Administration Of Ad Vectors Research Articles

Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals

Genome editing technology using the CRISPR-Cas system is attracting much attention not only as a promising experimental tool for analysis of genome functions, but also as a novel therapeutic approach for genetic disorders. Among the various types of Cas proteins, Cas12a is expected to be a promising gene editing tool due to its unique properties, including low off-target effects. As Cas proteins are of prokaryotic origin, they need to be fused with appropriate localization signals to perform their function in eukaryotic cells. Cas12a proteins fused with a nuclear localization signal (NLS) have been developed so far, but the relation between the nuclear localization activity and the genome editing efficiency has not been fully elucidated. Here, utilizing two Cas12a orthologs, AsCas12a and LbCas12a, with various number of NLSs derived from various origins, we revealed that the improved nuclear localization resulted in increased genome editing efficiencies when expressed using adenovirus (Ad) vector in cultured cells. However, when they were expressed in mouse liver, the improvement of the nuclear localization activity was not necessarily required to achieve the maximum genome editing efficiency four weeks after Ad vector administration. These data indicated that the optimized NLS modification of Cas12a proteins in in vitro situations differed from that in in vivo.

NF-κB promotes leaky expression of adenovirus genes in a replication-incompetent adenovirus vector.

The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes.

111. Efficient Transduction in Neonatal Mice via Systemic Administration of an Adenovirus Vector

An adenovirus (Ad) vector is widely used as a gene delivery vector due to their various advantages. An Ad vector is applicable to innate genetic diseases, which are crucial targets of gene therapy. Patients of several innate genetic disorders often display symptoms in their early stage of life and are danger of death without any treatments. Radical treatments in infant ages are required in such patients. Gene therapy in neonates and infants is promising for such innate genetic disorders. In addition, although immune responses to an Ad vector hamper Ad vector-mediated transduction in adults, it is expected that such inhibitions do not occur in neonates and infants because of their immature immunity; however, transduction properties of Ad vectors in neonates and infants have not been fully examined. In this study, Ad vector-mediated transduction properties in neonatal mice were examined.A first-generation Ad vector containing a CMV promoter-driven firefly luciferase expression cassette was administered to newborn mice on the second day of life at a dose of 5.9×1011 infectious unit (IFU)/kg (this titer is equal to 1010 IFU/5-weeks old mice) via retro-orbital sinus. First, biodistribution of an Ad vector in neonatal mice was investigated 2 days after administration. As is the case with adult mice, the highest copy numbers of Ad vector genome were found in the liver among the organs. The liver also showed the highest levels of luciferase expression among the organs in neonates, although the transduction efficiency in the neonatal liver was approximately 5-fold lower than that in the adult liver. On the other hand, neonatal mice exhibited 10-fold higher levels of luciferase expression in the heart than adult mice. While approximately more than 1000-fold higher transduction efficiency was found in the adult liver, compared with the adult heart, the neonatal liver exhibited only 30-fold higher transduction efficiency than the neonatal heart. In order to achieve liver-specific transduction, a liver-specific promoter (a fusion promoter composed of apolipoprotein E enhancer, the hepatocyte control region, and human alpha 1-antitrypsin promoter) was incorporated into an Ad vector. The Ad vector containing the liver-specific promoter mediated almost 100-fold higher transduction efficiencies in the liver, compared with those in the other organs. Second, Ad vector-induced hepatotoxicity in neonatal mice was evaluated. Obvious elevation in serum alanine aminotransferase (ALT) activity in neonatal mice was not observed within 2 weeks following Ad vector administration, indicating that this dose of Ad vectors did not provoke significant hepatotoxicity in neonatal mice. In addition, while anti-Ad antibodies were strongly induced in the serum of adult mice on 21 days post Ad vector injection, almost background levels of anti-Ad antibodies were detected in neonatal mice. These results provide crucial information for gene therapy in neonates and infants.

Evaluation of Transduction Properties of an Adenovirus Vector in Neonatal Mice

In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of life via retro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants.

Intratracheal instillation of high dose adenoviral vectors is sufficient to induce lung injury and fibrosis in mice.

RationaleReplication deficient adenoviruses (Ad) vectors are common tools in gene therapy. Since Ad vectors are known to activate innate and adaptive immunity, we investigated whether intratracheal administration of Ad vectors alone is sufficient to induce lung injury and pulmonary fibrosis.MethodsWe instilled Ad viruses ranging from 107 to 1.625×109 ifu/mouse as well as the same volume of PBS and bleomycin. 14 and 21 days after administration, we collected bronchoalveolar lavage fluid (BALF) and mouse lung tissues. We measured the protein concentration, total and differential cell counts, and TGF-β1 production, performed Trichrome staining and Sircol assay, determined gene and protein levels of profibrotic cytokines, MMPs, and Wnt signaling proteins, and conducted TUNEL staining and co-immunofluorescence for GFP and α-SMA staining.ResultsInstillation of high dose Ad vectors (1.625×109 ifu/mouse) into mouse lungs induced high levels of protein content, inflammatory cells, and TGF-β1 in BALF, comparable to those in bleomycin-instilled lungs. The collagen content and mRNA levels of Col1a1, Col1a2, PCNA, and α-SMA were also increased in the lungs. Instillation of both bleomycin and Ad vectors increased expression levels of TNFα and IL-1β but not IL-10. Instillation of bleomycin but not Ad increased the expression of IL-1α, IL-13 and IL-16. Treatment with bleomycin or Ad vectors increased expression levels of integrin α1, α5, and αv, MMP9, whereas treatment with bleomycin but not Ad vectors induced MMP2 expression levels. Both bleomycin and Ad vectors induced mRNA levels of Wnt2, 2b, 5b, and Lrp6. Intratracheal instillation of Ad viruses also induced DNA damages and Ad viral infection-mediated fibrosis is not limited to the infection sites.ConclusionsOur results suggest that administration of Ad vectors induces an inflammatory response, lung injury, and pulmonary fibrosis in a dose dependent manner.

Evaluation of miR-122-regulated suicide gene therapy for hepatocellular carcinoma in an orthotopic mouse model.

Intratumoral administration of adenoviral vector encoding herpes simplex virus (HSV) thymidine kinase (TK) gene (Ad-TK) followed by systemic ganciclovir (GCV) is an effective approach in treating experimental hepatocellular carcinoma (HCC). However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy. miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. By inserting miR-122 target sequences (miR-122T) in the 3' untranslated region (UTR) of TK gene, we constructed adenovirus (Ad) vectors expressing miR-122-regulated TK (Ad-TK-122T) and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.

Adenoviral Vector Immunity: Its Implications and Circumvention Strategies

Adenoviral (Ad) vectors have emerged as a promising gene delivery platform for a variety of therapeutic and vaccine purposes during last two decades. However, the presence of preexisting Ad immunity and the rapid development of Ad vector immunity still pose significant challenges to the clinical use of these vectors. Innate inflammatory response following Ad vector administration may lead to systemic toxicity, drastically limit vector transduction efficiency and significantly abbreviate the duration of transgene expression. Currently, a number of approaches are being extensively pursued to overcome these drawbacks by strategies that target either the host or the Ad vector. In addition, significant progress has been made in the development of novel Ad vectors based on less prevalent human Ad serotypes and nonhuman Ad. This review provides an update on our current understanding of immune responses to Ad vectors and delineates various approaches for eluding Ad vector immunity. Approaches targeting the host and those targeting the vector are discussed in light of their promises and limitations.

Adenovirus infection results in alterations of insulin signaling and glucose homeostasis

Recombinant adenovirus (Ad) vectors can initiate an inflammatory response, limiting its use in gene therapy and basic research. Despite increased efforts to better understand Ad infection, little is known about how it affects cellular metabolic responses. In the current studies, we explored the effects of Ad vectors on insulin signaling molecules and glucose homeostasis. Nonreplicative Ad vectors were injected into rats through the tail vein, and at 4-13 days postinjection insulin signaling and glucose tolerance were examined. Ad vector infection significantly reduced total levels of the insulin receptor (IR), and insulin receptor substrates 1 and 2 (IRS-1, IRS-2) in the liver of rats, resulting in decreased insulin-induced tyrosine phosphorylation of IR, IRS-1, and IRS-2, and decreased interaction of IRS-1 and IRS-2 with phosphoinositide 3-kinase (PI3K). In addition, Ad infection resulted in impaired systemic glucose homeostasis, which recovered by 13 days, after the protein levels of IR, IRS-1, and IRS-2 had started to normalize. Expression of a TNF inhibitor or Kupffer cell depletion attenuated the Ad vector-induced decreases of insulin signaling molecules, indicating a potential role of Kupffer cell activation in this process. These studies provide evidence that systemic administration of Ad vectors can impair insulin signaling in liver, resulting in altered systemic glucose metabolism. Thus, effects of Ad vector infection on insulin action and glucose metabolism need to be considered when Ad vectors are used in research or gene therapy and may be more broadly applicable to other viral agents.

Prevention of hepatic ischemia–reperfusion injury by pre-administration of catalase-expressing adenovirus vectors

Liver ischemia/reperfusion (I/R) injury, which is mainly caused by the generation of reactive oxygen species (ROS) during the reperfusion, remains an important clinical problem associated with liver transplantation and major liver surgery. Therefore, ROS should be detoxified to prevent hepatic I/R-induced injury. Delivery of antioxidant genes into liver is considered to be promising for prevention of hepatic I/R injury; however, therapeutic effects of antioxidant gene transfer to the liver have not been fully examined. The aim of this study was to examine whether adenovirus (Ad) vector-mediated catalase gene transfer in the liver is an effective approach for scavenging ROS and preventing hepatic I/R injury. Intravenous administration of Ad vectors expressing catalase, which is an antioxidant enzyme scavenging H 2O 2, resulted in a significant increase in catalase activity in the liver. Pre-injection of catalase-expressing Ad vectors dramatically prevented I/R-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and hepatic necrosis. The livers were also protected in another liver injury model, CCl 4-induced liver injury, by catalase-expressing Ad vectors. Furthermore, the survival rates of mice subjected to both partial hepatectomy and I/R treatment were improved by pre-injection of catalase-expressing Ad vectors. On the other hand, control Ad vectors expressing β-galactosidase did not show any significant preventive effects in the liver on the models of I/R-induced or CCl 4-induced hepatic injury described above. These results indicate that hepatic delivery of the catalase gene by Ad vectors is a promising approach for the prevention of oxidative stress-induced liver injury.

Transient Pretreatment With Glucocorticoid Ablates Innate Toxicity of Systemically Delivered Adenoviral Vectors Without Reducing Efficacy

More than 300 human clinical trials utilize recombinant adenoviruses (rAds) as a gene transfer vector, confirming that rAds continue to be of high clinical interest. A primary weakness of rAds is their known propensity to trigger an innate, proinflammatory immune response rapidly after high-dose, systemic administration. In this study, we investigated what affects that pre-emptive treatment with anti-inflammatory glucocorticoids might have upon Ad vector-triggered inflammatory immune responses. We found that a simple pretreatment regimen with Dexamethasone (DEX) can significantly reduce most Ad-induced innate immune responses. DEX prevented rAd induction of systemic cytokine/chemokine releases in a dose-dependent fashion, with higher dosages preventing rAd induction of acute thrombocytopenia, endothelial cell activation, proinflammatory gene induction, and leukocyte infiltration into transduced organs. Transient glucocorticoid pretreatment also significantly reduced rAd-induced adaptive immune responses, including a decreased induction of Ad-neutralizing antibodies (NAbs). Importantly, use of DEX did not reduce the efficacy of rAd-mediated gene transduction nor rAd-derived transgene expression. Our results demonstrate that a simple, pre-emptive and transient glucocorticoid pretreatment is a viable approach to reduce rAd-associated acute toxicities that currently limit the use of Ad vectors in systemic clinical applications.

Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors

Unresectable hepatic colorectal cancer (CRC) metastases are a leading cause of cancer mortality. These tumors and other epithelial tumors often express both cyclooxygenase-2 (COX-2) and carcinoembryonic antigen (CEA). Because adenovirus (Ad) vectors infect the liver and lack tumor tropism, they cannot be used for systemic therapy of hepatic metastases. We used COX-2 transcriptional restriction, in combination with transductional Ad hepatic untargeting and tumor retargeting by a bispecific adapter, sCARhMFE, composed of sCAR [the coxsackie/Ad receptor (CAR) ectodomain] and MFE-23 (a single-chain anti-CEA antibody), to untarget liver after i.v. administration of Ad vectors expressing firefly luciferase and to retarget virus to hepatic colorectal tumor xenografts and non-small cell lung tumor xenografts. To improve both liver untargeting and tumor retargeting, we developed sCARfMFE, a trimerized sCARhMFE adapter. Trimerization greatly improves both untargeting of CAR-dependent Ad infection and CEA-dependent virus retargeting in culture and in vivo. Combining sCARfMFE bispecific adapter transductional liver untargeting and transductional tumor retargeting with COX-2 transcriptional tumor-restricted transgene expression increases systemically administered Ad therapeutic efficacy for hepatic CRC tumors, using herpes virus type 1 thymidine kinase (HSV1-tk) as a therapeutic gene in conjunction with the prodrug ganciclovir (GCV). Both transductional untargeting and COX-2 transcriptional restriction also reduce HSV1-tk/GCV hepatic toxicity. In addition, transductional sCARfMFE untargeting reduces the innate immune response to systemic Ad administration. Combined transductional liver Ad untargeting, transductional tumor retargeting, and transcriptional transgene restriction suggests a means to engineer practical, effective therapeutic agents for hepatic CRC metastases in particular, as well as hepatic metastases of other epithelial cancers.

Sorting of Transgenic Secretory Proteins in Rhesus Macaque Parotid Glands After Adenovirus-Mediated Gene Transfer

We have previously used viral vectors encoding either human growth hormone (hGH) or erythropoietin (hEPO) to study the sorting of transgenic proteins in mouse and minipig salivary glands. Whereas hGH (a regulated secretory pathway [RSP] protein) is secreted predominantly into saliva in both species, hEPO (a constitutive secretory pathway [CSP] protein) is found primarily in the bloodstream with mice, but overwhelmingly in saliva with minipigs. In view of the hEPO sorting difference, we have conducted a similar study in nonhuman primates. Specifically, we examined hGH and hEPO sorting after adenoviral (Ad) vector-mediated gene transfer to parotid glands of rhesus macaques, another large and important animal model. Two groups (n = 2 per dose group; total n = 8) of male macaques received either 10(10) particles per gland (low-dose group) or 10(11) particles per gland (high-dose group) of adenoviral (Ad) vectors encoding either hGH (AdhGH) or hEPO (Ad-hEPO) via intraoral cannulation of both parotid glands. All macaques tolerated administration of Ad vectors well, with no clinically significant changes observed in any hematological and serum chemistry parameters. In AdhGH-treated animals, hGH was secreted exclusively into saliva. In contrast, after AdhEPO delivery, hEPO was secreted both in serum and saliva, at levels intermediate between mice and minipigs. We conclude that RSP proteins are faithfully secreted into saliva in all model species tested, whereas patterns of CSP protein secretion are variable.

Adenoviral Expression of Suppressor of Cytokine Signaling-1 Reduces Adenovirus Vector-Induced Innate Immune Responses

Adenovirus (Ad) vectors are among the most commonly used viral vectors in gene therapy clinical trials. However, the application of Ad vectors has been limited to local injection in many cases, because the systemic administration of Ad vectors triggers innate immune responses such as inflammatory cytokine production and tissue damage. To overcome this limitation, it will be necessary to develop safer Ad vectors less likely to induce the innate immune response. In the present study, we demonstrated that a suppressor of cytokine signaling-1 (SOCS1)-expressing Ad vector, Ad-SOCS1, reduces the innate immune response induced by Ad vectors. RAW264.7-SOCS1, a macrophage-like cell line that stably expresses SOCS1, was shown to produce lower levels of inflammatory cytokines after the transduction of Ad vectors. The systemic administration of Ad-SOCS1 into mice elicited the reduced production of inflammatory cytokines, as compared with that elicited by control Ad vectors, i.e., luciferase-expressing Ad vector, Ad-L2. Furthermore, the coadministration of Ad-L2 with Ad-SOCS1 attenuated inflammatory cytokine production and liver toxicity as compared with injection with Ad-L2 alone, and this was achieved without the suppression of luciferase production in various organs. The JAK/STAT pathway was involved in Ad vector-mediated cytokine production, which was impaired by the overexpression of SOCS1. These findings indicate that Ad-SOCS1 could be useful for reducing Ad vector-mediated innate immunity.

Development of PEGylated adenovirus vector with targeting ligand

For effective gene therapy, a vector system that transduces the therapeutic gene into target cells efficiently and safely is essential. Adenovirus (Ad) vectors frequently are used for gene therapy research, especially cancer gene therapy, because of their high transduction efficiency. However, broad clinical utility of Ad vectors have not yet been achieved owing to problems related to several properties inherent to Ads. Systemic administration of Ad vectors leads to acute virus accumulation and undesirable transgene expression in the liver, with subsequent inefficient systemic cancer-targeted therapy and pronounced hepatotoxicity. Furthermore, most people have Ad-neutralizing antibodies, which hamper gene expression efficiency. Chemical conjugation of Ad surface with polyethylene glycol (PEG) (PEGylation) is one of the promising strategies to overcome these problems. Furthermore, PEGylation of Ad vectors with targeting ligands on the tip of PEG, which alter the transfection range of Ad vectors will improve the safety and efficiency of Ad gene-delivery vectors. In this review, we describe the molecular biology of Ads and outline this PEGylation approach including our data.

72. Intravascular Interaction between Adenovirus and Platelets Promotes Adenovirus Sequestration, Degradation and Toxicity

Intravascular (iv) delivery of adenovirus (Ad) vectors is hindered by acute toxicity that is triggered by trapping of virus in the reticuloendothelial system (RES) within minutes of administration, and subsequent production of pro-inflammatory cytokines/chemokines. While Ad localizes to tissue macrophages and endothelial cells of the liver, spleen and lung within minutes of iv delivery, the mechanism of this reticuloendothelial targeting is unknown. We have identified a novel pathway by which platelets mediate trapping of Ad in the RES. Rapidly after iv administration fiber independent interaction between Ad and circulating platelets can be seen by Southern blot, by transmission electron microscopy (TEM) of isolated platelets and blood cell fractionation of mice receiving radiolabeled Ad. Blood platelet levels drop after Ad administration, while serum levels of the platelet activation marker soluble CD62p rise. Within 5 minutes of administration, Ad and platelets co-localize in the liver sinusoids along with endothelial and Kupffer cells. TEM showed Ad particles inside degranulated platelets and platelet aggregates within the liver sinusoids. Ad particles could also be seen inside Kupffer cells. Immunohistochemistry (IHC) showed widespread co-labelling of platelets (CD41), Kupffer cells (F4/80) and Ad (hexon) throughout the liver. In the spleen, Ad genomes were found within 5 minutes and Ad particles were detected in CD41 and F4/80 rich regions of the marginal zone by IHC. In the lung, Ad genomes were found within 5 minutes. Platelet accumulation was seen by IHC after delivery of Ad vectors, with platelet-endothelial interactions visible in the pulmonary vasculature by TEM. To determine the contribution of Ad-platelet interactions towards toxicity, mice were pre-injected with a platelet depleting antibody prior to virus delivery. At 6 hours post virus delivery, vector genomes in liver, lung and spleen were detected by Southern blot, and circulating serum cytokine levels were determined. Serum levels of the pro-inflammatory cytokines/chemokines IFN-|[gamma]|, IL-6 and MCP- 1 were lower in platelet depleted mice. The effect of Ad neutralizing antibodies on Ad-platelet interactions was also determined in mice that had been pre-immunized with the same vector prior to iv Ad vector administration. Early after vector administration significantly lower levels of Ad genomes were associated with circulating platelets in pre-immunized mice. While the levels of Ad deposition in the liver were unchanged in pre-immunized mice, there was reduced Ad deposition within the lungs of pre-immunized mice. Taken together these data suggest that rapid interaction between Ad and platelets occurs after iv administration. Platelet-Ad interactions apparently play a role in RES sequestration and degradation of Ad, and subsequent toxicity. We are currently attempting to identify the structural components involved in Ad-platelet interactions in vivo and investigating the contribution of this interaction to sequestration, degradation and toxicity, after Ad administration.

Interference with the IL-1-Signaling Pathway Improves the Toxicity Profile of Systemically Applied Adenovirus Vectors

The safety of gene therapy vectors is a major concern when novel viral or nonviral therapeutics are proposed for applications in humans. Adenovirus (Ad) vectors have been extensively used as efficient gene delivery vehicles in vitro over the last two decades. However, upon i.v. application, they elicit robust innate and inflammatory responses that may be fatal for the host. To date, the primary cytokines and chemokines involved in the initiation of these host responses remain illusive. In this study, we demonstrate that IL-1 is a major mediator involved in the initiation of immediate host responses toward i.v. applied Ad vectors. Using mice in which IL-1 signaling was genetically eliminated (IL-1RI-KO), or wild-type animals for which signaling was blocked by anti-IL-1 Abs, we found that i.v. applied Ad vectors elicited dramatically reduced acute inflammatory responses when compared with control animals. Importantly, the efficiency of Ad gene transfer in vivo was not significantly affected by interfering with IL-1 signaling. Using an in situ hybridization technique, we found that hepatocytes and Kupffer cells trigger IL-1 transcription in liver tissue after i.v. Ad vector administration. We also found that expression of the MIP-2 chemokine gene (which is responsible for recruitment of neutrophils to the liver) depends on IL-1 activation. Our data indicate that immediate innate and inflammatory host responses toward i.v. applied Ad vectors can be pharmacologically controlled through interference with IL-1 signaling pathways.

Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands

The treatment of most head and neck cancer patients includes ionizing radiation (IR). Salivary glands in the IR field suffer irreversible damage. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat submandibular glands following IR restored salivary flow to near normal levels. It is unclear if this strategy is useful in larger animals. Herein, we evaluated AdhAQP1-mediated gene transfer after parotid gland IR (20 Gy) in the miniature pig. Sixteen weeks following IR, salivation from the targeted gland was decreased by >80%. AdhAQP1 administration resulted in a dose-dependent increase in parotid salivary flow to approximately 80% of pre-IR levels on day 3. A control Ad vector was without significant effect. The effective AdhAQP1 dose was 2.5 x 10(5) pfu/microl infusate, a dose that leads to comparable transgene expression in murine and minipig salivary glands. Three days after Ad vector administration little change was observed in clinical chemistry and hematology values. These findings demonstrate that localized delivery of AdhAQP1 to IR-damaged salivary glands increases salivary secretion, without significant general adverse events, in a large animal model.

Inhibition of costimulation allows for repeated systemic administration of adenoviral vector in rhesus monkeys.

Immunogenicity of recombinant adenoviral (Ad) vectors severely hampers the clinical development of gene therapy protocols using repeated vector administrations. Inhibition of costimulation by APCs was explored as a strategy to circumvent the immune response against Ad particles. This strategy was tested in rhesus monkeys, treated transiently with chimeric anti-human CD40 and anti-human CD86 antagonist monoclonal antibodies (MAbs) at the time of systemic administration of a recombinant Ad vector. After Ad vector administration in the absence of immunosuppressive treatment, transgene expression in the serum lasted about 3-4 weeks. All control animals developed a strong neutralizing antibody (NAb) response to the Ad particles, which totally prevented efficient administration of a second vector, as shown by the lack of transgene expression. Treatment with anti-CD40 and anti-CD86 chimeric MAbs delayed or blocked the development of a humoral response against Ad and the infiltration of CD8(+) lymphocytes into the liver. This resulted in (i) increased persistence of Ad-transduced cells after injection of a first vector encoding a nonimmunogenic transgene, and (ii) the possibility of readministering a second Ad vector with significant efficacy. In both respects, the combined blockade of CD40 and CD86 was more efficient than treatment with anti-CD40 alone. This study shows for the first time in non-human primates that blocking CD40 and CD86 costimulatory molecules represents a promising strategy to inhibit immune responses against an Ad vector injected systemically.

The Role of Capsid–Endothelial Interactions in the Innate Immune Response to Adenovirus Vectors

Adenovirus (Ad) vectors can produce inflammatory responses at high doses. Intravenous administration of an Ad vector expressing green fluorescent protein (AdGFP) to naive mice induced a biphasic pattern of liver cytokine/chemokine gene expression over 7 days. Tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 2 (MIP-2), and interferon gamma-inducible protein 10 (IP-10) genes were upregulated, with two distinct peaks of mRNA expression occurring at 6 hr and 5 days. The administration of transcription-defective AdGFP particles induced the early but not the late peak of chemokine/cytokine gene expression, confirming that Ad vector-induced inflammation is capsid dependent in the early phase and transcription dependent in the late phase. To determine the role of adenoviral capsid motifs in the early phase, capsid-modified Ad vectors were employed. The intravenous administration of the RGD-deleted Ad vector AdL.PB*, the fiber mutant AdL.F*, or the double mutant AdL.F*PB* induced similar levels of cytokine/chemokine expression compared with the wild-type vector AdLuc. Kupffer cell blockade significantly reduced liver TNF-alpha, MIP-2, and IP-10 gene expression and liver inflammation after the administration of AdL.PB* or AdL.F*PB*. Fluorescence microscopy of AdLuc- and AdL.PB*-transduced liver at 1 hr revealed localization of Ad vectors to liver sinusoids in Kupffer cell-depleted mice. AdL.PB* induced less E-selectin and VCAM-1 gene expression in liver, confirming reduced endothelial activation in mice receiving RGD-deleted Ad vectors. In vitro studies of endothelial cells demonstrated reduced transduction and endothelial activation by AdL.PB* compared with AdLuc. These results demonstrate that adenovirus capsid RGD motifs are required for efficient transduction and endothelial cell activation. Altering vector tropism represents a feasible strategy to modulate the innate response to Ad vectors in nontargeted tissues.

1171. Detection of Human Prostate Cancer Metastasis in Nude Mice after Intravenous Administration of Ad Vector: Combined Fluorescence and Bioluminescence Imaging

1171. Detection of Human Prostate Cancer Metastasis in Nude Mice after Intravenous Administration of Ad Vector: Combined Fluorescence and Bioluminescence Imaging