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P. Seshidhar Reddy Neeraja Idamakanti Yan Chen Tyler Whale Lorne A. Babiuk Majid Mehtali Suresh Kumar Tikoo 《Journal of virology》1999,73(11):9137-9144
Although recombinant human adenovirus (HAV)-based vectors offer several advantages for somatic gene therapy and vaccination over other viral vectors, it would be desirable to develop alternative vectors with prolonged expression and decreased toxicity. Toward this objective, a replication-defective bovine adenovirus type 3 (BAV-3) was developed as an expression vector. Bovine cell lines designated VIDO R2 (HAV-5 E1A/B-transformed fetal bovine retina cell [FBRC] line) and 6.93.9 (Madin-Darby bovine kidney [MDBK] cell line expressing E1 proteins) were developed and found to complement the E1A deletion in BAV-3. Replication-defective BAV-3 with a 1.7-kb deletion removing most of the E1A and E3 regions was constructed. This virus could be grown in VIDO R2 or 6.93.9 cells but not in FBRC or MDBK cells. The results demonstrated that the E1 region of HAV-5 has the capacity to transform bovine retina cells and that the E1A region of HAV-5 can complement that of BAV-3. A replication-defective BAV-3 vector expressing bovine herpesvirus type 1 glycoprotein D from the E1A region was made. A similar replication-defective vector expressing the hemagglutinin-esterase gene of bovine coronavirus from the E3 region was isolated. Although these viruses grew less efficiently than the replication-competent recombinant BAV-3 (E3 deleted), they are suitable for detailed studies with animals to evaluate the safety, duration of foreign gene expression, and ability to induce immune responses. In addition, a replication-competent recombinant BAV-3 expressing green fluorescent protein was constructed and used to evaluate the host range of BAV-3 under cell culture conditions. The development of bovine E1A-complementing cell lines and the generation of replication-defective BAV-3 vectors is a major technical advancement for defining the use of BAV-3 as vector for vaccination against diseases of cattle and somatic gene therapy in humans. 相似文献
3.
犬2型腺病毒通用载体的构建及鉴定 总被引：4，自引：0，他引：4
为了获得能够携带较大外源基因的犬2型腺病毒E3区缺失性载体,以犬2型腺病毒全基因组质粒pPolyⅡ-CAV-2及E3区重组质粒pVAX-E3为基础,缺失1381bp的E3区片段(92.6%的E3区全序列),插入Linker-NF(内含NotⅠ、ClaⅠ、FseⅠ多克隆位点),获得重组载体质粒pPolyⅡ-CAV-2-ΔE3(NF)(31.9kb)。以AscⅠ和PmeⅠ双酶切,游离重组基因组,在脂质体LipofectamineTM2000介导下,转染MDCK细胞系,获得了E3区缺失的重组病毒CAV-2-ΔE3(NF)。通过病毒的形态学观察,血凝性、生长特性、感染性实验证明,该重组病毒与母源病毒没有差异。重组病毒CAV-2-ΔE3(NF)可以作为载体表达外源基因,其外源基因插入片段不小于3.3kb。 相似文献
4.
为了获得能够携带较大外源基因的犬2型腺病毒E3区缺失性载体,以犬2型腺病毒全基因组质粒pPolyⅡ-CAV-2及E3区重组质粒pVAX-E3为基础,缺失1381bp的E3区片段(92.6%的E3区全序列),插入Linker-NF(内含NotⅠ、ClaⅠ、FseⅠ多克隆位点),获得重组载体质粒pPolyⅡ-CAV-2-ΔE3（NF）(31.9kb)。以AscⅠ和PmeⅠ双酶切,游离重组基因组,在脂质体LipofectamineTM 2000介导下,转染MDCK细胞系,获得了E3区缺失的重组病毒CAV-2-ΔE3（NF）。通过病毒的形态学观察,血凝性、生长特性、感染性实验证明,该重组病毒与母源病毒没有差异。重组病毒CAV-2-ΔE3（NF）可以作为载体表达外源基因,其外源基因插入片段不小于3.3kb。 相似文献
5.
Monika Zochowska Agnieszka Paca Guy Schoehn Jean-Pierre Andrieu Jadwiga Chroboczek Bernard Dublet Ewa Szolajska 《PloS one》2009,4(5)
Background
Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro.Principal Findings
Dodecahedron (Dd) structure is preserved at up to about 50°C at pH 7–8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37°C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin.Conclusions/Significance
Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs. 相似文献6.
7.
Replication-Defective Adenovirus Vectors with Multiple Deletions Do Not Induce Measurable Vector-Specific T Cells in Human Trials 
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下载免费PDF全文 Richard A. Koup Laurie Lamoreaux David Zarkowsky Robert T. Bailer C. Richter King Jason G. D. Gall Douglas E. Brough Barney S. Graham Mario Roederer 《Journal of virology》2009,83(12):6318-6322
The magnitude and character of adenovirus serotype 5 (Ad5)-specific T cells were determined in volunteers with and without preexisting neutralizing antibodies (NAs) to Ad5 who received replication-defective Ad5 (rAd5)-based human immunodeficiency virus vaccines. There was no correlation between T-cell responses and NAs to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4+ T cells at time points where antibodies to Ad5 and T-cell responses to the recombinant gene products could be measured. These data indicate that rAd5-based vaccines containing deletions in the E1, E3, and E4 regions do not induce appreciable expansion of vector-specific CD4+ T cells.Replication-defective adenoviruses (rAd) have been engineered to provide high levels of expression of foreign inserts with minimum expression of adenovirus proteins, making them excellent candidates for vaccine and gene therapy applications (3, 16). Despite promising immunogenicity, a prophylactic vaccine trial of a serotype 5 rAd (rAd5) vector expressing human immunodeficiency virus (HIV) Gag, Pol, and Nef genes (Step trial) was recently halted due to an increase in HIV infections among volunteers who had preexisting neutralizing antibodies (NAs) to Ad5 (7). This finding raises the possibility that the presence of Ad5-specific T-cell responses (specifically CD4+ T-cell responses) in subjects with preexisting Ad5 NAs could be boosted by rAd5 vaccines, thereby providing an expanded susceptible target cell population that could be more easily infected by HIV. If this mechanism were operative, it would have broad implications for the future use of rAd viruses, and indeed other virus vectors, as vaccines or therapeutic agents within HIV-susceptible populations (2, 12, 15). We therefore measured the frequency, magnitude, and activation status of rAd5-specific T cells in HIV-uninfected volunteers who had received rAd5-based HIV vaccines in the presence or absence of preexisting NAs to Ad5.We studied 31 volunteers enrolled in two NIAID Institutional Review Board-approved phase I clinical trials of rAd5-based HIV vaccines. VRC 006 was a dose escalation study evaluating a single inoculation of a rAd5 mixture expressing EnvA, EnvB, EnvC, and fusion protein Gag/PolB at 109, 1010, and 1011 total particle units (10). VRC 008 evaluated DNA priming by needle and syringe or Biojector, followed by rAd5 boosting. Both studies enrolled healthy, HIV-uninfected adults; used the same rAd5 products; and evaluated immunogenicity on the day of and 4 weeks after rAd5 immunization. Both of these trials involved rAd5 products that contained deletions in the E1, E3, and E4 regions (8, 10).NAs to Ad5 were determined for all volunteers as previously described (19). A 90% NA titer of 12 or more was considered positive and taken as evidence of preexisting humoral immunity to Ad5. Volunteers were chosen for assessment of Ad5-specific T-cell responses based upon the availability of peripheral blood mononuclear cell samples at key time points and the presence or absence of preexisting NAs to Ad5. Only volunteers who received the vaccine (not the placebo) were included. Table Table11 lists the volunteers who were tested for Ad5-specific T-cell responses and their NA titers to Ad5 before and after rAd5 vaccination. All volunteers, except for one (volunteer 12) who had a less-than-maximum NA titer to Ad5 before vaccination, had an increase in titer by 4 weeks after vaccination, indicating the successful “take” of the rAd5-based vaccine. There was no correlation between rAd5 dose and increase in Ad5 NA titer.
Open in a separate windowaPU, particle units.HIV-specific T-cell responses were measured by multiparameter flow cytometry after 6 h of stimulation with peptides (15-mers overlapping by 11) corresponding to the HIV EnvA protein (one of the vaccine inserts expressed in the Ad5 vectors), as previously described (13). Overlapping peptides corresponding to the major Ad5 surface protein (hexon), the Ad5 early regulatory protein (E2A), and Ad5 ORF1, -2, and -3 proteins were used to assess Ad5-specific T-cell responses, and additional markers of cell viability (ViViD), T-cell memory (CD45RO and CD27), and activation/division (CCR5, CD38, HLA-DR, and Ki67) were added to the panel for these assessments. Antibodies and fluorochromes used in this panel were CCR5-Cy7-phycoerythrin (PE), CD38-allophycocyanin, Ki67-fluorescein isothiocyanate, and CD3-Cy7-allophycocyanin, all from BD PharMingen; CD8-Cy55-PE from BD Biosciences; CD27-Cy5-PE and CD45RO-Texas Red-PE, both from Beckman Coulter; CD4-Cy5.5-PE from Caltag; CD14- and CD19-PacificBlue, CD57-QDot545, and HLA-DR-Alexa680, conjugated according to standard protocols [http://drmr.com/abcon/index.html]); gamma interferon-PE and interleukin-2-PE from BD Biosciences; and a violet amine dye from Invitrogen. Cells were analyzed on an LSRII instrument (Becton Dickinson), and data analysis was performed using FlowJo, version 8.1.1 (TreeStar). The gating strategy is shown in Fig. Fig.1A1A.Open in a separate windowFIG. 1.CD4+ and CD8+ T-cell responses to Ad5. (A) Gating tree used to determine antigen-specific T-cell frequencies. Single CD3+ ViViD− CD14− CD19− cells were gated on CD4 or CD8 cells. Naïve CD27+ CD45RO− cells were gated out, and the frequency of cells expressing gamma interferon (IFNg) and/or interleukin-2 (IL2) was determined. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area. (B) Frequencies of CD4+ and CD8+ T-cell responses after stimulation with Ad5 hexon or E2A peptides were plotted against the prevaccination Ad5 NA titer. The prevaccine T-cell response was used. (C) Frequencies of CD4+ and CD8+ T-cell responses to Ad5 hexon, E2A, and HIV EnvA before and 4 weeks after rAd5 vaccination are shown for subjects with (Ad5 NA titer of >12) and without (Ad5 NA titer of <12) preexisting NAs to Ad5. Boxed areas represent interquartile ranges, and horizontal lines represent medians.Previously, we had found no T-cell responses to Ad5 ORF1, -2, or -3, so data from these antigen stimulations are not shown. As shown in Fig. Fig.1B,1B, T-cell responses to Ad5 hexon and E2A were detected, but there was no association between the NA response to Ad5 and the T-cell responses to these Ad5 proteins. Volunteers with an absence of NAs to Ad5 often had very strong CD4+ and CD8+ T-cell responses to Ad5 proteins. This probably reflects the degree of protein sequence homology between different adenovirus serotypes (11) and suggests that T-cell responses to adenoviruses may be significantly cross-reactive, while NAs are serotype specific. It also indicates that the NA response to Ad5 cannot be used as a surrogate for either a CD4+ or a CD8+ T-cell response to that adenovirus serotype.We next asked whether Ad5-specific T-cell responses were boosted by a single rAd5 vaccination in subjects with or without preexisting NAs to Ad5. At the time point 4 weeks after vaccination, there was clear evidence of boosting of the insert-specific (EnvA) CD4+ and CD8+ T-cell responses in volunteers with and without preexisting NAs to Ad5 (Fig. (Fig.1C).1C). The results of the Ad5-specific responses were consistent across volunteers who had received prior DNA immunization (VRC 008) and those who had not (VRC 006), so the results are combined in Fig. Fig.1C1C and show no increase in Ad5 hexon- or E2A-specific CD4+ T-cell responses after rAd5 immunization irrespective of Ad5 NA status. There was evidence of an increase in the CD8+ T-cell response to Ad5 hexon (P = 0.004 by paired t test), but not that to E2A, after rAd5 vaccination. These results, while showing evidence of adenovirus-specific CD8+ T-cell boosting by rAd5 vaccination, do not indicate an expansion of Ad5-specific CD4+ T cells that could serve as a substrate for HIV infection in subjects with or without NAs to Ad5.Having failed to demonstrate an expansion of Ad5-specific CD4+ T cells after vaccination, we assessed whether the activation profile of the unexpanded Ad5-specific CD4+ T cells was changed by vaccination. The gating tree is shown in Fig. Fig.2A.2A. Ad5 hexon- and E2A-specific CD4+ T cells expressed activation markers CCR5, CD38, and HLA-DR and a marker of recent cell division, Ki67, more frequently than did total memory CD4+ T cells (Fig. (Fig.2B).2B). However, none of these markers were significantly increased on total or Ad5-specific CD4+ T cells after vaccination in volunteers with or without preexisting NAs to Ad5.Open in a separate windowFIG. 2.Vaccine-induced activation of Ad5-specific CD4+ T cells. (A) Total CD4+ memory cells or Ad5-specific CD4+ memory cells (as gated in Fig. Fig.1A)1A) were further defined by expression of Ki67, CD38, CCR5, and HLA-DR. (B) Percentages of Ad5 hexon-specific cells, E2A-specific cells, or total memory CD4+ T cells that express CCR5, CD38, HLA-DR, or Ki67 before and 4 weeks after rAd5 vaccination are shown for subjects with (Ad5 NA titer of >12) (left) and without (Ad5 NA titer of >12) (right) preexisting NAs to Ad5. The phenotype was assessed only for those responders for whom at least 10 cytokine-positive events were counted. None of the comparisons of pre- and postvaccination marker expression were significant at a P value of 0.02 by paired t test. Boxed areas represent interquartile ranges, and horizontal lines represent medians.Expansion of Ad5-specific T cells after rAd5-based vaccination or gene therapy has been reported by others (14, 20, 21). Those studies evaluated Ad5-specific responses to rAd5 vectors with only the adenovirus E1 gene deleted (as used in the Step trial vaccines). The vectors used here contained deletions of the adenovirus E1, E3, and E4 genes (8, 10). While adenovirus gene deletions can render the vectors replication defective (6, 9), they do not necessarily completely shut off all adenovirus protein expression (20, 21). To demonstrate the importance of E4 deletions in limiting expression of adenovirus gene products, we measured the level of adenovirus protein synthesis in infected A549 cells as previously described (1, 4, 5). Cells were infected with adenovirus vectors with E1 and E3 deletions or with E1, E3, and E4 deletions at the same multiplicity of infection (10 focus-forming units per cell). At 24 h postinfection, [35S]methionine was added for 1 h. Levels of total and adenovirus protein synthesis in the infected and mock-infected cells were compared (Fig. (Fig.3).3). Adenovirus early protein single-stranded DNA binding protein, as well as late gene products hexon, penton, and fiber, was immunoprecipitated, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and resolved by autoradiography. The results show that the amount of newly synthesized adenovirus proteins in cells infected with adenovirus with E1, E3, and E4 deletions is significantly lower than that for an adenovirus vector with E1 and E3 deletions. Therefore, our inability to detect a vaccine-induced increase in the frequency and character of the Ad5-specific T-cell response could relate to the very low levels of adenovirus proteins that were probably expressed in vivo by the rAd5 vectors with multiple deletions.Open in a separate windowFIG. 3.Ad5 protein expression in vitro after infection with different Ad5 vectors. A549 cells were infected with adenovirus vectors with E1 and E3 deletions or with E1, E3, and E4 deletions and [35S]methionine labeled, and levels of total and adenovirus protein synthesis in the infected and mock-infected cells were compared after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Markers for the adenovirus early protein single-stranded DNA binding protein (DBP) and capsid proteins hexon, penton base, and fiber are shown.We were therefore unable to demonstrate (i) that Ad5-specific CD4+ T cells were restricted to subjects with preexisting Ad5 NAs, (ii) that rAd5 vaccination expanded or increased the activation of Ad5-specific CD4+ T cells, or (iii) that there was a substantial effect on the magnitude or character of the Ad5-specific CD4+ T-cell response to vaccination based upon preexisting NAs to Ad5. While the kinetics of Ad5-specific T-cell responses after rAd5-based vaccination are not known, it is clear that insert-specific responses are increased at 4 weeks after vaccination and subsequently contract (10). It is therefore reasonable to assume that if Ad5-specific responses were similarly affected, they would be detected at the 4-week-postvaccination time point.It is possible that rAd5 vaccines expand a preexisting mucosal T-cell response to Ad5 that is not reflected within the blood. While we do not have mucosal samples from our vaccine volunteers to directly address this possibility, it is likely that expansion of a mucosal response would be reflected to some degree within the blood.The mechanism underlying the increase in HIV infections in vaccinees with NAs to Ad5 in the Step trial is yet to be determined (2, 7, 12, 15, 17). Confounding factors and alternative hypotheses have recently been proposed to account for the increased acquisition (7, 12, 15, 18). Until there is a better understanding of the processes involved, future studies of rAd5-based products should proceed with appropriate safety considerations and monitoring of adenovirus-specific responses. In addition, the use of vaccine regimens involving single injections of vectors with multiple deletions may help mitigate risk. 相似文献
TABLE 1.
Ad5 serostatus before and after vaccination| Volunteer | Prior DNA immunization | rAd5 dose (PUa) | Ad5 NA titer
| |
|---|---|---|---|---|
| Prevaccine | Postvaccine | |||
| 1 | No | 1011 | <12 | 739 |
| 2 | No | 1011 | <12 | 834 |
| 3 | No | 1011 | <12 | 4,787 |
| 4 | No | 1011 | <12 | 806 |
| 5 | No | 1011 | <12 | 1,033 |
| 6 | No | 1010 | <12 | 130 |
| 7 | No | 1010 | <12 | 1,354 |
| 8 | Yes | 1010 | <12 | 1,387 |
| 9 | Yes | 1010 | <12 | 575 |
| 10 | Yes | 1010 | <12 | 170 |
| 11 | Yes | 1010 | <12 | >8,748 |
| 12 | Yes | 1010 | <12 | <12 |
| 13 | No | 1011 | 30 | >8,748 |
| 14 | No | 109 | 46 | >8,748 |
| 15 | No | 109 | 70 | 328 |
| 16 | No | 1010 | 176 | >8,748 |
| 17 | No | 1010 | 478 | 6,198 |
| 18 | No | 109 | 2,472 | >8,748 |
| 19 | No | 109 | 3,502 | >8,748 |
| 20 | No | 1010 | 4,820 | >8,748 |
| 21 | No | 109 | 5,078 | >8,748 |
| 22 | No | 1011 | 6,162 | >8,748 |
| 23 | No | 109 | >8,748 | >8,748 |
| 24 | No | 1011 | >8,748 | >8,748 |
| 25 | Yes | 1010 | 643 | >8,748 |
| 26 | Yes | 1010 | 942 | >8,748 |
| 27 | Yes | 1010 | 1,510 | >8,748 |
| 28 | Yes | 1010 | 1,611 | >8,748 |
| 29 | Yes | 1010 | 2,934 | >8,748 |
| 30 | Yes | 1010 | >8,748 | >8,748 |
| 31 | Yes | 1010 | >8,748 | >8,748 |
8.
目的:采用一种简便方法在复制缺陷型腺病毒载体纤突结(fibre knob)的HI loop插入精氨酸-甘氨酸-天冬氨酸(arginine-glycine-aspartate,RGD)肽,构建纤突结修饰的腺病毒载体,观察其对RD和T24肿瘤细胞的感染效率以及机体针对外源基因的抗体反应.方法:以PCR方法将RGD插入到pA... 相似文献
9.
Briana Jill Williams Shilpa Bhatia Lisa K. Adams Susan Boling Jennifer L. Carroll Xiao-Lin Li Donna L. Rogers Nikolay Korokhov Imre Kovesdi Alexander V. Pereboev David T. Curiel J. Michael Mathis 《PloS one》2012,7(10)
Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy. 相似文献
10.
腺病毒（ａｄｅｎｏｖｉｒｕｓ,Ａｄ）载体广泛应用于基因治疗,其主要特征之一是能高效地进行各种组织如肺上皮、肝、肌肉、胰岛、胆囊等的体内（ｉｎｖｉｖｏ）基因转移。然而,因其混杂的向性而引起的全身体内基因转导后治疗基因不受组织限制的表达,限制了它在基因治疗中的应用,因此希望改变腺病毒的天然向性,而使治疗基因导向转移到选择的细胞类型中。目前腺病毒载体细胞导向性的基因转移可通过（１）修饰腺病毒载体的细胞表面识别成分,限制腺病毒导向性感染选择的细胞类型;（２）腺病毒载体中导入组织特异性启动子,控制治疗基因… 相似文献
11.
Recombinant, Replication-Defective Adenovirus Gene Transfer Vectors Induce Cell Cycle Dysregulation and Inappropriate Expression of Cyclin Proteins 总被引：6，自引：1，他引：6 下载免费PDF全文
下载免费PDF全文 Robert P. Wersto Eugene R. Rosenthal Prem K. Seth N. Tony Eissa Robert E. Donahue 《Journal of virology》1998,72(12):9491-9502
First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (ΔE1) or lacking the Ad E3 region in addition to E1 sequences (ΔE1ΔE3) induced G2 cell cycle arrest and inhibited traverse across G1/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34cdc2 protein levels. In some instances, infection with ΔE1 or ΔE1ΔE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation ΔE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in ΔE1 or ΔE1ΔE3 Ad vectors induce G2 growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy. 相似文献
12.
13.
绵羊腺病毒287(OAdV287) 具有独特的基因组结构,全长29 576bp。基因组最大的特点是有很高的A/T含量(66.4%)和缺少典型的E1区,以及含有一段较长的冗余片段。已识别出其基因组中有3个外源基因插入区,理论上最多可容纳6.3kb的外源DNA。OAdV287的纤维和五邻体结构结构独特,衣壳蛋白上缺少可识别的整合蛋白结合域。OAdV287能感染一系列人和动物的细胞系,但是不能在非绵羊源细胞内成功复制。OAdV287载体可以避免人腺病毒的免疫干扰,具有很高的转染效率。目前,OAdV287已成为研究最热的动物源腺病毒载体之一。 相似文献
14.
腺病毒载体是基因治疗的常用载体之一,已经广泛应用于肿瘤和遗传疾病等的基因治疗研究中.但是临床发现腺病毒载体有较高的免疫原性,同时缺乏组织或细胞特异性,制约了其在临床上的应用.通过共价键结合或者静电力作用,将高分子修饰到病毒衣壳上,利用高分子的特殊性质可以降低载体的免疫原性和提高载体的靶向性,同时载体保持了较高的转染能力... 相似文献
15.
Background
Successful gene therapy will require targeted delivery vectors capable of self-directed localization. In this regard, the use of antibodies or single chain antibody fragments (scFv) in conjunction with adenovirus (Ad) vectors remains an attractive means to achieve cell-specific targeting. However, a longstanding barrier to the development of Ad vectors with genetically incorporated scFvs has been the biosynthetic incompatibility between Ad capsid proteins and antibody-derived species. Specifically, scFv require posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during protein synthesis and virion assembly.Methodology/Principal Findings
We have therefore sought to develop scFv-targeted Ad vectors using a secreted scFv that undergoes the requisite posttranslational modifications and is trafficked for secretion. Formation of the scFv-targeted Ad vector is achieved via highly specific association of the Ad virion and a targeting scFv employing synthetic leucine zipper-like dimerization domains (zippers) that have been optimized for structural compatibility with the Ad capsid and for association with the secreted scFv. Our results show that zipper-containing Ad fiber molecules trimerize and incorporate into mature virions and that zippers can be genetically fused to scFv without ablating target recognition. Most importantly, we show that zipper-tagged virions and scFv provide target-specific gene transfer.Conclusions/Significance
This work describes a new approach to produce targeted Ad vectors using a secreted scFv molecule, thereby avoiding the problem of structural and biosynthetic incompatibility between Ad and a complex targeting ligand. This approach may facilitate Ad targeting using a wide variety of targeting ligands directed towards a variety of cellular receptors. 相似文献16.
携带PTEN基因的重组腺病毒表达载体构建的研究 总被引：2，自引：0，他引：2
构建携带抑癌基因PTEN(Phosphatase and temin homolog deleted on chromosome ten)的重组腺病毒表达裁体,为研究PTEN的功能和作用机制奠定基础.采用RT-PCR法从大鼠海马神经元扩增目的基因PTEN,克隆人含绿色荧光蛋白(Green fluorescence protein),GFP基因的pAdTrack-CMV穿梭质粒,在含有腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌内进行同源重组;获得重组腺病毒质粒,经Pacl线性化后,转染AD293细胞.结果表明,感染腺病毒载体的AD293细胞表达GFP基因,随着时间逐渐增强,并且出现明显的细胞病变效应(Cytopathic effect,CPE),经PCR对传代的Ad-PTEN分析证实得到目的基因.成功构建了携带PTEN基因的腺病毒表达载体,为研究PTEN的功能和作用机制奠定了基础. 相似文献
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A case of alopecia totalis in a chimpanzee is reported. The disease was probably due to an autoimmune phenomenon and was successfully treated with immunosuppressive therapy. However, possible side-effects outweighed benefits. 相似文献
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An Adenovirus Vector with Genetically Modified Fibers Demonstrates Expanded Tropism via Utilization of a Coxsackievirus and Adenovirus Receptor-Independent Cell Entry Mechanism 总被引：18，自引：6，他引：18 下载免费PDF全文
下载免费PDF全文 Igor Dmitriev Victor Krasnykh C. Ryan Miller Minghui Wang Elena Kashentseva Galina Mikheeva Natalya Belousova David T. Curiel 《Journal of virology》1998,72(12):9706-9713
Recombinant adenoviruses (Ad) have become the vector system of choice for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to cells expressing marginal levels of the coxsackievirus and adenovirus receptor (CAR). In order to achieve CAR-independent gene transfer by Ad vectors in clinically important contexts, we proposed modification of viral tropism via genetic alterations to the viral fiber protein. We have shown that incorporation of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. We have also demonstrated that due to its expanded tissue tropism, this novel vector is capable of efficient transduction of primary tumor cells. An increase in gene transfer to ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector, suggesting that recombinant Ad containing fibers with an incorporated RGD peptide may be of great utility for treatment of neoplasms characterized by deficiency of the primary Ad type 5 receptor. 相似文献