmid integration events, we constructed some AAV plasmids, introduced them into HeLa cells by lipofection, and isolated chromosomal integrants. One of such plasmids, pTH-5, contained the rep and neomycin-resistant (neo(r)) genes flanked by the 5'- and 3'-inverted terminal repeats of AAV and the hygromycin-resistant (hyg(r)) gene located in the plasmid backbone. Southern blot analysis revealed that among 36 G418-resistant (G418(r)) clones isolated, 22 (61%) showed site-specific integration into AAVS1. Further structural and functional analyses on the expression of the hyg(r) gene in the site-specific clones and the LacZ gene in clones generated with plasmid pTH-2 indicated that, together with the AAV sequence, the plasmid backbone was integrated into the AAVS1 site and thus the neo(r) and hyg(r) genes remained linked at high frequencies in the targeted integrants compared with random integrants. Sequence analysis of integration junctions between pTH-5 and AAVS1 revealed that the junctions occurred in the p5 promoter region of the plasmid while mainly in the partial cDNA coding region of the AAVS1 site. We also found that plasmid pTH-1 linearized in the backbone before lipofection gave a significantly lower frequency of site-specific integration (26%) than the circular form (60%). This finding may support the involvement of the double-stranded, circular form of infected AAV in the integration process. Our results may help to understand the process and mechanism of site-specific integration of lipofected AAV plasmid vectors. 相似文献
2.
Persistent infection with adeno-associated virus (AAV) has been demonstrated in human tissues, most frequently in the female and male genital tract. The clinical significance of latent AAV infection remains, however, uncertain to date. The mode of latency of AAV is not known, i.e., it is unclear whether the viral genome is integrated in the cellular genome, and if integration occurs site-specifically in chromosome 19 as has been observed in cell culture. Therefore we investigated if viral DNA in AAV DNA-positive human testis samples from two patients, is integrated in the cellular genome. Using two different molecular approaches, uni-directional PCR and Walking Primer PCR, we could demonstrate that AAV DNA is present in an integrated form in testis tissue. Virus-cell DNA junction fragments were cloned and sequenced. A detailed analysis revealed integration within sequences of the so-called AAVS1 region on chromosome 19. These data demonstrate that AAV DNA can integrate also after natural infection, and that integration occurs within the AAVS1 region, at least in some cases. 相似文献
3.
Chimeric or hybrid herpes simplex virus type 1/adeno-associated virus amplicon vectors combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and stable transgene expression of AAV. These chimeric vectors have been demonstrated to support transgene expression for significantly longer periods than standard HSV-1 amplicons. Moreover, HSV/AAV hybrid vectors can mediate integration at the AAVS1 pre-integration site on human chromosome 19 at a relatively high rate, although random integration has also been observed. One major remaining hurdle of HSV/AAV hybrid vectors is the low packaging efficiency and titers when AAV rep sequences are included in the amplicon vector. In the conditions prevalent during the replication/packaging of HSV/AAV hybrid amplicons into HSV-1 virions, in particular the presence of HSV-1 replication factors and AAV Rep protein, at least three different viral origins of DNA replication are active: the HSV-1 ori, the AAV inverted terminal repeats (ITRs), and the p5 promoter/ori driving expression of the AAV rep gene. A detailed understanding of the properties of these origins of DNA replication and the molecular mechanisms of interactions between them, may allow designing novel hybrid vectors that allow the efficient and precise integration of large transgenes in the human genome. 相似文献
4.
5.
目的构建含腺病毒伴随病毒(AAV)基因组两端的反向重复序列(ITRs)和表达必须元件如启动子、多克隆位点和PolyA信号的通用型载体质粒pACR-Neo,并获得重组AAV(rVV/ACR-Neo)。方法通过DNA重组技术,将SV40PolyA、Neo基因、CMV-IE启动子和多克隆位点组成表达盒子,取代含AAV全基因组质粒pSSV9中AAV结构基因部分,构建成质粒pACR-Neo。用pACR-Neo转染5型腺病毒(Ad5)感染的重组AAV包装细胞系AE1201,能获得重组病毒rAAV/ACR-Neo。提取rAAV/ACR-Neo感染细胞的染色体,用Southem杂交分析重组病毒基因组在感染细胞中的存在情况。结果质粒pACR-Neo转染包装细胞系后所得rAAV/ACR-Neo滴度为4.2×105CFU/ml,并且rAAV/ACR-Neo能在转导细胞内实现其基因组与细胞染色体的整合。结论成功地构建了通用型AAV载体,为今后的AAV载体研究、基因治疗和临床应用打下了基础 相似文献
6.
Smith JR Maguire S Davis LA Alexander M Yang F Chandran S ffrench-Constant C Pedersen RA 《Stem cells (Dayton, Ohio)》2008,26(2):496-504
Silencing and variegated transgene expression are poorly understood problems that can interfere with gene function studies in human embryonic stem cells (hESCs). We show that transgene expression (enhanced green fluorescent protein [EGFP]) from random integration sites in hESCs is affected by variegation and silencing, with only half of hESCs expressing the transgene, which is gradually lost after withdrawal of selection and differentiation. We tested the hypothesis that a transgene integrated into the adeno-associated virus type 2 (AAV2) target region on chromosome 19, known as the AAVS1 locus, would maintain transgene expression in hESCs. When we used AAV2 technology to target the AAVS1 locus, 4.16% of hESC clones achieved AAVS1-targeted integration. Targeted clones expressed Oct-4, stage-specific embryonic antigen-3 (SSEA3), and Tra-1-60 and differentiated into all three primary germ layers. EGFP expression from the AAVS1 locus showed significantly reduced variegated expression when in selection, with 90% +/- 4% of cells expressing EGFP compared with 57% +/- 32% for randomly integrated controls, and reduced tendency to undergo silencing, with 86% +/- 7% hESCs expressing EGFP 25 days after withdrawal of selection compared with 39% +/- 31% for randomly integrated clones. In addition, quantitative polymerase chain reaction analysis of hESCs also indicated significantly higher levels of EGFP mRNA in AAVS1-targeted clones as compared with randomly integrated clones. Transgene expression from the AAVS1 locus was shown to be stable during hESC differentiation, with more than 90% of cells expressing EGFP after 15 days of differentiation, as compared with approximately 30% for randomly integrated clones. These results demonstrate the utility of transgene integration at the AAVS1 locus in hESCs and its potential clinical application. 相似文献
7.
Inserting genetic information at precise locations into the human genome has been the goal of gene transfer technology for almost two decades. The spectacular progress of mammalian genetics has led to the development of technology for genome editing and homologous recombination in human somatic cells that is finally approaching efficiency compatible with clinical application. Site-specific integration, or the insertion of genes at known locations by enzymes with target recognition capacity, has progressed slowly but steadily in recent years, and could very well be the basis of the next generation of gene transfer technology. This review focuses on the use of Rep, the replicase/integrase of the adeno-associated virus (AAV), to insert genes at the natural AAV integration site on human chromosome 19. This region (AAVS1) has characteristics that make it an ideal target for somatic transgenesis. 相似文献
8.
New recombinant serotypes of AAV vectors 总被引:12,自引:0,他引:12
AAV based vectors can achieve stable gene transfer with minimal vector related toxicities. AAV serotype 2 (AAV2) is the first AAV that was vectored for gene transfer applications. However, the restricted tissue tropism of AAV and its low transduction efficiency have limited its further development as vector. Recent studies using vectors derived from alternative AAV serotypes such as AAV1, 4, 5 and 6 have shown improved potency and broadened tropism of the AAV vector by packaging the same vector genome with different AAV capsids. In an attempt to search for potent AAV vectors with enhanced performance profiles, molecular techniques were employed for the detection and isolation of endogenous AAVs from a variety of human and non-human primate (NHP) tissues. A family of novel primate AAVs consisting of 110 non-redundant species of proviral sequences was discovered and turned to be prevalent in 18-19% of the tissues evaluated. Phylogenetic and functional analyses revealed that primate AAVs are segregated into clades based on phylogenetic relatedness. The members within a clade share functional and serological properties. Initial evaluation in mouse models of vectors based on these novel AAVs for tissue tropism and gene transfer potency led to the identification of some vector with improved gene transfer to different target tissues. Gene therapy treatment of several mouse and canine models with novel AAV vectors achieved long term phenotypic corrections. Vectors based on new primate AAVs could become the next generation of efficient gene transfer vehicles for various gene therapy applications. 相似文献
9.
目的 研究腺病毒伴随病毒(AAV)载体介导杀肿瘤瘤基因的转移及其在肿瘤治疗方面的应用。方法 应用作者构建的腺病毒伴随病毒载体,克隆Ⅰ型单纯疱疹病毒胸苷激酶(HSVI-TK)基因,构建质粒pACTK-19。用pACTK-19转染5型腺病毒(Ad5)感染的重组AAV包装细胞系AE1201,获得重组病毒rAAV/ACTK。再用此重组病毒感染肺癌细胞系A549,并联合现氧鸟苷(GCV)作用,研究其体外对肺 相似文献
10.
Vandenberghe LH Bell P Maguire AM Cearley CN Xiao R Calcedo R Wang L Castle MJ Maguire AC Grant R Wolfe JH Wilson JM Bennett J 《Science translational medicine》2011,3(88):88ra54
Gene therapy is emerging as a therapeutic modality for treating disorders of the retina. Photoreceptor cells are the primary cell type affected in many inherited diseases of retinal degeneration. Successfully treating these diseases with gene therapy requires the identification of efficient and safe targeting vectors that can transduce photoreceptor cells. One serotype of adeno-associated virus, AAV2, has been used successfully in clinical trials to treat a form of congenital blindness that requires transduction of the supporting cells of the retina in the retinal pigment epithelium (RPE). Here, we determined the dose required to achieve targeting of AAV2 and AAV8 vectors to photoreceptors in nonhuman primates. Transgene expression in animals injected subretinally with various doses of AAV2 or AAV8 vectors carrying a green fluorescent protein transgene was correlated with surgical, clinical, and immunological observations. Both AAV2 and AAV8 demonstrated efficient transduction of RPE, but AAV8 was markedly better at targeting photoreceptor cells. These preclinical results provide guidance for optimal vector and dose selection in future human gene therapy trials to treat retinal diseases caused by loss of photoreceptors. 相似文献
11.
Feifei Wang Can Huang Jinjing Cao Xiaomei Liu Dongxin Wang Chun Zhang Qin Chen 《Virus genes》2018,54(2):165-171
Adeno-associated virus has been gaining prominence in its use as a highly secure virus gene vector with low immunogenicity in the field of human gene therapy. However, wild-type adeno-associated virus sometimes has low transduction efficiency for certain tissues or cells both in vivo and in vitro. Thus, achieving the desired level of expression often requires a large dose. Large doses of viral injection in clinical applications will not only trigger the body’s immune response but will come at a high production cost. To improve the transduction efficiency of adeno-associated virus 6 (AAV6), we herein used fusion PCR to mutate a specific amino acid of the VP2 region of the wild-type AAV6 (AAV6-WT) and obtained AAV6-S663L, AAV6Y705 + 731F + T492A, AAV6Y705 + 731F + T492 V + S663 V and so on. We concluded that AAV6-S663L was the most efficient AAV6 mutant. When HEK293 cells were infected in vitro with a virus at a multiplicity of infection value of 1000, the transduction rate of AAV6-WT was only 43.8%, while that of AAV6-S663L was 83.9%. This highly efficient AAV6 mutant is highly significant for the future use of AAV6 in gene therapy. 相似文献
12.
Gene transfer into hematopoietic progenitor cells mediated by an adeno-associated virus vector 总被引:8,自引:0,他引:8
We describe here the transduction of murine hematopoietic progenitor cells with the dominant selectable neomycin drug-resistance (Neo) gene using a recombinant adeno-associated virus (AAV) vector. Successful transformation of progenitor cells to drug resistance was determined to be approximately 1.5% by colony formation in the presence of geneticin sulfate (G-418). The value of AAV as an alternative to the retrovirus vector systems is discussed. 相似文献
13.
Findings in the first clinical trial in which an adeno-associated virus (AAV) vector was introduced into the liver of human subjects highlighted an issue not previously identified in animal studies. Upon AAV gene transfer to liver, two subjects developed transient elevation of liver enzymes, likely as a consequence of immune rejection of transduced hepatocytes mediated by AAV capsid-specific CD8(+) T cells. Studies in healthy donors showed that humans carry a population of antigen-specific memory CD8(+) T cells probably arising from wild-type AAV infections. The hypothesis formulated at that time was that these cells expanded upon re-exposure to capsid, i.e. upon AAV-2 hepatic gene transfer, and cleared AAV epitope-bearing transduced hepatocytes. Other hypotheses have been formulated which include specific receptor-binding properties of AAV-2 capsid, presence of capsid-expressing DNA in AAV vector preparations, and expression of alternate open reading frames from the transgene; emerging data from clinical trials however fail to support these competing hypotheses. Possible solutions to the problem are discussed, including the administration of a short-term immunosuppression regimen concomitant with gene transfer, or the development of more efficient vectors that can be administered at lower doses. While more studies will be necessary to define mechanisms and risks associated with capsid-specific immune responses in humans, monitoring of these responses in clinical trials will be essential to achieving the goal of long-term therapeutic gene transfer in humans. 相似文献
14.
Owens RA 《Current gene therapy》2002,2(2):145-159
Adeno-associated virus type 2 (AAV-2) is a non-pathogenic human parvovirus that is being developed as a gene therapy vector for the treatment of numerous diseases. One property of wild-type AAV-2, that is highly desirable in a gene therapy vector, is its ability to preferentially integrate its DNA into a 4 kilobase region of human chromosome 19, designated AAVS1. One disadvantage of AAV-2 is its relatively small packaging capacity, approximately 4.7 kilobases. Because of this size limitation, the AAV-2 rep and cap genes were removed from first-generation AAV-2-based gene therapy vectors to make room for the therapeutic or marker gene. It was later discovered that the rep gene, or at least one of its products, the Rep68 or Rep78 protein, is required for preferential integration of AAV-2. Recent developments in AAV-2 gene therapy vector construction allow the inclusion of the rep gene into a second generation of AAV-2-based gene therapy systems. These new systems fall into four major categories: plasmid-based systems, co-transduction with multiple AAV-2 vectors, incorporation of the AAV-2 vector into a larger virus, and in vitro packaging. These systems not only allow the inclusion of the rep gene, they also allow the delivery of larger therapeutic genes. 相似文献
15.
16.
Zhong L Li B Jayandharan G Mah CS Govindasamy L Agbandje-McKenna M Herzog RW Weigel-Van Aken KA Hobbs JA Zolotukhin S Muzyczka N Srivastava A 《Virology》2008,381(2):194-202
We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy. 相似文献
17.
Adeno-associated virus (AAV) vectors in the CNS 总被引:10,自引:0,他引:10
McCown TJ 《Current gene therapy》2005,5(3):333-338
Adeno-associated virus (AAV) vectors exhibit a number of properties that have made this vector system an excellent choice for both CNS gene therapy and basic neurobiological investigations. In vivo, the preponderance of AAV vector transduction occurs in neurons where it is possible to obtain long-term, stable gene expression with very little accompanying toxicity. Promoter selection, however, significantly influences the pattern and longevity of neuronal transduction distinct from the tropism inherent to AAV vectors. AAV vectors have successfully manipulated CNS function using a wide variety of approaches including expression of foreign genes, expression of endogenous genes, expression of antisense RNA and expression of RNAi. With the discovery and characterization of different AAV serotypes, the potential patterns of in vivo vector transduction have been expanded substantially, offering alternatives to the more studied AAV 2 serotype. Furthermore, the development of specific AAV chimeras offers the potential to further refine targeting strategies. These different AAV serotypes also provide a solution to the immune silencing that proves to be a realistic likelihood given broad exposure of the human population to the AAV 2 serotype. These advantageous CNS properties of AAV vectors have fostered a wide range of clinically relevant applications including Parkinson's disease, lysosomal storage diseases, Canavan's disease, epilepsy, Huntington's disease and ALS. Each individual application, however, presents a unique set of challenges that must be solved in order to attain clinically effective gene therapies. 相似文献
18.
We used rep+ and rep- recombinant AAV-plasmid vectors containing the nonselectable marker chloramphenicol acetyltransferase (CAT) driven by the AAV p40 promoter, and having a selectable marker, neo, inserted in the plasmid genome, and driven by a herpesvirus thymidine kinase gene promoter. Each vector was transfected into human 293 cells or HeLa cells and the neo gene was used to select geneticin-resistant (genr) cells containing integrated vectors. The genr cells were then screened for expression of the unselected marker CAT. For 293 cells, most clones from the rep- vector gave high CAT expression whereas only 50% of those from the rep+ vector expressed CAT, generally at low level. For HeLa cells about 25% of the clones derived from either the rep+ or rep- vector expressed CAT, and several clones from the rep+ vector gave very high yields. We also analyzed integrated rep+ vectors by rescue after superinfection with adenovirus and by Southern blotting. The AAV-CAT genome could be rescued from 50% of HeLa cell clones but not from 293 cell clones. Lack of rescuability reflected rearrangement of the AAV genome termini or the rep gene. Western blotting showed low level constitutive expression of rep protein in one 293 cell clone and two HeLa cell clones. Thus, the AAV p40 promoter (as well as p5 and p19) can function in integrated vectors to express unselected markers which can subsequently be rescued. Expression and rescue depended upon several parameters including the cell type, the initial structure of the vector (rep+ or rep-) but not continued expression of rep, and possibly global effects of the surrounding chromatin. 相似文献
19.
Recombinant AAV efficacy has been demonstrated in numerous gene therapy preclinical studies. As this vector is increasingly applied to human clinical trials, it is a priority to evaluate the risks of its use for workers involved in research and clinical trials as well as for the patients and their descendants. At high multiplicity of infection, wild-type AAV integrates into human chromosome 19 in approximately 60% of latently infected cell lines. However, it has been recently demonstrated that only approximately 1 out of 1000 infectious units can integrate. The mechanism of this site-specific integration involves AAV Rep proteins which are absent in vectors. Accordingly, recombinant AAV (rAAV) do not integrate site-specifically. Random integration of vector sequences has been demonstrated in established cell lines but only in some cases and at low frequency in primary cultures and in vivo. In contrast, episomal concatemers predominate.Therefore, the risks of insertional mutagenesis and activation of oncogenes are considered low. Biodistribution studies in non-human primates after intramuscular, intrabronchial, hepatic artery and subretinal administration showed low and transient levels of vector DNA in body fluids and distal organs. Analysis of patients body fluids revealed rAAV sequences in urine, saliva and serum at short-term. Transient shedding into the semen has been observed after delivery to the hepatic artery. However, motile germ cells seemed refractory to rAAV infection even when directly exposed to the viral particles, suggesting that the risk of insertion of new genetic material into the germ line is absent or extremely low. Risks related to viral capsid-induced inflammation also seem to be absent since immune response is restricted to generation of antibodies. In contrast, transgene products can elicit both cellular and humoral immune responses, depending on the nature of the expressed protein and of the route of vector administration. Finally, a correlation between early abortion as well as male infertility and the presence of wt AAV DNA in the genital tract has been suggested. Although no causal relationship has been established, this issue stresses the importance of using rAAV stocks devoid of contaminating replication-competent AAV. This review comprehensively examines virus integration, biodistribution, immune interactions, and other safety concerns regarding the wild-type AAV and recombinant AAV vectors. 相似文献
20.
Cell and gene therapy using mesenchymal stem cells (MSCs) 总被引:4,自引:1,他引:3
Ozawa K Sato K Oh I Ozaki K Uchibori R Obara Y Kikuchi Y Ito T Okada T Urabe M Mizukami H Kume A 《Journal of autoimmunity》2008,30(3):121-127
Mesenchymal stem cells (MSCs) are considered to be a promising platform for cell and gene therapy for a variety of diseases. First, in the field of hematopoietic stem cell transplantation, there are two applications of MSCs: 1) the improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution based on the hematopoiesis-supporting ability; and 2) the treatment of severe graft-versus-host disease (GVHD) based on the immunomodulatory ability. Regarding the immunosuppressive ability, we found that nitric oxide (NO) is involved in the MSC-mediated suppression of T cell proliferation. Second, tumor-bearing nude mice were injected with luciferase-expressing MSCs. An in vivo imaging analysis showed the significant accumulation of the MSCs at the site of tumors. The findings suggest that MSCs can be utilized to target metastatic tumors and to deliver anti-cancer molecules locally. As the third application, MSCs may be utilized as a cellular vehicle for protein-supplement gene therapy. When long-term transgene expression is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of adeno-associated virus (AAV) would be particularly valuable. There will be wide-ranging applications of MSCs to frontier medical treatments in the near future. 相似文献