Use of a chimeric adenovirus vector enhances BMP2 production and bone formation

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.     

Efficient gene delivery in human and rodent mast cells using adenovirus vectors

Mast cells (MCs) have been shown to play an important role in immunoglobulin E (IgE)-associated immediate hypersensitivity and innate immunity by producing a variety of lipid mediators and cytokines. An efficient gene-delivery system is indispensable for elucidation of these mechanisms. In the present study, human and rodent MCs were transduced with various types of modified adenovirus (Ad) vectors. Fiber modification in Ad vectors significantly improved the transduction efficiencies in MCs. A fiber-substituted Ad serotype 5 (Ad5) vector containing Ad serotype 35 (Ad35) fiber proteins (Ad5F35) and an Ad35 vector, both of which transduce cells via human CD46, mediated 9.9-fold and 10.1-fold higher transduction efficiencies than conventional Ad5 vectors in the human mast cell line LAD2 among the Ad vectors. Ad5F35 and Ad35 vectors also efficiently transduced bone marrow-derived MCs (BMMCs) prepared from human CD46-transgenic (CD46TG) mice. The rat mast cell line RBL-2H3 were most efficiently transduced with a fiber-mutant Ad5 vector containing the Arg-Gly-Asp (RGD) peptide in the HI loop (Ad-RGD) of the fiber knob. Transduction with the Ad vectors did not induce degranulation or inflammatory cytokine production in the MCs. These results indicate that Ad vectors, including fiber-mutant Ad vectors, are effective gene-delivery tools for MCs.     

Comparison of the efficiency of transduction of leukemic cells by fiber-modified adenoviruses

Efficient gene transfer with adenoviral type 5 (Ad5) vectors depends on the initial attachment of their fiber, which binds the coxsackie-adenovirus receptor (CAR), and their subsequent internalization, mediated by the interaction of viral penton base with target cell alphav integrins. We previously demonstrated that human leukemic cells lack these receptors and are therefore resistant to Ad5 transduction, limiting efforts to genetically modify these cells. Human leukemic blasts are, however, susceptible to transduction with an adenovector made CAR independent by substitution of a chimeric Ad5/35 fiber [Yotnda et al. (2001). Gene Ther. 8, 930-937]. Other receptors can also be targeted with recombinant ligand moieties incorporated into adenovirus fiber. We have determined which of these fiber-modified adenovectors is most effective at modifying human primary leukemia cells, and lines. We used a replication-incompetent Ad5-beta-gal vector, in which the Ad5 fiber was replaced with fiber from various adenovirus serotypes (Ad35 and Ad11), or modified either with variable length polylysine (K4, K7, K21) or RGD-4C peptide. All the modified fiber vectors transduced primary leukemia cells and cell lines more efficiently than Ad5. Polylysine-substituted Ad5F/K21 and peptide-modified Ad5F/RGD vectors were most effective overall (up to 100% efficiency), whereas Ad5F/RGD was the most effective at transducing B cell acute lymphoblastic leukemia cells (90% efficiency). Ad5F/K21 and Ad5F/RGD should be of value for the genetic modification of human primary leukemia cells.     

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector

Efficient gene transfer into human hematopoietic stem cells (HSCs) is the most important requirement for gene therapy of hematopoietic disorders and for study of the hematopoietic system. An adenovirus (Ad) vector based on the Ad serotype 5 (Ad5) is known to transduce HSCs, including CD34(+) cells, with very low efficiency because of low-level expression of its primary receptor, coxsackievirus and adenovirus receptor (CAR). In the present study, we developed a recombinant Ad vector composed of the whole Ad serotype 35 (Ad35), which recognizes an unidentified receptor different from CAR for its infection. A transduction study showed that the Ad35-based vectors exhibit a higher transduction efficiency in human CD34(+) cells than the conventional Ad5 vectors and the Ad5F35 vectors, which are fiber-substituted Ad5 vectors containing Ad35 fiber proteins. The mean of fluorescence intensity in the CD34(+) cells transduced with the Ad35 vectors was 12-76 and 1.4-3 times higher than that in the cells transduced with the Ad5 and Ad5F35 vectors, respectively. The percentages of green fluorescent protein (GFP)-positive CD34(+) cells by transduction with Ad35, Ad5, and Ad5F35 vectors expressing GFP at 300 PFU/cell were 53%, 5%, and 52%, respectively, suggesting that Ad35 vectors mediate a more efficient gene transfer into human CD34(+) cells than Ad5 and Ad5F35 vectors, although the percentage of transduced cells was similar between Ad35 and Ad5F35 vectors. The Ad vector based on Ad35 could be very useful in gene therapy for blood disorders and gene transfer experiments using HSCs.     

Novel compound enables high-level adenovirus transduction in the absence of an adenovirus-specific receptor

Viral vectors are extensively used to deliver foreign DNA to cells for applications ranging from basic research to potential clinical therapies. A limiting step in this process is virus uptake and internalization into the target cells, which is mediated by membrane receptors. Although it is possible to modify viral capsid proteins to target the viruses, such procedures are complex and often unsuccessful. Here we present a rapid, inexpensive system for improving transduction of cells, including those that lack receptors for adenovirus fiber proteins. Addition of GeneJammer (Stratagene, La Jolla, CA) during the adenovirus transduction led to a significant increase in both the total number of transduced cells and the level of transgene expression per cell. Studies using cell lines deficient in adenovirus receptors demonstrated that addition of GeneJammer provided a novel cellular entry mechanism for the virus. These findings were tested in a cell-based gene therapy system for the induction of bone, which is contingent on high-level expression of the transgene. Inclusion of GeneJammer in either Ad5BMP2 or Ad5F35BMP2 transduction of a variety of cells demonstrated a correlating increase in bone formation. The results suggest a novel and versatile method for achieving high-level transduction using adenovirus.     

Efficient and rapid osteoinduction in an immune-competent host

Osteoinductive systems to induce targeted rapid bone formation hold clinical promise, but development of technologies for clinical use that must be tested in animal models is often a difficult challenge. We previously demonstrated that implantation of human cells transduced with Ad5F35BMP2 to express high levels of bone morphogenetic protein-2 (BMP2) resulted in rapid bone formation at targeted sites. Inclusion of human cells in this model precluded us from testing this system in an immune-competent animal model, thus limiting information about the efficacy of this approach. Here, for the first time we demonstrate the similarity between BMP2-induced endochondral bone formation in a system using human cells in an immune-incompetent mouse and a murine cell-based BMP2 gene therapy system in immune-competent animals. In both cases the delivery cells are rapidly cleared, within 5 days, and in neither case do they appear to contribute to any of the structures forming in the tissues. Endochondral bone formation progressed through a highly ordered series of stages that were both morphologically and temporally indistinguishable between the two models. Even longterm analysis of the heterotopic bone demonstrated similar bone volumes and the eventual remodeling to form similar structures. The results suggest that the ability of BMP2 to rapidly induce bone formation overrides contributions from either immune status or the nature of delivery cells.     

Evaluation of twenty human adenoviral types and one infectivity-enhanced adenovirus for the therapy of soft tissue sarcoma

The clinical course of sarcoma warrants the development of new therapeutic options, such as gene therapy. However, the lack of coxsackievirus-adenovirus receptor (CAR) on sarcoma cells limits the efficacy of adenovirus type 5 (Ad5)-based gene therapy. In this study we evaluated 20 different adenoviral types and 1 Ad5 vector with RGD-containing fiber for their internalization efficiency in sarcoma cells. We demonstrated that adenovirus types 35, 3, 7, 11, 9, and 22 and Ad5lucRGD virions (ranked in descending order) have significantly higher internalization efficiency in the tested sarcoma cells when compared with Ad5. On the basis of these results we developed a conditionally replication-competent adenoviral vector, Ad5Delta24.Ki.COX, and compared its oncolytic efficacy with that of Ad5/35Delta24.Ki.COX, an Ad5-based vector with the Ad35 fiber shaft and knob domains. Because both vectors differed only in the fiber, we were able to assess whether the adenoviral type with the most efficient internalization resulted also in enhanced treatment efficacy. We evaluated the antineoplastic activity of the oncolytic adenoviral vectors alone or in combination with the expression of measles virus fusogenic membrane glycoproteins and/or ifosfamide. The findings of our xenograft model were as follows: animals that received Ad5/35-based therapy had significantly smaller tumors than animals treated with the homologous Ad5-based vectors. In addition, we demonstrated that the combination of virotherapy, intratumoral expression of fusogenic membrane glycoproteins, and ifosfamide was clearly superior compared with treatment with individual components alone or as combinations of two components. In conclusion, Ad35-based vectors are promising for the treatment of sarcoma.     

Rapid clearance of syngeneic transplanted hepatocytes following transduction with E-1-deleted adenovirus indicates early host immune responses and offers novel ways for studying viral vector, target cell and host interactions.

To distinguish between transduced cell clearance and transgene regulation following adenoviral gene transfer, we infected F344 rat hepatocytes with an E-1-deleted adenovirus (Ad beta gal) and studied cell survival in the liver of dipeptidyl peptidase IV-deficient (DPPIV-) F344 rats. Transplanted cells were localized with histochemical staining for DPPIV and transgene expression localized with staining for beta-galactosidase (lacZ). The transgene was expressed in 90-100% hepatocytes without impairment in cell viability in vitro, although transplanted cells were cleared mostly within 1 day by infiltrates containing activated macrophages, CD4+ or CD8+ lymphocytes, and phagocytes. When Ad beta gal-transduced hepatocytes were transplanted repeatedly at 14-day intervals, transplanted cells were cleared rapidly each time. LacZ expression following Ad beta gal administration to intact animals was associated with apoptosis and unscheduled DNA synthesis in the liver. To determine whether adenoviral antigen expression activated consequential MHC-restricted liver injury, we transplanted Ad beta gal-hepatocytes followed subsequently by transplantation of nontransduced hepatocytes. Transplanted cells expressing Ad beta gal were rapidly cleared as before, whereas nontransduced hepatocytes engrafted with progressive liver repopulation. The findings indicated that adenovirally transduced cells are cleared early in the host liver. Use of ex vivo strategies will facilitate analysis of modified adenoviral vectors in the context of immunoregulatory, cellular and viral mechanisms.     

Human adenovirus type 35: nucleotide sequence and vector development

In this report, we describe the complete 34,794 base pair genomic sequence of the human adenovirus serotype 35 (Ad35) Holden strain. The viral genome exhibits a compact organization similar to other adenoviral serotypes, with overlapping genes on both strands. In all, 47 open reading frames (ORFs) were identified, including early (E1, 2, 3, 4) and late (L1, 2, 3, 4, 5) regions conserved among the adenoviridae family. In addition, 14 ORFs were identified that do not encode known adenoviral genes. Comparison of the predicted translational products of the conserved genes with those of other adenoviruses revealed that Ad35 has high homology to Ad7, Ad3, Ad21, Ad17, and simian Ads25. Based on the complete Ad35 DNA sequence, E3-, E1-, and E1/E3-deleted Ad35-based vector systems were developed. An HEK293-derived cell line was established for the propagation of the E1-deleted Ad35 vector, avoiding the emergence of replication-competent adenovirus. Moreover, production of the E1-deleted recombinant Ad35 vector was achieved by transient transduction of a plasmid encoding the Ad35 E1B gene in HEK293 cells. Testing showed that the Ad35-based vector efficiently infects both human and rhesus macaque dendritic cells. Our novel Ad35-based vectors and their corresponding packaging cell lines will provide a versatile and powerful system for DNA-based vaccine development and gene therapy applications.     

腺病毒介导的BMP-2基因转染骨髓基质干细胞及种植异种骨支架的体外观察

目的　用人骨形态发生蛋白 2腺病毒表达载体 (Ad BMP 2 )转染的兔骨髓基质干细胞 (BMSC) ,种植BCB(去抗原牛松质骨 )支架体外构建组织工程骨。方法　蛋白印迹法检测转染后细胞BMP 2的表达 ,ALP活性检测分析基因转染对细胞分化的影响。然后将转染后细胞接种到BCB支架上 ,扫描电镜观察细胞贴附、生长状况。结果　转染后 ,BMSC表达BMP 2 ,ALP活性明显增高。扫描电镜见转染细胞分布均匀 ,伸展良好。结论　Ad BMP 2可高效转染BM SC ,且促进细胞分化。转染后细胞在BCB上生长良好 ,BMP 2基因治疗的组织工程骨构建成功     

Efficient infection of primitive hematopoietic stem cells by modified adenovirus.

Almost all studies of adenoviral vector-mediated gene transfer have made use of the adenovirus type 5 (Ad5). Unfortunately, Ad5 has been ineffective at infecting hematopoietic progenitor cells (HPC). Chimeric Ad5/F35 vectors that have been engineered to substitute the shorter-shafted fiber protein from Ad35 can efficiently infect committed hematopoietic cells and we now show highly effective gene transfer to primitive progenitor subsets. An Ad5GFP and Ad5/F35GFP vector was added to CD34(+) and CD34(-)lineage(-) (lin(-)) HPC. Only 5-20% of CD34(+) and CD34(-)lin(-) cells expressed GFP after Ad5 exposure. In contrast, with the Ad5/F35 vector, 30-70% of the CD34(+), 50-70% of the CD34(-)lin(-) and up to 60% of the CD38(-) HPC expressed GFP and there was little evident cellular toxicity. Because of these improved results, we also analyzed the ability of Ad5/F35 virus to infect the hoechst negative 'side population' (SP) of marrow cells, which appear to be among the very earliest multipotent HPC. Between 51% and 80% of marrow SP cells expressed GFP. The infected populations retained their ability to form colonies in two short-term culture systems, with no loss of viability. We also studied the transfer and expression of immunomodulatory genes, CD40L (cell surface expression) and interleukin-2 (secreted). Both were expressed at immunomodulatory levels for >5 days. The ability of Ad5/F35 to deliver transgenes to primitive HPC with high efficiency and low toxicity in the absence of growth factors provides an improved means of studying the consequences of transient gene expression in these cells.     

Fiber-modified adenovirus vectors containing the TAT peptide derived from HIV-1 in the fiber knob have efficient gene transfer activity

The interaction between viral capsid proteins and specific molecules exposed on the plasma membrane of the cells is involved in the viral tropism. A human adenovirus (Ad) belonging to subgroups A, C, D, E and F infects cells via the interaction between the fiber knob and the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Conventional human adenovirus type 5 (hAd5) vectors show efficient transduction in CAR-positive cells; in contrast, hAd5 vector application is limited by poor transduction into cells lacking CAR expression. In the present study, to broaden the tropism of hAd5 vectors, we generated hAd5 vectors containing the TAT peptide, which is a protein transduction domain derived from human immunodeficiency virus, in the HI loop of the fiber knob (Ad-TAT(HI)-L2) or the C-terminus of the fiber knob (Ad-TAT(C)-L2). In CAR-negative adherent cells, Ad-TAT(HI)-L2 and Ad-TAT(C)-L2 showed approximately 50- to 500-fold higher gene expression than the conventional hAd5 vector (Ad-L2). Ad-TAT(HI)-L2 was also more efficient than Ad-L2 in blood cell lines and in two types of primary cultured human vascular smooth muscle cells, which are almost refractory to Ad-L2. Furthermore, Ad-TAT(HI)-L2 was more efficient than other types of fiber-modified Ad vectors, which harbor an RGD (Arg-Gly-Asp) or a poly-lysine (KKKKKKK;K7) peptide in the HI loop or the C-terminus of the fiber knob, respectively. Ad-TAT(HI)-L2 efficiently transduced the organs in levels and patterns that were roughly similar to those of Ad-L2 after being systemically injected into mice. To the best of our knowledge, this study is the first report showing that hAd5 vectors containing the TAT peptide in the fiber knob could efficiently transduce cells independently of CAR. These Ad vectors should be useful for gene functional analysis and gene therapy.     

Mesenchymal stem cell-mediated gene delivery of bone morphogenetic protein-2 in an articular fracture model.

In articular fractures, both bone and cartilage are injured. We tested whether stem cells transduced with bone morphogenetic protein 2 (BMP2) can promote bone and cartilage repair. Distal femoral articular osteotomies in nude rats were treated with stem cells, either wild-type or transduced with an adenoviral (Ad) BMP2. Cells were delivered in alginate (ALG) carrier or by direct injection in saline solution. Gene expression of these cells at the osteotomy site was confirmed by in vivo imaging. At day 14, only the group that received AdBMP2 stem cells by direct injection showed completely healed osteotomies, while other groups remained unhealed (P < 0.0003). In ALG groups, bone healing was impeded by the development of a chondroid mass, most pronounced in the AdBMP2 ALG group (P < 0.002). We were successful in achieving repair of both bone and cartilage in vivousing direct stem cell injection. Our data suggests that BMP2 augmentation might be critically important in achieving this effect.     

Dendritic cell function after gene transfer with adenovirus-calcium phosphate co-precipitates.

Dendritic cells (DCs) are essential for initiating and directing antigen-specific T-cell responses. Genetic modification of DC is under study for cancer immunotherapy, vaccine development, and antigen-targeted immunosuppression. Adenovirus (Ad) type 5 (Ad5)-mediated gene transfer to mouse bone marrow DCs and human monocyte-derived DCs is inefficient because neither express the cognate high-affinity Ads receptor. We show that co-precipitating adenoviral vectors with calcium phosphate (CaPi) increased gene expression (2000-fold) and transduction efficiency (50-fold) in mouse DC, primarily owing to receptor-independent viral uptake. Moreover, Ad5:CaPi-treated DCs were activated to express the maturation surface molecules CD40 and CD86, and to secrete proinflammatory cytokines tumor necrosis factor-alpha and interleukin 6. However, neither DC transduction nor maturation was dependent on viral protein interactions with cell surface integrin. Ad5:CaPi also transduced human DC more efficiently than Ad5 alone, similar to a genetically modified vector (Ad5f35) targeted to the CD46 receptor. As such, this approach combines the efficiency of adenoviral-mediated endosomal escape and nuclear trafficking with the receptor independence of nonviral gene delivery. Importantly, CaPi co-precipitation could be used to functionally modify DC to activate and expand cytomegalovirus-specific memory cytotoxic T lymphocytes. This study identifies a simple technique to improve the efficacy of current Ad5 gene transfer, in support of clinical adoptive immunotherapy.     

不同重组病毒载体转染大鼠骨间充质干细胞的效率及其基因表达

背景:利用基因工程细胞进行替代治疗和基因治疗中合适的供体细胞、靶细胞以及相应载体是细胞和基因治疗最为困难的部分.目的:检测重组腺病毒Ad5和Ad5F35、腺相关病毒rAAV1/2和rAAV2、慢病毒LV对大鼠骨髓间充质干细胞的感染效率和外源基因表达水平,拟筛选能够高效转导骨髓间充质干细胞的载体.设计、时间及地点:细胞基因工程对照观察,于2006-10/2007-03在上海市第一人民医院中心实验室完成.材料:清洁级SD大鼠10只用于制备骨髓间充质干细胞.所有重组病毒均携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因,AdS由本实验室制备,Ad5F35由解放军第二军医大学钱其军教授惠赠,rAAV2及rAAV1/2为本元正阳基因技术有限公司产品,LV由中科院上海生化与细胞生物学研究所郭礼和教授惠赠.方法:采用淋巴细胞分离液密度梯度离心法体外分离培养大鼠骨髓间充质干细胞,传至第4代用于实验.按1×105/孔接种于24孔细胞培养板中,1 d后细胞贴壁,设立5组:第1组向细胞培养液中加入10,100,1 000 MOI Ad5-EGFP,第2组加入10,100,1 000 MOIAdSF35-EGFP,第3组加入1×104,1×105vg rAAV1/2-EGFP,第4组加入1×104,1×105vg rAAV2-EGFP,第5组加入30 TU LV-EGFP.Ad病毒感染2d,rAAV及LV病毒感染6d.主要观察指标:EGFP阳性表达及荧光强度.结果:Ad3-EGFP感染24 h后镜下可见EGFP阳性细胞,随着病毒用量的增加EGFP阳性细胞数目增多,荧光亮度增强,12d后阳性细胞开始逐渐减少,荧光亮度减弱.AdSF35-EGFP感染情况与Ad5-EGFP基本相似,但EGFP阳性细胞数和荧光亮度均增加.rAAV1/2-EGFP或rAAV2-EGFP感染6 d后,EGFP阳性细胞数和荧光亮度均较弱.LV-EGFP感染第2天即可见少量EGFP阳性细胞,随着时间延长EGFP阳性细胞数目逐渐增多,至第6天表达EGFP荧光的细胞数量及亮度不再有明显变化.结论:腺病毒Ad5、AdSF35与慢病毒LV能够有效感染体外培养的骨髓间充质干细胞,并表达外源基因,感染效率与病毒用量之间存在量效关系.腺相关病毒rAAV1/2和rAAV2体外感染效果不佳.     

Adenovirus vectors based on human adenovirus type 19a have high potential for human muscle-directed gene therapy

Until recently, adenovirus-based gene therapy has been almost exclusively based on human adenovirus serotype 5 (Ad5). The aim of this study was to systematically compare the efficiency of transduction of primary muscle cells from various species by two adenoviral vectors from subgroups C and D. Transduction of a panel of myoblasts demonstrated a striking specificity of an Ad19a-based replication-defective E1-deleted vector (Ad19aEGFP) for human cells, whereas the Ad5-based vector had high affinity for nonhuman primate myoblasts. Transgene expression correlated well with cell-associated vector genomes. Up to 6.59% of the initially applied Ad19aEGFP vector particles were taken up by human myoblasts, as compared with 0.1% of the corresponding Ad5 vector. Remarkably, Ad19aEGFP but not Ad5EGFP efficiently transduced differentiated human myotubes, an in vitro model for skeletal muscle transduction. Uptake of Ad19aEGFP vector particles in human myotubes was 12-fold more efficient than that of Ad5EGFP. Moreover, both vectors demonstrated an early block at the level of vector uptake in mouse myoblasts and rat L6 cells. Investigation of the underlying mechanism for binding and uptake of the two vectors by human myoblasts showed high susceptibility for Ad19a to neuraminidase and wheat germ agglutinin (WGA) lectin, whereas Ad5-mediated transduction was dependent on binding to the coxsackie-adenovirus receptor (CAR) and sensitive to soluble RGD peptide and heparin. Our study offers insights into species-dependent factors that determine Ad tropism and, moreover, provides a basis for application of the novel Ad19a-based vector for gene transfer into human skeletal muscle.     

Adenovirus serotype 35 vector-mediated transduction into human CD46-transgenic mice

We previously demonstrated that systemic administration of adenovirus serotype 35 (Ad35) vectors to mice does not mediate efficient transduction in organs, probably because expression of the mouse analog of the subgroup B Ad receptor, human CD46 (membrane cofactor protein), is limited to the testis. Here, we describe the in vitro and in vivo transduction characteristics of Ad35 vectors by using homozygous and hemizygous human CD46-transgenic (CD46TG) mice, which ubiquitously express human CD46. An Ad35 vector more efficiently transduced the primary dendritic cells and macrophages prepared from CD46TG mice than those from wild-type mice. In vivo transduction experiments demonstrated that CD46TG mice are more susceptible to Ad35 vector-mediated in vivo transduction than are wild-type mice. In particular, homozygous CD46TG mice, which express higher levels of CD46 in the organs than hemizygous CD46TG mice, tend to exhibit higher transduction efficiencies after intraperitoneal administration than hemizygous CD46TG mice. Intraperitoneal administration of Ad35 vectors resulted in efficient transduction into the mesothelial cells of the peritoneal organs in homozygous CD46TG mice. These results indicate that an Ad35 vector recognizes human CD46 as a cellular receptor in CD46TG mice. However, the in vivo transduction efficiencies of Ad35 vectors in CD46TG mice are much lower than those of conventional Ad5 vectors in wild-type mice.     

Downregulation of human CD46 by adenovirus serotype 35 vectors

Human CD46 (membrane cofactor protein), which serves as a receptor for a variety of pathogens, including strains of measles virus, human herpesvirus type 6 and Neisseria, is rapidly downregulated from the cell surface following infection by these pathogens. Here, we report that replication-incompetent adenovirus (Ad) serotype 35 (Ad35) vectors, which belong to subgroup B and recognize human CD46 as a receptor, downregulate CD46 following infection. A decline in the surface expression of CD46 in human peripheral blood mononuclear cells was detectable 6 h after infection, and reached maximum (72%) 12 h after infection. Ad35 vector-induced downregulation of surface CD46 levels gradually recovered after the removal of Ad35 vectors, however, complete recovery of CD46 expression was not observed even at 96 h after removal. The surface expression of CD46 was also reduced after incubation with fiber-substituted Ad serotype 5 (Ad5) vectors bearing Ad35 fiber proteins, ultraviolet-irradiated Ad35, vectors and recombinant Ad35 fiber knob proteins; in contrast, conventional Ad5 vectors did not induce surface CD46 downregulation, suggesting that the fiber knob protein of Ad35 plays a crucial role in the downregulation of surface CD46 density. These results have important implications for gene therapy using CD46-utilizing Ad vectors and for the pathogenesis of Ads that interact with CD46.     

Transgene expression is increased by photochemically mediated transduction of polycation-complexed adenoviruses

Poor efficiency of adenoviral gene transfer to target cells is a major limitation to adenoviral gene therapy. Inefficient gene transfer occurs in the absence of coxsackie- and adenovirus receptor (CAR) on the cell surface, and can be overcome by enhancing viral entry with cationic molecules. Recombinant adenovirus (Ad) noncovalently complexed with polycations imply a lack of transduction specificity. Therefore, we have investigated the potential of a novel light-specific treatment, named photochemical internalization (PCI), to enhance gene delivery of adenovirus serotype 5 (Ad5) complexed with the cationic agents poly-L-lysine (PLL) and SuperFect trade mark. Cell lines differing in their receptiveness to Ad5 were infected with amounts of virus transducing about 2% of the cells by conventional Ad infection. The combination of polycations and photochemical treatment enabled a substantial increase in reporter gene expression, resulting in up to 75% positive cells. The effect was most prominent in cell lines expressing moderate to low levels of CAR. Furthermore, we show that PCI enables proper gene delivery of fiberless Ad5 at viral concentrations and infection times where transduction of photochemically untreated cells was negligible, both in the absence and presence of PLL. Thus, we conclude that the photochemically induced transduction by adenoviral vectors complexed with polycations present an opportunity to obtain high cell-infectivity levels with low viral doses, also without the fiber-CAR interaction.     

构建能转染人骨髓间充质干细胞的Ad5 GM-CSF-IL-2腺病毒载体

背景：如何将树突状细胞和T细胞活化因子运送至肿瘤局部是有待解决的难题之一。利用人骨髓问充质干细胞具有肿瘤趋向性和低免疫原性的特性,将树突状细胞和T细胞活化因子导入骨髓间充质干细胞后运送至肿瘤局部表达可能是解决上述难题的方案之一。目的：构建共表达粒细胞-巨噬细胞集落刺激因子和白细胞介素2的5型腺病毒载体（Ad5GM-CSF-IL-2）,感染人骨髓间充质干细胞,检测粒细胞一巨噬细胞集落刺激因子和白细胞介素2的表达水平和持续时间,为体内活化树突状细胞和T细胞提供实验依据。方法：提取人外周血单个核细胞总RNA,以此为模板用RT-PCR方法扩增粒细胞-巨噬细胞集落刺激因子和白细胞介素2基因cDNA,PCR扩增片段分别插入pcDNA3．1／Myc-His（-）B真核表达载体中,再利用脑心肌炎病毒内部核酸进入位点（IRES）序列,构建腺病毒载体穿梭质粒pDC515GM-CSF-IRES-IL-2。采用AdMax^TMFLP重组腺病毒载体体系,将穿梭质粒pDC515GM-CSF-IRES-IL-2与pBGHfrt△E1,3FLP骨架质粒共转染至293细胞,通过重组酶位点特异性重组获得Ad5GM-CSF-IL-2。Ad5GM-CSF-IL-2感染人骨髓间充质干细胞后经v射线照射使其失去增殖能力,分别用粒细胞-巨噬细胞集落刺激因子和白细胞介素2ELISA试剂盒在不同的时间点检测细胞上清中粒细胞-巨噬细胞集落刺激因子和白细胞介素2蛋白的水平。结果与结论：经测序证实,克隆获得的粒细胞-巨噬细胞集落刺激因子、白细胞介素2的cDNA序列分别与GenBank NM_000758（435bp）和GenBank NM_000586（462bp）提供的序列完全一致。用AdMax^TMFLP重组腺病毒载体可高效、快捷地获得所要包装的腺病毒载体,通过IRES连接粒细胞-巨噬细胞集落刺激因子和白细胞介素2两个基因,均获得高效表达。Ad5GM-CSF-IL-2感染人骨髓间充质干细胞后可连续7d高水平地分泌粒细胞-巨噬细胞集落刺激因子和白细胞介素2。