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1.  Key Role of Splenic Myeloid DCs in the IFN-αβ Response to Adenoviruses In Vivo 
PLoS Pathogens  2008;4(11):e1000208.
The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.
Author Summary
Adenoviruses (Ads) are important pathogens and promising vectors for gene therapy applications. In the course of adenoviral infections innate immune responses are activated, which can be beneficial for the antiviral host defense but also detrimental if activated in a deregulated manner. Type I IFNs are crucial for the innate immune control of various viral infections in the mammalian host. So far, the early, systemic release of IFN-αβ during viral infections has been attributed to specialized immune cells, the plasmacytoid dendritic cells. Here, in a mouse infection model, we show that wild type Ads, as well as adenoviral vectors, elicit rapid IFN-αβ production almost exclusively in another cell population, the splenic myeloid dendritic cells. This IFN-αβ storm depends on viral escape from endosomes to the cytosol and the requirements of the response are suggestive of a novel viral induction pathway. Furthermore, we show that virus induced IFN-αβ is the key mediator of Ad-induced hypersensitivity to the cytokine-inducing and toxic activity of lipopolysaccharide, a common constituent of Gram-negative bacteria. Since these bacteria comprise several commensals and pathogens, enhanced susceptibility to lipopolysaccharide may contribute to toxic reactions observed during adenoviral gene therapy and to the clinical symptoms of adenoviral diseases.
doi:10.1371/journal.ppat.1000208
PMCID: PMC2576454  PMID: 19008951
2.  A crucial role for tumor necrosis factor receptor 1 in synovial lining cells and the reticuloendothelial system in mediating experimental arthritis 
Introduction
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that mainly affects synovial joints. Biologics directed against tumor-necrosis-factor (TNF)-α are efficacious in the treatment of RA. However, the role of TNF receptor-1 (TNFR1) in mediating the TNFα effects in RA has not been elucidated and conflicting data exist in experimental arthritis models. The objective is to investigate the role of TNFR1 in the synovial lining cells (SLC) and the reticuloendothelial system (RES) during experimental arthritis.
Methods
Third generation of adenovirus serotype 5 were either injected locally in the knee joint cavity or systemically by intravenous injection into the retro-orbital venous sinus to specifically target SLC and RES, respectively. Transduction of organs was detected by immunohistochemistry of the eGFP transgene. An adenoviral vector containing a short hairpin (sh) RNA directed against TNFR1 (HpTNFR1) was constructed and functionally evaluated in vitro using a nuclear factor-kappaB (NF-κB) reporter assay and in vivo in streptococcal cell wall-induced arthritis (SCW) and collagen-induced arthritis (CIA). Adenoviruses were administered before onset of CIA, and the effect of TNFR1 targeting on the clinical development of arthritis, histology, quantitative polymerase chain reaction (qPCR), cytokine analyses and T-cell assays was evaluated.
Results
Systemic delivery of Ad5.CMV-eGFP predominantly transduced the RES in liver and spleen. Local delivery transduced the synovium and not the RES in liver, spleen and draining lymph nodes. In vitro, HpTNFR1 reduced the TNFR1 mRNA expression by three-fold resulting in a 70% reduction of TNFα-induced NF-κB activation. Local treatment with HpTNFR1 markedly reduced mRNA and protein levels of interleukin (IL)-1β and IL-6 in SLC during SCW arthritis and ameliorated CIA. Systemic targeting of TNFR1 in RES of liver and spleen by systemic delivery of Ad5 virus encoding for a small hairpin RNA against TNFR1 markedly ameliorated CIA and simultaneously reduced the mRNA expression of IL-1β, IL-6 and Saa1 (75%), in the liver and that of Th1/2/17-specific transcription factors T-bet, GATA-3 and RORγT in the spleen. Flow cytometry confirmed that HpTNFR1 reduced the numbers of interferon (IFN)γ (Th1)-, IL-4 (Th2)- and IL-17 (Th17)-producing cells in spleen.
Conclusions
TNFR1-mediated signaling in both synovial lining cells and the reticuloendothelial system independently played a major pro-inflammatory and immunoregulatory role in the development of experimental arthritis.
doi:10.1186/ar2974
PMCID: PMC2888212  PMID: 20370892
3.  Efficient Transduction of Vascular Endothelial Cells with Recombinant Adeno-Associated Virus Serotype 1 and 5 Vectors 
Human gene therapy  2005;16(2):235-247.
Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human α1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding β-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.
OVERVIEW SUMMARY
Gene delivery to the vasculature has significant potential as a therapeutic strategy for several cardiovascular disorders including atherosclerosis, hypertension, angiogenesis, and chronic vascular rejection of transplanted organs. However, limited advances have been made in achieving successful vascular endothelial cell gene transfer. The results of the present study demonstrate the superior efficacy of recombinant adeno-associated virus (rAAV) serotype 1 and 5 vectors in comparison to the traditionally used rAAV serotype 2 in transduction of primary vascular endothelial and smooth muscle cells in vitro. Our results have identified sialic acid residues for rAAV1 transduction in endothelial cells, similar to rAAV5. Transduction of rat aortic segments demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors both ex vivo and in vivo, while rAAV2 showed no significant transduction. These results suggest significant advantages of using alternative rAAV serotypes 1 and 5 for vascular-targeted gene delivery.
doi:10.1089/hum.2005.16.235
PMCID: PMC1364465  PMID: 15761263
4.  Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4 
Arthritis Research  2000;2(4):293-302.
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Introduction:
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
Objective:
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
Results:
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Discussion:
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
PMCID: PMC17812  PMID: 11056670
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
5.  Coronary restenosis and gene therapy. 
Texas Heart Institute Journal  1994;21(1):104-111.
Restenosis continues to limit the efficacy of coronary angioplasty, despite the various mechanical and pharmaceutical interventions that have been employed. The migration, proliferation, and extracellular matrix production by vascular smooth muscle cells are processes integral to restenosis, and sustained local delivery of drugs at high concentration should curtail these vascular responses to balloon angioplasty. Our laboratory and others are exploring the potential of using somatic cell gene therapy to provide such treatment and thereby prevent restenosis. However, conventional methods of gene transfer fail to produce physiologic levels of recombinant protein in vivo. This obstacle might be overcome by using adenoviral vectors to mediate efficient direct gene transfer. Herein we summarize these developments and focus upon our laboratory's progress towards evaluating adenovirus-mediated gene therapy in porcine coronary arteries. Recombinant adenoviruses directing the expression of the beta-galactosidase and luciferase reporter genes were evaluated in cultured coronary vascular smooth muscle cells in vitro and in porcine coronary arteries in vivo. Following percutaneous transluminal gene transfer in vivo, recombinant adenoviruses were shown to produce 70- to 240-fold more reporter protein than that produced by Lipofectin-DNA complexes. Furthermore, the high levels of adenovirus-mediated gene expression were shown to persist for at least 14 days following catheterization. Additional histologic studies will be required to determine the cellular distribution of gene expression and to elucidate potential interactions between adenovirus and the host's immune system, but recombinant adenovirus appears to be a promising vector for evaluating gene therapy against coronary restenosis.
PMCID: PMC325139  PMID: 8180504
6.  Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids 
PLoS ONE  2014;9(4):e93608.
Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.
doi:10.1371/journal.pone.0093608
PMCID: PMC3973564  PMID: 24695466
7.  Platelet-Derived Growth Factor (PDGF) Gene Delivery for Application in Periodontal Tissue Engineering 
Journal of periodontology  2001;72(6):815-823.
Background
A challenge in the reconstruction of periodontal structures is the targeted delivery of growth-promoting molecules to the tooth root surface. Polypeptide growth factors such as platelet-derived growth factor (PDGF) stimulate both cementogenesis and osteogenesis. Recent advances in gene therapy offer the advantage of delivering recombinant proteins to tissues for extended periods of time in vivo.
Methods
Recombinant adenoviral vectors encoding for the PDGF-A gene were constructed to allow delivery of PDGF transgenes to cells. The recombinant adenoviruses were assembled using the viral backbone of Ad2/CMV/EGFP and replacing GFP (reporter gene encoding green fluorescent protein driven by the cytomegalovirus promoter [CMV] within adenovirus type 2) with the PDGF-A gene. Root lining cells (cloned cementoblasts) were transduced with Ad2/PDGF-A and evaluated for gene expression, DNA synthesis, and cell proliferation. PDGF-inducible genes, c-myc and osteopontin, were also evaluated following gene delivery of Ad2/PDGF-A.
Results
The results revealed high level transduction of cementoblasts by gene transfer for 7 days as evidenced by flow cytometry and Northern blotting. Cementoblast DNA synthesis and subsequent proliferation were stimulated by Ad2/PDGF-A at levels equal to or greater than continuous rhPDGF-AA application. Strong message for the PDGF-A gene and protein as evidenced by Northern blotting and immunocytochemistry was noted. Furthermore, the potent induction of c-myc and osteopontin mRNA was found after PDGF gene delivery to cementoblasts.
Conclusions
These findings demonstrate that gene delivery of platelet-derived growth factor stimulates cementoblast activity that is sustained above that of rhPDGF-AA application. The use of gene therapy as a mode of growth factor delivery offers a novel approach to periodontal tissue engineering.
PMCID: PMC2602862  PMID: 11453245
Growth factors, platelet-derived; periodontium/growth; osteogenesis, dental cementum/growth and development; gene therapy
8.  Recombinant Adenoviral Vectors Can Induce Expression of p73 via the E4-orf6/7 Protein 
Journal of Virology  2006;80(11):5349-5360.
Despite the utility of recombinant adenoviral vectors in basic research, their therapeutic promise remains unfulfilled. Most engineered adenoviral vectors use a heterologous promoter to transcribe a foreign gene. We show that adenoviruses containing the cytomegalovirus immediate-early promoter induce the expression of the proapoptotic cellular protein TAp73 via the cyclin-dependent kinase-retinoblastoma protein-E2F pathway in murine embryonic fibroblasts. Cells transduced with these vectors also expressed high levels of the adenoviral E4-orf6/7 and E2A proteins. By contrast, adenoviruses containing the ubiquitin C promoter failed to elicit these effects. E4-orf6/7 is necessary and sufficient for increased TAp73 expression, as shown by using retrovirus-mediated E4-orf6/7 expression and adenovirus with the E4-orf6/7 gene deleted. Activation of TAp73 likely occurs via E4-orf6/7-induced dimerization of E2F and subsequent binding to the inverted E2F-responsive elements within the TAp73 promoter. In addition, adenoviral vectors containing the cytomegalovirus immediate-early promoter, but not the ubiquitin C promoter, cooperated with chemotherapeutic agents to decrease cellularity in vitro. In contrast to murine embryonic fibroblasts, adenoviruses containing the ubiquitin C promoter, but not the cytomegalovirus immediate-early promoter, induced both E4-orf6/7 and TAp73 in human foreskin fibroblasts, emphasizing the importance of cellular context for promoter-dependent effects. Because TAp73 is important for the efficacy of chemotherapy, adenoviruses that increase TAp73 expression may enhance cancer therapies by promoting apoptosis. However, such adenoviruses may impair the long-term survival of transduced cells during gene replacement therapies. Our findings reveal previously unknown effects of foreign promoters in recombinant adenoviral vectors and suggest means to improve the utility of engineered adenoviruses by better controlling their impact on viral and cellular gene expression.
doi:10.1128/JVI.02016-05
PMCID: PMC1472169  PMID: 16699015
9.  Gene Transfer into Rat Brain Using Adenoviral Vectors 
Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc.
doi:10.1002/0471142301.ns0424s50
PMCID: PMC2883311  PMID: 20066657
viral vectors; gene therapy; immune response; T cells; B cells; brain inflammation; macrophages; gene transfer; immunocytochemistry; qPCR; ELISPOT assay; adenovirus
10.  Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure 
Gut  1999;44(6):800-807.
BACKGROUND/AIMS—Replication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed.
METHODS—An E1/E3 deleted recombinant adenovirus (denoted AdCMVβGal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both expressing the bacterial lacZ gene under the control of a human cytomegalovirus promoter were used for reporter gene expression in vitro and in vivo. β-Galactosidase activity was determined by specific chemiluminescent reporter gene assay.
RESULTS—Intravenous or intraperitoneal injection of AdCMVβGal into healthy Balb/c mice caused strong reporter gene expression in the liver and spleen but not in the colon. In contrast, local administration of AdCMVβGal resulted in high reporter gene expression in colonic epithelial cells and lamina propria mononuclear cells. A local route of adenovirus administration in mice with experimental colitis induced by the hapten reagent trinitrobenzenesulphonic acid was next evaluated. Interestingly, rectal administration of AdCMVβGal caused a higher β-galactosidase activity in isolated lamina propria cells from infected mice with experimental colitis than in those from controls. Furthermore, isolated lamina propria cells from mice with colitis infected in vitro showed a significant increase in reporter gene activity compared with controls. Finally, AdZ.F(pk7) adenoviruses with modified fibre structure produced 10- to 40-fold higher reporter gene activity in spleen T cells and lamina propria mononuclear cells of colitic mice compared with standard AdCMVβGal vectors.
CONCLUSIONS—Local administration of recombinant adenoviruses with normal or modified fibre structure could provide a new reliable method for targeted gene expression in the inflamed colon. Such gene delivery could be used to specifically express signal transduction proteins with therapeutic potential in inflamed colonic tissue. In particular, adenoviruses with modified fibre structure may be useful in T cell directed therapies in intestinal inflammation.


Keywords: adenovirus; gene transfer; colitis; colon
PMCID: PMC1727540  PMID: 10323880
11.  In vitro evaluation of a double-stranded self-complementary adeno-associated virus type2 vector in bone marrow stromal cells for bone healing 
Background
Both adenoviral and lentiviral vectors have been successfully used to induce bone repair by over-expression of human bone morphogenetic protein 2 (BMP-2) in primary rat bone marrow stromal cells in pre-clinical models of ex vivo regional gene therapy. Despite being a very efficient means of gene delivery, there are potential safety concerns that may limit the adaptation of these viral vectors for clinical use in humans. Recombinant adeno-associated viral (rAAV) vector is a promising viral vector without known pathogenicity in humans and has the potential to be an effective gene delivery vehicle to enhance bone repair. In this study, we investigated gene transfer in rat and human bone marrow stromal cells in order to evaluate the effectiveness of the self-complementary AAV vector (scAAV) system, which has higher efficiency than the single-stranded AAV vector (ssAAV) due to its unique viral genome that bypasses the rate-limiting conversion step necessary in ssAAV.
Methods
Self-complementaryAAV2 encoding GFP and BMP-2 (scAAV2-GFP and scAAV2-BMP-2) were used to transduce human and rat bone marrow stromal cells in vitro, and subsequently the levels of GFP and BMP-2 expression were assessed 48 hours after treatment. In parallel experiments, adenoviral and lentiviral vector mediated over-expression of GFP and BMP-2 were used for comparison.
Results
Our results demonstrate that the scAAV2 is not capable of inducing significant transgene expression in human and rat bone marrow stromal cells, which may be associated with its unique tropism.
Conclusions
In developing ex vivo gene therapy regimens, the ability of a vector to induce the appropriate level of transgene expression needs to be evaluated for each cell type and vector used.
doi:10.1186/1479-0556-9-4
PMCID: PMC3056728  PMID: 21352585
12.  Adenoviral-mediated gene transfer to fetal pulmonary epithelia in vitro and in vivo. 
Journal of Clinical Investigation  1995;95(6):2620-2632.
Vector-mediated gene transfer offers a direct method of correcting genetic pulmonary diseases and might also be used to correct temporary abnormalities associated with acquired, nongenetic disorders. Because the fetus or newborn may be a more immune tolerant host for gene transfer using viral vectors, we used replication defective recombinant adenoviral vectors to test the feasibility of gene transfer to the fetal pulmonary epithelium in vitro and in vivo. Both proximal and distal epithelial cells in cultured fetal lung tissues from rodents and humans diffusely expressed the lacZ transgene 3 d after viral infection. In vivo gene delivery experiments were performed in fetal mice and lambs. Delivery of Ad2/CMV-beta Gal to the amniotic fluid in mice produced intense transgene expression in the fetal epidermis and amniotic membranes, some gastrointestinal expression, but no significant airway epithelial expression. When we introduced the adenoviral vector directly into the trachea of fetal lambs, the lacZ gene was expressed in the tracheal, bronchial, and distal pulmonary epithelial cells 3 d after viral infection. Unexpectedly, reactive hyperplasia and squamous metaplasia were noted in epithelia expressing lacZ in the trachea, but not in the distal lung of fetal lambs. 1 wk after infection, adenovirus-treated fetuses developed inflammatory cell infiltrates in the lung tissue with CD4, CD8, IgM, and granulocyte/macrophage positive immune effector cells. Transgene expression faded coincident with inflammation and serologic evidence of antiadenoviral antibody production. While these studies document the feasibility of viral-mediated gene transfer in the prenatal lung, they indicate that immunologic responses to E1-deleted recombinant adenoviruses limit the duration of transgene expression.
Images
PMCID: PMC295945  PMID: 7539457
13.  Protein Transduction Domains Fused to Virus Receptors Improve Cellular Virus Uptake and Enhance Oncolysis by Tumor-Specific Replicating Vectors 
Journal of Virology  2004;78(24):13743-13754.
Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CARex) to attach PTDs to adenoviral fiber knobs. CARex-Tat and CARex-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CARex-PTD-mediated infection using CARex and inhibition experiments with heparin showed that binding of CARex-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CARex-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CARex-PTD to adenoviral particles. Consequently, the mechanism of CARex-PTD-mediated infection involves coating of the viral fiber knobs by CARex-PTD, rather than placement of CARex domains on cell surfaces. Expression of CARex-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CARex-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CARex-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.
doi:10.1128/JVI.78.24.13743-13754.2004
PMCID: PMC533898  PMID: 15564483
14.  Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates 
PLoS Pathogens  2009;5(7):e1000503.
Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors.
Author Summary
Adenoviruses were originally discovered as a causative agent for upper respiratory infections, especially in children. They are now recognized as being responsible for disease in several other settings, e.g., as the cause of infectious diarrheas or of kerato-conjunctivitis. In the 1960s, chimpanzees were found to shed adenoviruses in their stools. We now find that normal great apes (chimpanzees, gorillas, and orangutans) and monkeys frequently shed adenoviruses in their stools, corroborating the older finding. In contrast, data from humans have showed that adenoviral shedding in stools is fairly uncommon, being restricted to patients recovering from adenoviral infections, although adenoviruses can frequently be recovered from lymphoid tissue such as the adenoids from normal people. In the course of these investigations we have purified and characterized 30 different adenoviruses from great apes by genomic sequencing. We have compared the gene sequences of these adenoviruses to 22 human adenoviruses whose complete genome sequences are available. Even though human adenoviruses are not found in apes and vice versa, based on these sequence data we propose that it is possible that cross-species transmission events may have contributed to the natural evolution of adenoviruses found to infect humans today.
doi:10.1371/journal.ppat.1000503
PMCID: PMC2698151  PMID: 19578438
15.  α-Galactosidase A Expressed in the Salivary Glands Partially Corrects Organ Biochemical Deficits in the Fabry Mouse Through Endocrine Trafficking 
Human Gene Therapy  2010;22(3):293-301.
Abstract
Fabry disease is caused by an X-linked deficiency of the lysosomal enzyme α-galactosidase A (GLA) and has been treated successfully with enzyme replacement therapy (ERT). Gene therapy has been proposed as an alternative to ERT due to the presumed advantages of continuous, endogenous production of the therapeutic enzyme. GLA production in the liver and its therapeutic efficacy in the Fabry mouse have been demonstrated previously with various viral vector systems. In consideration of the potential advantages of using the salivary glands as endogenous GLA biosynthesis sites, we explored the feasibility of this approach in the Fabry mouse. GLA −/0 or −/− mice received an adenoviral vector (2 × 1010 or 1 × 109 viral particles) expressing GLA to the right submandibular gland via oral cannulation of the submandibular duct. Four days later, animals were sacrificed; saliva, plasma, kidney, liver, and brain were collected and assayed using ELISA, Western blot, and a GLA enzymatic activity assay using both traditional fluorescence methods and isotope dilution mass spectrometry by following the U.S. EPA Method 6800. GLA activity was significantly elevated in the serum and liver of both treatment groups, and improvement in the kidney was marginally significant (P < 0.069) in the high-dose group. Notably, we found that liver and salivary gland produce different glycoforms of the GLA transgene. Only small numbers of adenoviral genomes were observed in the livers of treated animals, but in four of 14 in the high-dose groups, liver levels of adenovirus exceeded 20 copies/μg, indicating that the sequestration in the salivary gland was imperfect at high doses. Taken together, these results indicate that the salivary gland-based gene therapy for Fabry disease is promising, and further studies with advanced viral vector gene delivery systems (e.g., adeno-associated virus) for long-term treatment appear to be warranted.
Fabry disease is caused by an X-linked deficiency of the lysosomal enzyme α-galactosidase A (GLA) and has been treated with enzyme replacement therapy (ERT). Here, Passineau and colleagues report results from a gene therapy study in the GLA-knockout mouse intended to provide a proof-of-concept that the salivary glands might be targets for gene transfer to serve as a depot for delivery of enzyme in Fabry disease and possibly other lysosomal storage disorders.
doi:10.1089/hum.2010.069
PMCID: PMC3057212  PMID: 20858137
16.  A novel and simple method for construction of recombinant adenoviruses 
Nucleic Acids Research  2006;34(12):e89.
Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy™ adenoviral vector system. The recombinant adenoviral plasmid can be generated by a direct and seamless substitution, which replaces the stuff fragment in a full-length adenoviral genome with the gene of interest in a small plasmid in Escherichia coli. Recombinant adenoviral plasmids can be rapidly constructed in vivo by using the new method, without manipulations of the large adenoviral genome. In contrast to other traditional systems, it reduces the need for multiple in vitro manipulations, such as endonuclease cleavage, ligation and transformation, thus achieving a higher efficiency with negligible background. This strategy has been proven to be suitable for constructing an adenoviral cDNA expression library. In summary, the new method is highly efficient, technically less demanding and less labor-intensive for constructing recombinant adenoviruses, which will be beneficial for functional genomic and proteomic researches in mammalian cells.
doi:10.1093/nar/gkl449
PMCID: PMC1524918  PMID: 16855284
17.  Retargeting Adenoviral Vectors to Improve Gene Transfer into Tumors 
Cancer gene therapy  2010;18(4):275-287.
Gene targeting to tumors using adenoviral vectors holds great potential for cancer imaging and therapy, but the limited efficacy of current methods used to improve delivery to target tissues and reduce unwanted interactions remain substantial barriers to further development. Progress in characterizing the set of molecular interactions used by adenoviral vectors to infect particular tissues has aided the development of novel strategies for retargeting vectors to tumor cells. One method is chemical retargeting of adenovirus using bispecific antibodies against both viral capsid proteins and tumor-specific cell surface molecules. This approach can be combined either with competitive inhibitors designed to reduce viral tropism in undesired tissues, or with traditional therapeutics to increase the expression of surface molecules for improved tumor targeting. Ablating liver cell-specific interactions through mutation of capsid proteins or chemical means are promising strategies for reducing adenovirus-induced liver toxicity. The nature of tumor neovasculature also influences adenoviral delivery, and the use of vascular disrupting agents such as combretastatin can help elucidate these contributions. In this investigation, we evaluate a variety of these methods for retargeting adenoviral vectors to tumor cells in vitro and in vivo, and assess the contributions of specific molecular and tissue interactions that affect adenoviral transgene delivery.
doi:10.1038/cgt.2010.78
PMCID: PMC3060954  PMID: 21183946
adenovirus; Bavituximab; bispecific antibody; fiber; targeting
18.  Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy 
Human Gene Therapy  2014;25(11):929-941.
Abstract
Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.
doi:10.1089/hum.2014.041
PMCID: PMC4236031  PMID: 25072415
19.  Inflammation and Immune Response of Intra-Articular Serotype 2 Adeno-Associated Virus or Adenovirus Vectors in a Large Animal Model 
Arthritis  2012;2012:735472.
Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad), serotype 2 adeno-associated virus vectors (rAAV2), or self-complementary (sc) AAV2 vectors carrying green fluorescent protein (GFP). Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb) titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.
doi:10.1155/2012/735472
PMCID: PMC3263587  PMID: 22288012
20.  Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System 
PLoS ONE  2008;3(8):e3084.
A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes).
doi:10.1371/journal.pone.0003084
PMCID: PMC2518115  PMID: 18769728
21.  Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes 
Journal of Virology  1999;73(11):9314-9324.
Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus–adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (ΔAd.AAV) and stimulate transgene integration. We demonstrate here that ΔAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. ΔAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. ΔAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The ΔAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with ΔAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that ΔAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. ΔAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.
PMCID: PMC112966  PMID: 10516040
22.  Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5 
PLoS ONE  2010;5(9):e13103.
Background
Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important.
Methods/Results
We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly.
Conclusion
In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline.
doi:10.1371/journal.pone.0013103
PMCID: PMC2948038  PMID: 20927353
23.  Helper-dependent adenoviral vectors in experimental gene therapy* 
Acta biochimica Polonica  2005;52(3):589-599.
In the majority of potential applications gene therapy will require an effective transfer of a transgene in vivo resulting in high-level and long-term transgene expression, all in the absence of significant toxicity or inflammatory responses. The most efficient vehicles for delivery of foreign genes to the target tissues are modified adenoviruses. Adenoviral vectors of the first generation, despite the high infection efficacy, have an essential drawback: they induce strong immune response, which leads to short term expression of the transgene, and limits their usefulness in clinical trials. In contrast, helper-dependent adenoviral vectors (HdAd) lacking all viral coding sequences display only minimal immunogenicity and negligible side-effects, allowing for long-term transgene expression. Thus, HdAd vehicles have become the carrier of choice for adenoviral vector-mediated experimental gene therapy, effectively used in animal models for delivery of transgenes into the liver, skeletal muscle, myocardium or brain. Strong and long-lasting expression of therapeutic genes has allowed for successful treatment of dyslipidemias, muscular dystrophy, obesity, hemophilia, and diabetes. Additionally, the large cloning capacity of HdAd, up to 37 kb, facilitates the use of physiologically regulated, endogenous promoters, instead of artificial viral promoter sequences. This enables also generation of the single vectors expressing multiple genes, which can be potentially useful for treatment of polygenic diseases. In this review we characterize the basic features of HdAd vectors and describe some of their experimental and potential clinical applications.
PMCID: PMC1383728  PMID: 16082408
gene therapy; adenoviruses; helper-dependent adenoviral vectors; AAV, adeno-associated viruses; apo, apolipoprotein; CTL, cytotoxic T lymphocyte; DMD, Duchenne muscular dystrophy; HdAd, helper-dependent adenoviral vectors; ITR, inverted terminal repeat
24.  Innate Immune Response to Adenoviral Vectors Is Mediated by both Toll-Like Receptor-Dependent and -Independent Pathways▿  
Journal of Virology  2007;81(7):3170-3180.
Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.
doi:10.1128/JVI.02192-06
PMCID: PMC1866082  PMID: 17229689
25.  Efficient generation of double heterologous promoter controlled oncolytic adenovirus vectors by a single homologous recombination step in Escherichia coli 
BMC Biotechnology  2006;6:36.
Background
Oncolytic adenoviruses are promising agents for the multimodal treatment of cancer. However, tumor-selectivity is crucial for their applicability in patients. Recent studies by several groups demonstrated that oncolytic adenoviruses with tumor-/tissue-specific expression of the E1 and E4 genes, which are pivotal for adenoviral replication, have a specificity profile that is superior to viruses that solely target the expression of E1 or E4 genes. Presently the E1 and E4 regions are modified in a time consuming sequential fashion.
Results
Based on the widely used adenoviral cloning system AdEasy we generated a novel transfer vector that allows efficient and rapid generation of conditionally replication-competent adenovirus type 5 based vectors with the viral E1 and E4 genes under the transcriptional control of heterologous promoters. For insertion of the promoters of interest our transfer vector has two unique multiple cloning sites. Additionally, our shuttle plasmid allows encoding of a transgene within the E1A transcription unit. The modifications, including E1 mutations, are introduced into the adenoviral genome by a single homologous recombination step in Escherichia coli. Subsequently infectious viruses are rescued from plasmids. As a proof-of-concept we generated two conditionally replication-competent adenoviruses Ad.Ki•COX and Ad.COX•Ki with the promoters of the Ki-67 protein and the cyclooxygenase-2 (COX-2) driving E1 and E4 and vice versa.
Conclusion
We demonstrated with our cloning system efficient generation of double heterologous promoter controlled oncolytic adenoviral vectors by a single homologous recombination step in bacteria. The generated viruses showed preferential replication in tumor cells and in a subcutaneous HT-29 colon cancer xenograft model the viruses demonstrated significant oncolytic activity comparable with dl327.
doi:10.1186/1472-6750-6-36
PMCID: PMC1557486  PMID: 16887042

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