Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.     

Development of a recombinant adenovirus vector production system free of replication-competent adenovirus by utilizing a packaging size limit of the viral genome

In a conventional adenovirus (Ad) vector production method using 293 cells, homologous recombination between Ad vector DNA and 293 cell-derived Ad E1 DNA occurs with low efficiency, resulting in the generation of replication-competent adenovirus (RCA). RCA can induce the spread of replication-incompetent Ad vectors, leading to unexpected tissue damage. In order to overcome this problem, we developed an Ad vector production system free of RCA generation by utilizing the Ad packaging size limit of the viral genome. It is well known that up to approximately 105% (37.7 kb) of the wild-type genome (35.9 kb) can be packaged in the Ad virion. We designed the Ad vector genome by insertion of a transgene expression cassette into the E3 region, such that homologous recombination between the Ad vector DNA and 293 cell-derived Ad E1 DNA would produce an Ad vector genome that exceeds in the size of the packaging limit. In accord with our strategy, no RCA generation was observed during the passages when we used the E1 (3.2kb)-deleted Ad vectors containing a more than 3.0-kb transgene expression cassette in the E3 region. In contrast, the E1 (3.2kb)-deleted Ad vectors, which retain 37.7 kb of the viral genome and have an insertion of a 2.1-kb transgene expression cassette in the E3 region, generated RCA, although RCA derived from this Ad vector exceeded the packaging size limit (105.0%). These results suggest that RCA generation can be avoided when the genome size of RCA is more than 108.3% (38.9 kb) of the wild-type Ad genome. This Ad vector production system generates safe, easy, and efficient Ad vector stock for both basic study as well as clinical research.     

Generation of a replication-deficient recombinant human adenovirus type 35 vector using bacteria-mediated homologous recombination

The use of adenovirus type 35 (Ad35) as a vector in vaccine and gene therapy studies is promising due to its broad cell tropism and low seroprevalence in humans. However, to date, a simple and effective system for producing recombinant Ad35 (rAd35) has not been well developed. This report describes a two-plasmid Ad35-Easy system to facilitate the production of recombinant Ad35 (rAd35). The system employed the pAd35-shuttle vector for foreign gene transfer and the pAd35-backbone vector to provide the Ad35 genomic backbone. A 293-Ad35E1B cell line was used to trans-complement rAd35 replication. rAd35 plasmids were obtained through homologous recombination following co-transformation of E. coli BJ5183 cells with recombinant pAd35-shuttle vectors harboring foreign genes. rAd35 viruses were obtained directly by transfecting 293-Ad35E1B cells with foreign gene-containing rAd35 plasmids and the pAd35-backbone vector. The production of E1 deficient rAd35 was evaluated by transfecting the 293-Ad35E1B cells with the rAd35 plasmid containing the enhanced green fluorescent protein (EGFP) gene. The virus grew effectively at a yield comparable to that of wild type Ad35 in HEp2 cells, indicating that the Ad35-Easy system is an efficient method for rapid production of rAd35 in sufficient quantities for vaccine development or gene therapy.     

Spontaneous mobilization of integrated recombinant adenoassociated virus in a cell culture model of virus latency

A cell line containing integrated recombinant adenoassociated virus (AAV) was investigated for spontaneous mobilization of vector sequence. Detection of these rare events was facilitated by using a vector design that allowed the circular rescue product (cAAV) to be individually scored by bacterial transformation. Restriction and sequence analysis of captured clones revealed five highly ordered classes of cAAV, each of which contained a defined segment of the integrated vector locus. A common feature of all cAAV classes was the presence of a modified inverted terminal repeat that joined the ends of the liberated sequence. Assembly of extrachromosomal vector genomes was accompanied by deletions in the integration locus that could be mapped to one of the five cAAV classes, suggesting an excision-type mechanism. We propose that the spontaneous deletion and mobilization of vector sequence from the recombinant adenoassociated virus (rAAV) integration locus is mediated by a recombination event between the inverted terminal repeats that define the boundaries of the individual genome subunits.     

A simplified cloning strategy for the generation of an endothelial cell selective recombinant adenovirus vector

Specifically targeting adenoviral vectors to particular cell/tissue types can be achieved by genetically modifying the adenovirus fiber protein. Two common strategies are: (1) directly modifying the fiber gene in the adenovirus genome and (2) in trans supply of the modified fiber. The former however, suffers from difficulties in directly manipulating large adenoviral genomic DNA. Although the latter allows easy manipulation of the small fiber gene, our studies show that the in trans supplement of the modified fiber causes incomplete fiber assimilation in the virus. Thus an alternate cloning strategy was devised to facilitate the insertion of cell-targeting sequences into the HI loop of a CAR binding-ablated fiber gene in the Ad5 genomic backbone. Our approach retains the advantage of easily modifying the fiber with the additional benefit of genetic re-insertion into the Ad genomic backbone to ensure complete modified fiber incorporation. Using this strategy, an endothelial cell binding peptide sequence (Asn-Gly-Arg) was introduced into the Ad fiber and showed that the generated Ad vector displayed selective transduction of endothelial cells both in vitro and in vivo compared to the conventional vector. Furthermore, this Ad vector cloning strategy can be adapted to introduce other peptide sequences to target other cell types.     

一组可提供AAV载体复制和包装功能的重组HSV-1

目的 构建一组具有AAV载体复制和包装功能的重组HSV-1，从中选出功能较强者用于重组AAV的生产。方法 采用一套含有HSV-1基因组的粘粒系统构建重组HSV-1。首先将2型腺病毒伴随病毒（AAV-2）rep基因的起始密码子ATG人工定点突变成ACG；然后将自身启动子控制下的AAV-2rep(起始密码子突变或未突变）和cap基因分别插入粘粒上HSV-1的UL2基因或UL44基因，构建成重组粘粒cos6-rmc/△UL2,cos56-rc/△UL44cos56-rmc/△UL44；将重组粘粒上的HSV-1片段分别与HSV-1的其余片段进行同源重组，得到3株重组HSV-1，连同过去报道的一株重组HSV-1一起，分别命名为HSV1-rc/△UL2,HSV1-rmc/△UL2,HSV1-rc/△UL44,HSV1-rmc/△UL44。结果 4株重组HSV-1经PCR鉴定均含有rep基因，分别感染携带绿色荧光蛋白（GFP）基因的AAV载体细胞株后均能产生重组AAV，重组AAV可在BHK-21细胞中表达GFP，HSV1-rc/△UL2和HSV1-rmc/△UL2对AAV载体的包装能力明显强于HSV1-rc/△UL44和HSV1-rmc/△UL44。结论 构建的4株重组HSV-1均具有复制和包装重组AAV的能力，其中HSV1-rc/△UL2和HSV1-rmc/△UL2功能较强，有望用于重组AAV的大规模生产。     

Promoter strength in adenovirus transducing vectors: down-regulation of the adenovirus E1A promoter in 293 cells facilitates vector construction

Most adenovirus transducing vectors have the cytomegalovirus major immediate-early (CMV) or the Rous sarcoma virus long terminal repeat (RSV) promoter driving expression of the transgene. Both of these promoters are highly active in transfection and transduction assays in 293 cells, in which transducing vectors are constructed and grown, and in HeLa cells. The CMV promoter exhibits rapid activation while the RSV promoter exhibits a lag prior to the onset of viral DNA replication in transduction assays. While the use of very strong promoters facilitates expression of the transgene, high-level expression of certain gene products hinders virus construction and growth. For such genes, the use of the adenovirus type 5 E1A promoter offers advantages. The E1A promoter exhibits modest activity in HeLa cells after transfection or transduction, but very little activity in 293 cells, suggesting that the E1A promoter would permit construction and growth of vectors encoding deleterious gene products that could not be constructed with the CMV and RSV promoters. This idea was tested through attempts to construct viruses encoding the immunoglobulin loop 6 and transmembrane regions of the prostaglandin F2alpha receptor regulatory protein (FPRP), a product that inhibits adenovirus vector construction for reasons that are not clear. Only the E1A promoter permitted construction and growth of the transducing vector encoding the fragment of FPRP.     

细菌内同源重组高效制备含绿色荧光蛋白和抗凋亡基因bcl-XL的重组腺病毒载体

目的:制备含绿色荧光蛋白和抗凋亡基因bcl-X_L的重组腺病毒载体。方法:采用PCR方法从质粒pEGFP-C3-bcl-X_L中扩增出bcl-X_L基因,再亚克隆至腺病毒穿梭质粒中,形成转移质粒pAdTrack-CMV-bcl-X_L;然后与pAdEasy-1共转化BJ5183菌,采用细菌内同源重组法构建重组腺病毒质粒pAdEasy-1-gfp-bcl-X_L,筛选同源重组的阳性克隆;抽提的pAdEasy-1-gfp-bcl-X_L经PacI线性化后,再用LIPOFECTAMINE^TM2000介导转染至人胚肾293细胞内包装扩增出重组腺病毒颗粒;采用PCR方法对重组腺病毒进行鉴定;用氯化铯超高速梯度离心纯化获取高滴度的重组腺病毒rAd-gfp-bcl-X_L。结果:由pAdTrack-CMV-bcl-X_L和pAdEasy-1共转化BJ5183菌16-20h后,可获得35%的阳性重组体细菌克隆。由重组质粒DNA经293细胞包装后产生的重组腺病毒,经PCR检测表明含有目的基因bcl-X_L。氯化铯纯化所得重组腺病毒滴度约为65×10¹²PFU/L。结论:用细菌内同源重组法可以高效、简便、快捷地制备出重组腺病毒质粒载体,重组体病毒质粒经293细胞包装、扩增和氯化铯纯化后可制备出高滴度的重组腺病毒颗粒rAd-gfp-bcl-X_L,为人类相关疾病的基因治疗提供良好的基因转染载体。     

Recombinant adenovirus vector vaccine induces stronger cytotoxic T-cell responses than recombinant vaccinia virus vector, plasmid DNA, or a combination of these

The efficiency of prime-boost vaccinations on the induction of T-cell responses to Sin Nombre virus nucleocapsid protein expressed by recombinant vaccinia virus, replication-deficient adenovirus, and plasmid DNA in mice was quantitated by the number of epitope-specific interferon-gamma-producing T cells and cytotoxic T-lymphocyte activity induced. In prime-boost immunizations, all combinations that included the recombinant adenovirus induced a much higher number of epitope-specific interferon-gamma-producing T cells than did other combinations. A single immunization of the recombinant adenovirus was able to induce similarly high levels of epitope-specific interferon-gamma-producing cells, despite the fact that the recombinant adenovirus produces less amount of the Sin Nombre virus nucleocapsid protein.     

Differential amplification of adenovirus vectors by flanking the packaging signal with attB/attP-PhiC31 sequences: implications for helper-dependent adenovirus production

Current strategies to amplify helper-dependent adenovirus, based on excision of the packaging signal, do not routinely reduce helper adenovirus contamination below 1%. Here, we have tested if reducing the efficiency of the packaging process of the helper adenovirus could impair its packaging without affecting helper-dependent adenovirus production. Interestingly, insertion of attB/attP-PhiC31 sequences flanking the packaging signal significantly lengthens adenovirus cycle up to 60 h without reducing virus viability or production yield. This delay occurs in the absence of PhiC31 recombinase indicating that other mechanisms different from excision of packaging signal must be involved. In addition, at 36 h post-coinfection helper-dependent adenovirus are efficiently produced, while production levels of helper attB/attP-modified adenovirus are 100-1000 times lower than controls. Therefore, these results suggest that attB/attP-mediated packaging impairment of the adenovirus genome is an attractive strategy to significantly reduce helper adenovirus contamination in helper-dependent adenovirus preparations, which in turn would facilitate scaling-up processes for clinical grade preparations.     

CTL epitopes identified with a defective recombinant adenovirus expressing measles virus nucleoprotein and evaluation of their protective capacity in mice

Cytotoxic T-lymphocyte (CTL) responses to measles virus (MV) play an important role in recovery from infection, with one of the major target proteins for CTL activity being the nucleoprotein (Np). In this report, a replication-deficient adenovirus-5 recombinant, expressing for MV Np (Rad68) was tested for in vivo priming of MV Np-specific CTL responses in BALB/c and CBA mice. In both strains of mice strong Np-specific CTL responses were induced and these responses were shown to be MHC class I restricted. Using overlapping 15mer peptides spanning residues 1-505 of MV Np a single epitope comprising residues 281-295 was identified in BALB/c mice whereas, in CBA mice two epitopes comprising residues 51-65 and 81-95, were identified. These epitopes were found to contain class I motifs for H-2L(d) and H-2K(k) MHC molecules, respectively. Immunization of BALB/c and CBA mice with the respective CTL epitopes resulted in the in vivo induction of peptide-and MV Np-specific CTL responses. In addition, the identified H-2K(k) restricted CTL epitopes conferred some protection against encephalitis induced following intracerebral challenge with a lethal dose of canine distemper virus (the Np of which shares 70% sequence homology with MV Np). These findings highlight the potential of using well-defined CTL epitopes to control virus infection.     

人微小RNA-133a(miR-133a)重组腺病毒的构建和鉴定

目的: 构建人miR-133a重组腺病毒,研究其在人骨髓间充质干细胞(hMSCs)中的表达。方法: 从人基因组DNA中扩增包含miR-133a的PCR产物，并定向插入腺病毒穿梭载体pAdTrack-CMV。将经pmeⅠ线性化的重组穿梭载体与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183，通过同源重组获得重组腺病毒质粒rAd-mir-133a。将rAd-mir-133a在人胚肾293细胞（HEK293）中包装并扩增出miR-133a的重组腺病毒。将重组腺病毒感染hMSCs，利用RT-PCR和实时定量PCR分别检测初级miR-133a和成熟miR-133a的表达。结果: 限制性内切酶分析和DNA测序结果显示，重组腺病毒穿梭载体pAdTrack-CMV-miR-133a构建正确。在HEK293细胞中成功包装并扩增出重组腺病毒rAd-miR-133a。rAd-miR-133a感染的hMSCs中初级miR-133a转录子和成熟miR-133a表达显著增强。结论: 成功构建了人miR-133a 重组腺病毒，并在hMSCs中高效表达出miR-133a，为进行miR-133a的功能研究创造条件。     

人微小RNA-133a(miR-133a)重组腺病毒的构建和鉴定

目的:构建人miR-133a重组腺病毒,研究其在人骨髓间充质干细胞(hMSCs)中的表达。方法:从人基因组DNA中扩增包含miR-133a的PCR产物,并定向插入腺病毒穿梭载体pAdTrack-CMV。将经pmeⅠ线性化的重组穿梭载体与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,通过同源重组获得重组腺病毒质粒rAd-mir-133a。将rAd-mir-133a在人胚肾293细胞(HEK293)中包装并扩增出miR-133a的重组腺病毒。将重组腺病毒感染hMSCs,利用RT-PCR和实时定量PCR分别检测初级miR-133a和成熟miR-133a的表达。结果:限制性内切酶分析和DNA测序结果显示,重组腺病毒穿梭载体pAdTrack-CMV-miR-133a构建正确。在HEK293细胞中成功包装并扩增出重组腺病毒rAd-miR-133a。rAd-miR-133a感染的hMSCs中初级miR-133a转录子和成熟miR-133a表达显著增强。结论:成功构建了人miR-133a重组腺病毒,并在hMSCs中高效表达出miR-133a,为进行miR-133a的功能研究创造条件。     

Efficient cloning of hepatitis B virus DNA from single-stranded replicative intermediates and its application to S1 mapping of viral RNA in human liver tissue

An efficient method for cloning subgenomic fragments of the hepatitis B virus (HBV) was developed, utilizing its abundant single-stranded replicative intermediates. The total genomic DNA obtained from the liver tissue of patients with chronic HBV infection was treated by using the Klenow fragment of E. coli DNA polymerase I without adding any exogenous primers. Single-stranded replicative intermediates were efficiently converted to double-stranded linear DNA, one end of which terminated at (or near to) the direct repeat 1 (DR1) sequence of the HBV genome. By screening less than 1,000 recombinants from a DNA library after this treatment, we obtained a subgenomic HBV fragment of 2.0 kilobases. We then analysed HBV RNA in human liver tissue by S1 mapping. It was possible to map HBV RNA only when a DNA probe from the same tissue was used.     

Presence of viral replicative intermediates in the liver and serum of patients infected with hepatitis C virus

Hemophilic patients may present immunological dysfunctions resulting from either human immunodeficiency virus (HIV) infection, or other factors like impure factor VIII concentrate and other viral infections. We evaluated prospectively the serologic response to polio vaccination of Israeli hemophilic patients who were vaccinated during an outbreak of poliomyelitis. Eighty-two hemophilic patients, 43 seronegative and 39 seropositive for human immunodeficiency virus (HIV), were vaccinated with enhanced inactivated poliovirus (elPV). Titers of antibodies for poliovirus types 1–3 were determined before and 4 weeks after immunization. T helper and suppressor lymphocytes (T4 and T8), B and T lymphocyte mitogenic response, and natural killer cells were tested and correlated with the response to vaccination. Both groups responded to vaccination with increased titers of antibodies to the three viral types, 4 weeks after immunization. HIV-seronegative patients, however, exhibited higher titers than the HIV-seropositive group. The same pattern was found when 21 patients were tested 1 year after the exposure to elPV. HIV seropositive patients were grouped according to their T4 count (between 16/μ and 500/μ). There was no statistically significant difference in the response of these different groups to vaccination. No correlation was found between the response to vaccination and other immune parameters. These results suggest that asymptomatic HIV-seropositive hemophilic patients respond well to elPV, irrespective of their T4 count. © 1993 Wiley-Liss, Inc.     

Cloning of the herpes simplex virus ICP4 gene in an adenovirus vector: effects on adenovirus gene expression and replication

To assess the ability of the herpes simplex virus ICP4 protein to complement adenovirus E1a mutants we have constructed an adenovirus type 5 vector containing a temperature-sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in cells which are permissive (293) or nonpermissive (KB and R970-5) for viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus-encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus; however, it is incapable of complementing adenovirus E1a mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. Our results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators.     

hPDGF-BB腺病毒表达载体的构建及其在表皮干细胞中的表达

目的构建hPDGE-BB(human platelet-derived growth factor-BB)腺病毒表达载体并观察其在人表皮干细胞(human epidermal stem cells,hESCs)中PDGF-BB蛋白的表达情况。方法从质粒pCMV-SPORT6上通过PCR扩增hPDGF-BB基因,将其酶切后连接到穿梭质粒pAd-track-CMV上,线性化后在BJ5183细菌中和骨架质粒pAdeasy-1进行同源重组,筛选阳性克隆并酶切鉴定,线性化后转染入HEK293细胞中进行包装、扩增,得到重组腺病毒rAD-PDGF。原代培养hESCs,流式细胞术分析hESCs的纯度,用收集的腺病毒感染靶细胞hESCs后,Westornblot检测其表达PDGF-BB情况。结果成功使重组腺病毒载体rAD-PDGF携带PDGF-BB基因,流式细胞术检测hESCs的纯度达88%以上,rAD-PDGF成功导入hESCs后,Westorn blot检测hESCs能表达PDGF-BB蛋白。结论成功构建的重组腺病毒rAD-PDGF在hESCs中表达PDGF-BB蛋白。