E1-deleted Adenoviral Vectors Research Articles

Human myeloid dendritic cells transduced with an adenoviral interleukin-10 gene construct inhibit human skin graft rejection in humanized NOD-scid chimeric mice.

Human myeloid DC were generated from peripheral blood mononuclear cells by monocyte adhesion and subsequent culture with rhGM-CSF and rhIL-4. We transduced immature (day 5 of culture) myeloid DC with an E1-deleted replication-deficient adenoviral vector encoding the cytokine IL-10 (AdV IL-10) and a control adenovirus MX-17 (AdV MX 17). Human DC transduced with AdV IL-10 showed inhibition of the mixed leukocyte culture, reduced cell surface expression of co-stimulatory molecules (CD80/CD86) and were unable to produce the potent allo-stimulatory cytokine, interleukin-12. In order to test the in vivo properties of these cells a humanized immunodeficient mouse skin transplantation model was developed. Immunodeficient NOD-scid mice were engrafted with human skin, reconstituted via intraperitoneal injection with allogeneic mononuclear cells (MNC) mixed with 1 x 10(6) DC that were autologous to the skin donor and that had been transduced with either AdV IL-10 or AdV MX-17. Skin grafts were removed at day 7 and 14 after reconstitution and studied histologically for evidence of rejection. In animals that received DC modified with AdV IL-10 there was reduced skin graft rejection as characterized by reduced mononuclear cell infiltration and less dermo-epidermal junction destruction compared with those animals that received DC modified with the control virus alone. Injection of equivalent numbers of donor-derived fibroblasts transduced with AdV IL-10 were ineffective at modifying rejection of skin grafts. Immunosuppressive cytokine gene therapy targeting human DC is a novel means of inhibition of the alloimmune response.

Biology of E1-deleted adenovirus vectors in nonhuman primate muscle.

Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.

Murine IL-10 gene transfer inhibits established collagen-induced arthritis and reduces adenovirus-mediated inflammatory responses in mouse liver.

The effects of homologous IL-10 administration during an established autoimmune disease are controversial, given its reported immunostimulatory and immunosuppressive properties. Studies of collagen-induced arthritis have shown efficacy with repeated administrations of IL-10; however, when the EBV IL-10 homologue was administered via adenovirus gene transfer technology the results were equivocal. Therefore, the present study was undertaken to elucidate the effects of prolonged homologous IL-10 administration via adenovirus-mediated gene delivery on the progression of established arthritis. Collagen type II (CII)-immunized mice received i.v. injections of 10(7) or 10(8) PFU of an E1-deleted adenoviral vector containing the murine IL-10 gene (AdIL-10), after arthritis onset. Mice were monitored for 3 wk for disease progression, and gene transduction was assessed by quantification of serum mIL-10. CII-specific cell-mediated and humoral immune responses were analyzed by lymph node cell proliferation, cytokine production, and anti-CII Ab responses. Furthermore, because adenoviral vectors have been reported to induce organ dysfunction due to cell-mediated immune responses to the viral Ags, we have also evaluated delayed-type hypersensitivity responses and reactive hepatitis to the systemically delivered adenovirus and whether the IL-10 produced could influence those responses. Sustained suppression of autoimmune arthritis and elevated serum levels of IL-10 were achieved in our study. AdIL-10 treatment reduced cell-mediated immune reactivity, but did not affect humoral responses. Furthermore, IL-10 was able to reduce, but not totally abrogate, adenovirus-induced hepatic inflammation. These findings provide further insights into the diverse interplay of immune processes involved in autoimmune inflammation and the mechanism of cytokine immunotherapy.

Improved adenoviral vector for vascular gene therapy : beneficial effects on vascular function and inflammation.

First-generation, E1-deleted adenoviral vectors (E1-AV) can transduce the vascular endothelium with high efficiency, but their use is limited by the resulting acute endothelial injury and the long-term development of intimal hyperplasia. To reduce the impact of viral proteins on the gene-modified cells, a second-generation adenoviral vector with an additional pair of deletions in the E4 region was developed. To determine whether this E1/E4-AV vector would be useful for vascular gene transfer, we directly compared the efficiency of gene transfer to uninjured rabbit carotid arteries using either an E1/E4-AV or an E1-AV vector encoding beta-galactosidase. Both vectors efficiently transduced vascular endothelium; however, the E1/E4-AV vector gene-modified vessels showed higher beta-galactosidase expression 10 days after gene transfer. Importantly, the E1/E4-AV vector produced substantially less endothelial cell activation, less inflammation, and reduced neointimal hyperplasia compared with the E1-AV vector-treated vessels. The E1-AV vector-transduced vessels also demonstrated significantly impaired endothelium-dependent relaxation whereas the E1/E4-AV vector did not impact vasomotor function, even at doses of virus in 5-fold excess of the amount required for >90% transduction of the endothelium. We conclude that the E1/E4-AV vector is superior to the E1-AV vector for vascular gene therapy because of the prolonged transgene expression, reduced vascular inflammation, reduced intimal hyperplasia, and maintenance of normal vasomotor function.

A Cre-Expressing Cell Line and an E1/E2a Double-Deleted Virus for Preparation of Helper-Dependent Adenovirus Vector

Adenoviral vectors are attractive for the delivery of transgenes into mammalian cells because of their efficient transduction, high titer, and stability. The major concerns with using E1-deleted adenoviral vectors in gene therapy are the pathogenic potential of the virus backbone and the leaky viral protein synthesis that leads to host immune responses and a short duration of transgene expression. Helper-dependent (HD) adenoviral vectors that are devoid of all viral protein-coding sequences have significantly increased the safety and reduced the immunogenicity of these vectors. Currently available HD vectors depend on an E1-deleted adenovirus as a helper to provide viral proteins in trans. As a consequence, contamination with helper virus cannot be avoided in the HD vector preparation though it can be decreased to 0.01% using a Cre/loxP mechanism. Since the presence of E1-deleted helper virus may have substantial unwanted effects, we have developed a new Cre-expressing cell line based on an E1- and E2a-complementing cell. This new cell line can efficiently cleave the packaging region in the helper virus genome. We have also developed an E1 and E2a double-deleted helper virus. By using the CreE cell with the helper virus deleted in both the E1 and the E2a genes it may be possible to further improve the safety of the vectors.

An Immunomodulatory Procedure That Stabilizes Transgene Expression and Permits Readministration of E1-Deleted Adenovirus Vectors

Immune responses against E1-deleted adenovirus vectors and/or their transgene products result in the rapid elimination of vector-transduced cells and the generation of neutralizing antibodies. Different strategies of immunomodulation to stabilize transgene expression at therapeutic levels and to permit productive vector readministration have been examined. Our previous studies have shown that depletion of macrophages from spleen and liver decreases hepatic inflammation, significantly prolongs transgene expression, and delays the onset of humoral immune responses after systemic administration of an E1-deleted adenovirus vector. In the present study, we have examined the effects of macrophage depletion in combination with temporary blockade of CD40 ligation on E1-deleted adenovirus vector-mediated gene transfer. Alone, each of these treatments significantly inhibited the humoral immune response against the transgene product and prolonged its expression. Together, these treatments completely stabilized transgene expression and inhibited the production of neutralizing anti-adenovirus antibodies, permitting successful vector readministration. Animals rendered immunologically unresponsive to vector and transgene antigens regained their ability to mount productive immune responses against the vector after recovery of immune function, but remained unresponsive to the transgene product. These experiments demonstrate that this treatment is transient and antigen-specific.

Intravitreal adenoviral gene transfer evokes an immune response in the retina that is directed against the heterologous lacZ transgene product but does not limit transgene expression

Recombinant E1-deleted adenoviral vectors (ΔE1-Ad) are promising tools for in vivo gene transfer into the mammalian CNS including the retina. However, the duration of transgene expression is limited, and this limitation has partly been attributed to an immune response directed against vector-derived proteins. Here, we employed immunocytochemistry to assess the immune response to intravitreously injected ΔE1-Ad encoding the lacZ gene or various neurotrophins (NTs). β-Galactosidase was expressed by retinal cells for up to 4 weeks. Following intravitreal inoculation of AdCMV– lacZ, microglial and T cells were detected with a panel of antibodies in the retinal cell layers after 2 days (D2). The inflammatory response reached a maximum between D7 and D14. In contrast, no immune response was seen following injection of Ad encoding NTs. Yet, like with Ad–CMV–lacZ, their expression was also limited to approximately 4 weeks. Thus, β-galactosidase seems to trigger a host immune response following intravitreal adenoviral lacZ gene transfer, but immune responses are not the cause of limited NT transgene expression from the CMV promoter in the inner retina.

Direct adenovirus-mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function.

Cell-based cartilage-resurfacing procedures may be enhanced by the addition of insulin-like growth factor I (IGF-I) to the transplant biomatrix. Given the relatively short half-life of IGF-I in biological systems, however, maintenance of effective concentrations of this peptide necessitates either high initial doses, or repeated treatment. This study investigated IGF-I delivery via adenoviral gene therapy, targeting graftable articular chondrocytes. Cultured articular chondrocytes were infected with an E1-deleted adenoviral vector containing IGF-I-coding sequence under CMV promoter control. Increased adenovirus-IGF-I concentrations resulted in coordinate increase in IGF-I mRNA and ligand expression; however, chondrocyte matrix synthesis was maximized by the lower adenovirus-IGF-I concentration (100 MOI) without additional increase at 200 or 500 MOI. Using 100 MOI, infected monolayers produced medium IGF-I content of at least 10 ng/ml in each 48-hr period for 28 days, reaching a day 4 peak concentration of 66 +/- 4.0 ng/ml. These concentrations were sufficient to produce significant stimulation of normal cartilage matrix gene expression. The concentration of secreted matrix products in medium from infected monolayers was increased up to 8-fold over uninfected control cultures. Moreover, compared with uninfected cultures, cells in infected cultures were more resistant to de-differentiation over time under serum-starved conditions, maintaining a normal chondrocyte molecular phenotype for at least 28 days. These data indicate that cultured chondrocytes are readily transduced by recombinant adenoviral vectors. The adenoviral-IGF transgene is abundantly expressed and its product secreted at therapeutic concentrations for at least 28 days, resulting in increased matrix biosynthesis and maintenance of the chondrocytic phenotype. Combined, this information suggests that there may be significant value in preimplantation adenoviral-IGF gene therapy for chondrocytes destined for cartilage resurfacing.

Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells.

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells.

Insulinlike growth factor-I gene therapy applications for cartilage repair.

Cartilage function after resurfacing with cell-based transplantation procedures or during the early stages of arthritic disease may be bolstered by the addition of growth factor genes to the transplanted tissue. Insulinlike growth factor-I maintains chondrocyte metabolism in normal cartilage homeostasis and has been shown to improve cartilage healing in vivo. Given the relatively short half-life of insulinlike growth factor-I in biologic systems, however, maintenance of effective concentrations of this peptide has necessitated either very high initial doses or repeated treatment. Delivery of the insulinlike growth factor-I gene, using a deleted adenovirus vector, specifically targeting graftable articular chondrocytes, bone marrow-derived chondroprogenitor cells, or synovial lining cells, may provide more durable insulinlike growth factor-I fluxes to articular tissues. Cultured equine articular chondrocytes, mesenchymal stem cells, synovial explants, and synovial intimal cells were readily transfected with an E1-deleted adenoviral vector containing equine insulinlike growth factor-I coding sequence. Optimal viral concentrations for effective transduction were 100 multiplicities of infection in synoviocytes, 500 multiplicities of infection in chondrocytes, and 1000 multiplicities of infection in mesenchymal stem cells. Production of insulinlike growth factor-I ligand varied from 65 ng/mL to 246 ng/mL in medium from chondrocytes and synovial explants, respectively. For chondrocytes, these concentrations were sufficient to produce significant stimulation of cartilage matrix gene expression and subsequent proteoglycan production. Moreover, cells in infected cultures maintained a chondrocytic phenotype and continued to express elevated insulinlike growth factor-I levels during 28 days of monolayer culture. Minimal synthetic activity, other than insulinlike growth factor-I ligand synthesis, was evident in synovial cultures. These experiments suggest several avenues for insulinlike growth factor-I supplementation of articular cartilage, including preimplantation adenoviral-insulinlike growth factor gene transfer to chondrocytes or chondroprogenitor cells, and direct injection of adenoviral-insulinlike growth factor to transfect the synovial structures in situ.

Efficient transformation of primary human amniocytes by E1 functions of Ad5: generation of new cell lines for adenoviral vector production.

Primary human cells are relatively refractory to transformation by adenoviral E1 functions. For almost two decades, human embryonic kidney (HEK)-derived 293 cells have been the only E1-complementing cell line suitable for production of E1-deleted adenoviral vectors. More recently, new vector production cell lines have been derived from human embryonic retina (HER) cells, a cell type that is difficult to obtain. We were surprised to find that readily available primary human amniocytes are efficiently transformed by adenoviral E1 functions. We selected cell lines that allow high-titer production of recombinant adenoviral vectors. The generation of replication-competent adenovirus (RCA) during production, caused by homologous recombination between vector and cellular DNA, was excluded by designing the transforming plasmid to lack sequence overlap with current adenoviral vectors. In addition, we generated an infectious plasmid that can be used for convenient generation of first-generation adenoviral vectors in Escherichia coli and that matches the E1 complementation in the new production cell lines.

Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer.

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human beta-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells. GUSB secreted from the gene-transferred HAE cells were efficiently transported to murine fibroblasts with endocytosis mediated by mannose-6-phosphate receptors. The cells were administered into the spleen of the mice with the lysosomal storage disease mucopolysaccharidosis type VII (B6/MPSVII). Approximately 10-15% of the normal GUSB activity was detected in both liver and spleen 7 days after the cell administration. Histopathological examination showed that lysosomal enlargement in tissue macrophages in the liver and the spleen had disappeared by day 14. These results suggest that transplantation of the HAE cells transduced with adenoviral vectors can be employed for the treatment of congenital lysosomal storage disorders.

STIMULATORY AND INHIBITORY ACTION OF CYTOKINES ON THE REGULATION OF hCMV-IE PROMOTER ACTIVITY IN HUMAN ENDOTHELIAL CELLS

Viral promoters are commonly used as regulatory elements in gene therapy vectors due to their strong activity in various cell lines in vitro. However, transgene expression under the control of viral promoters in vivo has been shown to be limited to a short period of time. Several mechanisms for the transient expression of the delivered transgene may be important including deletion of transduced cells or promoter downregulation. Recently we reported that cytokines may either decrease or increase the activity of the human cytomegalovirus (hCMV) promoter in monocytes depending on the differentiation status of the transduced cells. For many applications, the gene of interest has to be delivered into an inflammatory milieu (tumour, ischaemia/reperfusion, vector-induced inflammation etc.). In this report we investigated the influence of various inflammatory cytokines on the hCMV-IE promoter activity in transduced human primary endothelial cells (Huvec) in vitro, which may be the first target cells after gene transfer into different organs. Cultured cells were infected with an E1-deleted adenoviral vector encoding for E. coliβ-galactosidase (Adβ-gal) driven by the hCMV-IE promoter and incubated either with or without various cytokines. Our results indicate that interferon-γ (IFN-γ) and interleukin-10 (IL-10) downregulate promoter activity in endothelial cells whereas, in contrast, tumour necrosis factor (TNF-α), interleukin 1β (IL-1β) and interleukin 4 (IL-4) increased the promoter activity. These results suggest that inflammatory processes influence the in vivo expression of transferred viral promoter controlled genes of interest.

Ischemia/reperfusion injury-mediated down-regulation of adenovirus-mediated gene expression in a rat heart transplantation model is inhibited by co-application of a TNFRp55-Ig chimeric construct.

E1-deleted adenoviral vectors are efficient vectors for somatic gene therapy. Recently, we have shown that intratracheal application of an adenoviral reporter construct leads to significant reporter gene expression in rat lungs within 24 h. In contrast, reporter gene expression in syngeneic rat heart transplants after adenovirus-mediated gene transfer was delayed. Since the adenovirus cannot replicate, down-regulation of the hCMV-IE promoter controlled reporter gene expression in initially infected cells by cytokines, which are released as a result of ischemia/reperfusion injury, might be involved. In order to investigate the role of proinflammatory cytokines, eg TNF-alpha in affecting hCMV-IE promoter-driven reporter gene expression, transient blockade of TNF-alpha was achieved by local co-application of an Ad-construct encoding for a soluble TNFRp55-Ig chimeric molecule in a syngeneic rat heart transplantation model. Co-application of the reporter construct together with the TNFRp55-Ig chimeric molecule significantly increased the early reporter gene expression after transplantation. Moreover, infiltration of inflammatory cells (T cells, macrophages, NK cells) and production of TNF-alpha in the transplant was markedly reduced. Our results indicate that: (1) proinflammatory cytokines are involved in down-regulation of reporter gene expression in ischemia/reperfusion injured tissues; and (2) inhibition of TNF-alpha might be a useful tool to increase early gene expression in gene therapy protocols, particularly in transplantation. Gene Therapy (2000) 7, 1238-1243.

Liver bypass significantly increases the transduction efficiency of recombinant adenoviral vectors in the lung, intestine, and kidney.

Recombinant adenoviruses have great potential as gene delivery systems because of their ability to infect a wide range of target cells. However, systemic delivery of viral vectors to tissues other than liver and spleen has been inefficient because of the rapid clearance of the circulating virus by the liver. In the present study we tested the hypothesis that a systemic administration of E1-deleted recombinant adenovirus vectors that bypasses the hepatic circulation will lead to enhanced expression of these vectors in extrahepatic tissues. The portal vein and hepatic artery in B6/129 F1 mice were clamped and an E1-deleted recombinant adenovirus carrying the beta-galactosidase gene (Ad.CBlacZ) was then administered through the retroorbital venous plexus. The clamp was released 30 min after viral injection with no major chronic ischemic consequences noted. High levels of LacZ expression were detected predominantly in the vessels and capillaries of the lung, intestinal wall, and renal glomeruli 7 days after viral infusion. The transgene expression persisted for at least 21 days. Intense LacZ staining was also observed in the liver, suggesting that liver infection occurred after the portal clamp was released. A retroorbital infusion of anti-adenovirus neutralizing antibodies 5 min before the release of the portal clamp significantly reduced postclamp viral infection to the liver, while LacZ expression in lung and intestine persisted after the antibody treatment. Taken together, these results suggest that liver bypass can significantly improve the transduction efficiency in the other target organs. This method could be used to develop animal models of human diseases that predominantly affect the vessels of the lung, intestine, and kidney.

Humoral immune responses to adenovirus vectors in the brain

We have investigated the humoral immune response to E1-deleted adenovirus vectors encoding the lacZ gene introduced into the brains of mice. Injection of these non-replicating vectors into the brain induced systemic antibody production to adenovirus vectors in dose dependent manner. Apparent antibody production to β-galactosidase, the product of the lacZ gene, was detected later than anti-adenovirus antibody. Neutralizing antibody was not detected. This study demonstrates that E1-deleted adenovirus vectors injected into the brain trigger humoral immune responses to the adenovirus and its gene products, but they are not sufficient to block the infection of cells by adenovirus upon repeat injection.

A Cell Line for High-Yield Production of E1-Deleted Adenovirus Vectors without the Emergence of Replication-Competent Virus

Production of E1-deleted adenovirus vectors for gene therapy has been plagued by the emergence of replication-competent adenovirus. A number of investigators have minimized homologous sequences between the vector and transfected E1 DNA in an attempt to avoid replication-competent adenovirus. We describe a HeLa-based cell line called GH329 that stably expresses the E1 locus from a promoter derived from the phosphoglycerate kinase gene. Overlap sequences with a standard E1-deleted vector that retains a full pIX transcriptional unit have been eliminated at the 5' end and minimized at the 3' end. The GH329 cell line plaques and produces E1-deleted adenovirus as well as 293 cells. Replication-competent virus has emerged after 5 passages of vector on 293 cells but was not detected after 20 passages on GH329 cells.

Efficient gene transfer and expression in islets by an adenoviral vector that lacks all viral genes.

Although adenoviral vector-mediated gene transfer has significant potential for gene therapy, host immune responses to virally expressed proteins and small insert capacity may limit its clinical application. In order to overcome these disadvantages, a new adenoviral vector that lacks all viral genes has been developed. Using the green fluorescent (GFP) gene as a reporter gene, we investigated the efficiency of gene transfer by this all-viral-genes-deleted and minimal cis-element remaining adenoviral vector (miniAd-GFP) in islets in vitro and ex vivo, and compared it with the E1-deleted adenoviral vector (E1-GFP). One day after in vitro infection, GFP was expressed in both miniAd-GFP- and E1-GFP-infected islets. The percentage of GFP-positive single cells was not significantly different between miniAd-GFP-infected islets and E1-GFP-infected islets. When these islets were transplanted into syngeneic diabetic mice, both miniAd-GFP- and E1-GFP-infected islet grafts reversed diabetes, and normal blood glucose levels were maintained for over 20 weeks posttransplantation. Mild lymphocyte infiltration was found in all E1-GFP-infected islet grafts at all time points. However, this was not seen in most miniAd-GFP-infected islet grafts. Our results indicate that gene transfer by an adenoviral vector that lacks all viral genes is as efficient as E1-deleted adenoviral vector-mediated gene transfer in islets. Furthermore, this adenoviral vector might be less immunogeneic than the E1-deleted adenoviral vector.

Local adenovirus-mediated CTLA4-immunoglobulin expression suppresses the immune responses to adenovirus vectors in the brain

The effect of local administration of two adenovirus vectors, one of which expressed CTLA4-immunoglobulin (AdCTLA), which blocks the B7-CD28 co-stimulatory pathway of T cell activation in the inflammatory response to adenovirus vectors was investigated. Mice injected with AdCTLA and an E1-deleted adenovirus vector that encodes the lacZ gene (AdRL) into the brain showed inflammatory cell infiltration from the early phase until day 6 after injection that was not different from that seen in control mice injected with an E1-deleted adenovirus vector containing no transgene (Ad0) and AdRL. After day 6 the inflammation in the control mice increased, peaked by day 15 and then decreased gradually but persisted until day 60. By contrast, in mice treated with AdCTLA and AdRL the inflammation, especially T cell infiltration, was suppressed after day 15. The anti-adenovirus antibody titer increased gradually until day 60 in the Ad0-AdRL control group, and whereas the mice injected with AdCTLA and AdRL showed lower anti-adenovirus antibody titers than the control group mice after day 15. Neutralizing antibody was not detected in either group. Expression of β-galactosidase, the gene product of AdRL, at the injection site in the striatum and corpus callosum peaked on day 6 and remained until day 60 although it was very low in both groups; β-galactosidase expression was similar in the two groups in spite of the difference in the degree and extent of the local immune response in the brain. This study demonstrated that the injection of an adenovirus vector expressing CTLA4-immunoglobulin into the brain suppressed not only local cell infiltration in the brain but also reduced the humoral immune response to adenovirus vectors.

Generation of adenovirus vectors devoid of all viral genes by recombination between inverted repeats.

Direct or inverse repeated sequences are important functional features of prokaryotic and eukaryotic genomes. Considering the unique mechanism, involving single-stranded genomic intermediates, by which adenovirus (Ad) replicates its genome, we investigated whether repetitive homologous sequences inserted into E1-deleted adenoviral vectors would affect replication of viral DNA. In these studies we found that inverted repeats (IRs) inserted into the E1 region could mediate predictable genomic rearrangements, resulting in vector genomes devoid of all viral genes. These genomes (termed DeltaAd.IR) contained only the transgene cassette flanked on both sides by precisely duplicated IRs, Ad packaging signals, and Ad inverted terminal repeat sequences. Generation of DeltaAd.IR genomes could also be achieved by coinfecting two viruses, each providing one inverse homology element. The formation of DeltaAd.IR genomes required Ad DNA replication and appeared to involve recombination between the homologous inverted sequences. The formation of DeltaAd. IR genomes did not depend on the sequence within or adjacent to the inverted repeat elements. The small DeltaAd.IR vector genomes were efficiently packaged into functional Ad particles. All functions for DeltaAd.IR replication and packaging were provided by the full-length genome amplified in the same cell. DeltaAd.IR vectors were produced at a yield of approximately 10(4) particles per cell, which could be separated from virions with full-length genomes based on their lighter buoyant density. DeltaAd.IR vectors infected cultured cells with the same efficiency as first-generation vectors; however, transgene expression was only transient due to the instability of deleted genomes within transduced cells. The finding that IRs present within Ad vector genomes can mediate precise genetic rearrangements has important implications for the development of new vectors for gene therapy approaches.