Regulatable gutless adenovirus vectors sustain inducible transgene expression in the brain in the presence of an immune response against adenoviruses

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding beta-galactosidase (beta-gal) driven by a TetOn system containing the rtTAS(s)M2 transactivator and the tTS(Kid) repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-beta-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-beta-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-beta-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-beta-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.     

Tissue-specific consequences of the anti-adenoviral immune response: implications for cardiac transplants.

The immune response to adenoviral vectors can induce inflammation and loss of transgene expression in transfected tissues. This would limit the use of adenovirus-mediated gene transfer in disease states in which long-term gene expression is required. While studying the effect of the anti-adenoviral immune response in transplantation, we found that transgene expression persisted in cardiac isografts transfected with an adenovirus encoding beta-galactosidase. Transfected grafts remained free of inflammation, despite the presence of an immune response to the vector. Thus, adenovirus-mediated gene transfer may have therapeutic value in cardiac transplantation and heart diseases. Furthermore, immunological limitations of adenoviral vectors for gene therapy are not universal for all tissue types.     

Preexisting immunity to adenovirus in rhesus monkeys fails to prevent vector-induced toxicity

In an earlier study we evaluated innate immune responses to a first-generation adenoviral vector infused into the portal vein of rhesus monkeys who had never been exposed to adenovirus previously. In these animals, the systemic administration of E1/E3-deleted adenoviral vectors resulted in immediate activation of innate immunity and serious toxicity caused by targeting of vector to antigen-presenting cells and systemic inflammation. We analyze here how these responses are affected by vector-specific preexisting immunity that was induced by intramuscular immunization 6 months prior to evaluation. Our results show that preexposure to the vector substantially diminishes the transgene expression in most tissues but has little effect on gene transfer. Significantly, preimmunization does not eliminate systemic vector-induced toxicity. These conclusions are based on the presence of clinical features of coagulopathy and elevated levels of proinflammatory cytokine interleukin-6 in the serum of animals treated with vector after intramuscular immunization. Furthermore, preexisting immunity appears to induce a vector-specific inhibitory effect on erythroid progenitor development in the bone marrow that is not found when naive animals are challenged with vector.     

Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody.

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.     

TNF-alpha receptor signaling and IL-10 gene therapy regulate the innate and humoral immune responses to recombinant adenovirus in the lung

Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-alpha and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. beta-Galactosidase expression in mice receiving intratracheal instillation of Adv/beta-gal (adenovirus construct expressing beta-galactosidase) was transient (less than 14 days), but a significant early increase of beta-galactosidase expression was seen in mice lacking either or both TNF-alpha receptors. Absence of TNF-alpha or the p55 receptor significantly attenuated the Ab response to both adenovirus and beta-galactosidase. Human IL-10 expression in the lung suppressed local TNF-alpha production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with beta-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-alpha signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with beta-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with beta-galactosidase, and is nonimmunogenic in the lung.     

Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways

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.     

New insights into stability of recombinant adenovirus vector genomes in mammalian cells

Recombinant adenoviruses are widely used in basic virology research, therapeutic applications, vaccination studies or simply as a tool for genetic manipulation of eukaryotic cells. Dependent on the application, transient or stable maintenance of the adenoviral genome and transgene expression are required. The newest generation of recombinant adenoviral vectors is represented by high-capacity adenoviral vectors (HC-AdVs) which lack all viral coding sequences. HC-AdVs were shown to result in long-term persistence of transgene expression and phenotypic correction in small and large animal models with negligible toxicity.Although there is evidence that adenoviral vectors predominantly persist as episomal DNA molecules with a low integration frequency into the host genome, detailed information about the nuclear fate and the molecular status of the HC-AdV genome once inside the nucleus is lacking. In recent years we have focused on analyzing and modifying the nuclear fate of HC-AdVs after infection of mammalian cells. We have focused on investigating the molecular DNA forms of HC-AdV genomes and we have designed strategies to excise and stably integrate a transgene from an episomal adenovirus vector genome into the host chromosomes by recombinases. This review article provides a state-of-the art overview of the current knowledge of episomal HC-AdV persistence and it discusses strategies for changing the nuclear fate of a transgene inserted into the HC-AdV genome by somatic integration into host chromosomes.     

Gene therapy for Parkinson's disease

Gene therapy is a potentially powerful approach to the treatment of neurological diseases. The discovery of neurotrophic factors inhibiting neurodegenerative processes and neurotransmitter-synthesizing enzymes provides the basis for current gene therapy strategies for Parkinson's disease. Genes can be transferred by viral or nonviral vectors. Of the various possible vectors, recombinant retroviruses are the most efficient for genetic modification of cells in vitro that can thereafter be used for transplantation (ex vivo gene therapy approach). Recently, in vivo gene transfer to the brain has been developed using adenovirus vectors. One of the advantages of recombinant adenovirus is that it can transduced both quiescent and actively dividing cells, thereby allowing both direct in vivo gene transfer and ex vivo gene transfer to neural cells. Probably because the brain is partially protected from the immune system, the expression of adenoviral vectors persists for several months with little inflammation. Novel therapeutic tools, such as vectors for gene therapy have to be evaluated in terms of efficacy and safety for future clinical trials. These vectors still need to be improved to allow long-term and possibly regulatable expression of the transgene.     

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo.

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.     

Toxicity associated with repeated administration of first-generation adenovirus vectors does not occur with a helper-dependent vector

BACKGROUND: Certain gene therapy protocols may require multiple administrations of vectors to achieve therapeutic benefit to the patient. This may be especially relevant for vectors such as adenoviral vectors that do not integrate into the host chromosome. Because immunocompetent animal models used for gene transfer studies develop neutralizing antibodies to adenoviral vectors after a single administration, little is known about how repeat administrations of vectors might affect transgene expression and vector toxicity. MATERIALS AND METHODS: We used mice deficient in the membrane spanning region of immunoglobulin (IgM), which do not develop antibodies, to evaluate the effect of repeated intravenous administration of first-generation and helper-dependent adenoviral vectors expressing human alpha 1-antitrypsin (hAAT). The duration and levels of transgene expression were evaluated after repeated administration of vectors. Toxicity was assessed by measuring the level of liver enzymes in the serum and the degrees of hepatocyte hypertrophy and proliferation. RESULTS: We found that previous administration of first-generation adenoviral vectors can alter the response to subsequent doses. These alterations included an increase in transgene expression early (within 1 and 3 days), followed by a rapid drop in expression by day 7. In addition, previous administrations of first-generation vectors led to an increase in toxicity of subsequent doses, as indicated by a rise in liver enzymes and an increase in hepatocyte proliferation. In contrast to first-generation vectors, use of the helper-dependent adenovirus vector, Ad-STK109, which contained no viral coding regions, did not lead to increased toxicity after multiple administrations. CONCLUSIONS: We conclude that the response of the host to adenoviral vectors can be altered after repeated administration, compared with the response after the initial vector dose. In addition, these experiments provide further evidence for the relative safety of helper-dependent adenoviral vectors for gene therapy, compared with first-generation vectors.     

Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers

E1-deleted adenoviral recombinants most commonly based on the human serotype 5 (AdHu5) have been shown thus far to induce unsurpassed transgene product-specific CD8(+) T cell responses. A large percentage of the adult human population carries neutralizing Abs due to natural exposures to AdHu5 virus. To circumvent reduction of the efficacy of adenovirus (Ad) vector-based vaccines by neutralizing Abs to the vaccine carrier, we developed E1-deleted adenoviral vaccine carriers based on simian serotypes. One of these carriers, termed AdC68, expressing a codon-optimized truncated form of gag of HIV-1 was shown previously to induce a potent transgene product-specific CD8(+) T cell response in mice. We constructed a second chimpanzee adenovirus vaccine vector, termed AdC6, also expressing the truncated gag of HIV-1. This vector, which belongs to a different serotype than the AdC68 virus, induces high frequencies of gag-specific CD8(+) T cells in mice including those pre-exposed to AdHu5 virus. Generation of an additional E1-deleted adenoviral vector of chimpanzee origin allows for sequential booster immunizations with heterologous vaccine carriers. In this study, we show that such heterologous prime boost regimens based on E1-deleted adenoviral vectors of different serotypes expressing the same transgene product are highly efficient in increasing the transgene product-specific CD8(+) T cell response. They are equivalent to sequential vaccinations with an E1-deleted Ad vector followed by booster immunization with a poxvirus vector and they surpass regimens based on DNA vaccine prime followed by a recombinant adenoviral vector boost.     

Helper-dependent adenoviral vectors in experimental gene therapy

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.     

Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector

To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1beta, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.     

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.     

Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro.

BACKGROUND: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consuming. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. MATERIALS AND METHODS: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle plasmid containing a transgene and adenovirus genomic DNA. RESULTS: After transfection of Cre-treated DNA into 293 cells, replication-defective viral vectors were rapidly obtained without detectable wild-type virus. CONCLUSION: This system facilitates the development of recombinant adenoviral vectors for basic and clinical research.     

An shRNA silencing a non‐toxic transgene reduces nutrient consumption and increases production of adenoviral vectors in a novel packaging cell

During production of an adenoviral vector in a packaging cell, transgene is expressed concomitantly with the adenoviral gene. Depending on the protein encoded by the transgene, the yield of an adenoviral vector can be reduced or blocked as a result of transgene expression in the packaging cell. We tested the effect of a short hairpin RNA (shRNA) inhibiting the expression of a transgene encoding hIcon, a therapeutic molecule that selectively destroys pathological angiogenic blood vessels, on the yield of an adenoviral vector containing the transgene. The results showed that the yield of infectious adenoviral vector particles was increased about 10‐fold in a novel packaging cell with stable production of an shRNA that can silence the transgene, as compared to the yield in standard packaging line, and the consumption of nutrient in the novel packaging cell line is decreased due to silence of adenoviral transgene expression. The study indicates that shRNA can increase the production of adenoviral vectors by silencing transgene and reducing nutrient consumption of the novel packaging cell. The use of shRNA silencing expression of transgenes encoding therapeutic molecules can reduce the time and cost of producing adenoviral vectors for clinical use. J. Cell. Physiol. 219: 365–371, 2009. © 2008 Wiley‐Liss, Inc.     

Neuron-restrictive silencer elements mediate neuron specificity of adenoviral gene expression.

Neuron-restrictive silencer elements (NRSEs) were used to target the gene expression of adenoviral vectors specifically to neuron cells in the central nervous system. By generating adenoviral constructs in which NRSE sequences were placed upstream from the ubiquitous phosphoglycerate kinase promoter, the specificity of expression of a luciferase reporter gene was tested in both cell lines and primary cultures. Whereas transgene expression was negligible in nonneuronal cells following infection with an adenovirus containing 12 NRSEs, neuronal cells strongly expressed luciferase when infected with the same adenovirus. The NRSEs restricted expression of the luciferase gene to neuronal cells in vivo when adenoviruses were injected both intramuscularly into mice and intracerebrally into rats. This NRSE strategy may avoid side effects resulting from the ectopic expression of therapeutic genes in the treatment of neurological diseases. In particular, it may allow the direct transfection of motor neurons without promoting transgene expression within inoculated muscles or the secretion of transgene products into the bloodstream.     

Induction of immunological tolerance to adenoviral vectors by using a novel dendritic cell-based strategy

The success of helper-dependent adenoviral (HD-Ad) vector-mediated lung gene therapy is hampered by the host immune response, which limits pulmonary transgene expression following multiple rounds of vector readminstration. Here, we show that HD-Ad-mediated pulmonary gene expression is sustained even upon three rounds of readministration to immunodeficient mice, highlighting the need to suppress the adaptive immune response for sustained gene expression following vector readministration. Therefore, we devised a dendritic cell (DC)-based strategy for induction of immunological tolerance toward HD-Ad vectors. DCs derived in the presence of interleukin-10 (IL-10) are refractory to HD-Ad-induced maturation and instead facilitate generation of IL-10-producing Tr1 regulatory T cells which suppress HD-Ad-induced T cell proliferation. Delivery of HD-Ad-pulsed, IL-10-modified DCs to mice induces long-lasting immunological tolerance to HD-Ad vectors, whereby pulmonary DC maturation, the T cell response, and antibody response to HD-Ad vectors are suppressed even after three rounds of pulmonary HD-Ad readministration. Moreover, sustained transgene expression is also observed in the lungs of mice immunized with HD-Ad-pulsed, IL-10-modified DCs even after three rounds of pulmonary HD-Ad delivery. Taken together, these studies identify the use of DCs generated in the presence of IL-10 as a novel strategy to induce long-lasting immune tolerance to HD-Ad vectors.