131 Versican Stimulates Adenoviral Transgene Expression in a JAK Dependent Manner Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S53 ADENOV[.]
ADENOVIRUS AND OTHER DNA VIRUS VECTORS I in host cells, they are not considered as high risk for insertional tumorigenesis To date, there has been no reported evidence for any association between adenoviral gene therapy and transformation of host cells and tumor development In our study, a 66 year subject with ornithine transcarbamlase deficiency (OTCD) participated in a phase I gene therapy trial This subject was the first to receive the rAd human ornithine transcarbamylase (hOTC) vector (2×109 particles/kg, of the H5.001CB.hOTC vector) in April 1997 at age 52 years However the subject developed hepatocellular carcinoma (HCC) 14 years later More recently, another subject in the same gene therapy trial, who received Ad hOTC vector at age 45 years in 1997, was also found to develop colon malignancy in 2012 Although, the tumor formation in these two cases is unlikely related to rAd vector administration more than a decade ago, it is necessary to exclude this possibility by further investigation Here, we developed, optimized and validated a nested PCR assay to target multiple regions of both rAd and wtAd genomes and recover the trace viral or transgene sequences in the tumor tissues isolated from those two subjects Under these conditions, the hOTC mutant allele in exon in patient was verified by PCR and sequencing With our highly sensitive assay, we can detect as low as GC of rAd or wtAd sequence in 200 ng cellular DNAs from frozen or paraffin embedded mouse liver tissue Sequencing data further confirmed that the PCR products were identical to the predicted sequences of spiked rAd or wtAd genome Using those optimized primers and PCR conditions, we then attempted to recover any possible rAd or wtAd sequences in the cellular DNAs from normal liver, liver tumor or colon tumor tissue isolated from those two patients No any PCR products with predicted sizes were detected in all groups Some PCR products with sizes closing to predicted band were subjected to cloning and sequencing The sequencing data confirmed that those products were non-specific and did not contain any rAd or wtAd sequences Our results indicated that neither normal tissues nor tumor tissues from those two patients contain any rAd or wtAd sequences from their vector treatment in 1990’s, suggesting that the previous adenovirus vector gene therapy is unlikely contributed to the HCC or colon cancer in these two patients 129 Oncolytic Adenovirus Expressing IL-23 and p35 Elicits IFN-γ- and TNF-a-Co-Producing T CellMediated Antitumor Immunity Il-Kyu Choi,1 Yan Li,1 Eonju Oh,1 Chae-Ok Yun.1 Department of Bioengineering, Hanyang University, Seoul, Republic of Korea Cytokine immunogene therapy is a promising strategy for cancer treatment Interleukin (IL)-12 boosts potent antitumor immunity by inducing Th cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35 Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model The levels of IL-12, IL-23, interferon- (IFN-), and tumor necrosis factor-a (TNF-a) were elevated in RdB/IL23/p35-treated tumors Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35 Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4+ and CD8+ T cells into the tumor as well as enhanced induction of tumor-specific immunity Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-- and TNF-a-co-producing Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy T cells in tumor microenvironment These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity 130 Wnt Decoy Receptor (sLRP6E1E2) Induces Antifibrotic Effect Via Inhibition of Wnt Signaling in Keloid Scars Jung-Sun Lee,1 Won Jai Lee,2 Chae-Ok Yun.1 Department of Bioengineering, Hanyang University, Seoul, Republic of Korea; 2Department of Plastic & Reconstructive Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea Keloid scars are pathologic proliferations of the dermal skin layer resulting from excessive collagen deposition The aberrant activation of the Wnt pathway signaling plays a critical role in keloid scars In this study, we evaluated the therapeutic potential of a soluble Wnt receptor decoy in treatment of keloid scars Therefore, we designed a Wnt antagonist sLRP6E1E2, and generated a replicationincompetent adenovirus (Ad), dE1-k35/sLRP6E1E2 We showed that -galactosidase expression confirmed the efficient transduction of dE1-K35/lacZ into human dermal fibroblasts In addition, dE1-k35/ sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic -catenin and decreased Wnt/-catenin signaling dE1-k35/ sLRP6E1E2 also inhibited Wnt-induced TGF-b up-regulation as well as Smad2/3 signaling pathway Consistent with these data, the expression of ECM proteins was significantly decreased in keloid spheroids transduced with dE1-k35/sLRP6E1E2 These results suggest that the antifibrotic effect of sLRP6E1E2-expressing adenovirus may have therapeutic effects on keloids 131 Versican Stimulates Adenoviral Transgene Expression in a JAK Dependent Manner Patricia Y Akinfenwa,1,4 Wesley S Bond,1,4 Mary Y Hurwitz,2,4 Richard L Hurwitz.1,4 Translational Biology and Molecular Medicine Program, Baylor College of Medicine, Houston, TX; 2Department of Pediatrics, Baylor College of Medicine, Houston, TX; 3Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 4Cancer and Hematology Centers, Texas Children’s Hospital, Houston, TX Enhancing the expression of transgenes delivered by adenoviral vectors (AdV) could result in increased gene therapy efficiency Our group reported the enhancement of AdV transgene expression (TGE) following transduction in the presence of vitreous, the gelatinous material that serves a major structural component of the posterior eye Treatment with vitreous has no effect on the efficiency of vector internalization but results in increased transgene mRNA levels We have determined that the interaction of hyaluronan (HA), a major vitreous component, with CD44 contributes to enhancement of AdV TGE, however CD44-independent effects have also been observed We hypothesize that the HA-binding proteoglycan, versican, which associates with HA and is abundant in vitreous, is responsible for regulating AdV TGE in both a CD44-dependent and a CD44independent manner To assess the effect of versican on AdV TGE, the proteoglycan was isolated from the supernatant of ACHN cells, a cell line known to secrete high levels of versican The supernatant was concentrated and enriched for high molecular weight components (>300kDa) using centrifugal filtration Cells transduced with AdV and treated with versican-enriched supernatant, exhibited a significant enhancement of TGE in both CD44-negative and CD44-positive cells Nanomolar concentrations of versican resulted in similar levels of TGE enhancement as that seen in vitreous-treated cells, suggesting that versican plays a significant role in modulating the expression of AdV transgenes TGE enhancement mediated by S53 ADENOVIRUS AND OTHER DNA VIRUS VECTORS I vitreous or versican was lost upon inhibition of Janus kinase (JAK) using the small molecule inhibitor ruxolitinib Our results support a model in which the extracellular binding of versican results in JAK activation, facilitating enhanced transcription of the vector transgene Both CD44-dependent and CD44-independent pathways appear to be regulated by JAK signaling Thus, modulation of these biochemical pathways could serve as a method of regulating AdV TGE in gene therapy protocols Ad5 became neutralized when FX in the serum was blocked using X-bp With some non-Ad5 viruses, in contrast, we found that mouse serum neutralized certain Ad serotypes in spite of the presence of FX On the other hand, a number of other Ad serotypes remained infectious in serum even when FX was blocked We conclude that Ad serotypes differ in their susceptibility to neutralization by mouse serum, as well as differing in whether they rely on FX for protection from neutralization 132 Replication of Human Adenoviruses in Non-Human Cells In Vitro and In Vivo 134 Non-Covalent Coating of Adenoviral Vectors with PAMAM Dendrimer Conjugates Allows for CAR Independent Virus Uptake and Targeting to the EGF Receptor Catherine M Crosby,1 Mallory A Turner,1 Michael A Barry.1 Mayo Clinic, Rochester, MN Human adenoviruses (hAd) are widely studied for their utility as vaccine, gene therapy, and oncolytic vectors While most work has focused on testing replication-defective (RD) hAds, the use of replication-competent (RC) hAds has increased interest in virus replication in non-human cells and models While mice are an ideal model for many preclinical tests, they are reportedly poor models for viral DNA replication In contrast, hamsters and cotton rats are thought to be better models of viral replication, but are limited models for most preclinical work Screening hAds from species B, C, D, and E Ads on non-human cells revealed that a variety of cells are susceptible to killing in Ad species-specific fashion Interestingly, only species C Ad5 and Ad6 were able to infect and kill hamster HaK and CHO cells, whereas these cells were non-permissive for other hAds Complementation of these cells with CAR or CD46 failed to rescue this phenotype Species D Ads infected and killed human and mouse B cell lines, but not other cell types Species C hAds infected many mouse and non-human cells In the current study, we directly studied the replication of hAd in mice, as well as various other non-human cells We engineered RC-hAd6 and RD-hAd6 expressing a GFPluciferase transgene to test the biology of this lower seroprevalence species C virus In vitro, hAd6 infects, replicates its genome, and produces infectious burst in mouse, hamster, and rhesus macaque cells In vivo, hAd6 infects, replicates its genome, and amplifies luciferase expression in BALB/c mice after intravenous and intranasal delivery These data suggest that non-human animal models can support human adenovirus genome replication for use in preclinical testing of replication-competent vectors 133 Adenovirus Serotypes Differ in Their Susceptibility to Neutralization by Mouse Serum: Role of Coagulation Factor X Jie Tian,1 Zhili Xu,1 Andrew P Byrnes.1 Division of Cellular and Gene Therapies, FDA Center for Biologics Evaluation and Research, Bethesda, MD When adenoviral vectors are injected systemically, they immediately come into contact with plasma proteins such as antibodies, complement and coagulation factors Ad5 vectors have the ability to bind coagulation factor X (FX), and we have recently shown that FX protects Ad5 from neutralization by complement When Ad5 is prevented from binding FX, we have found that naive mouse serum can neutralize Ad5 through natural IgM and the classical complement pathway Although Ad5 is the most widely-used vector serotype, additional serotypes are increasingly being used as gene therapy vectors and vaccines In the current study, we have examined whether other Ad serotypes are neutralized by naive mouse serum, and whether FX plays a role in protecting non-Ad5 viruses from neutralization Methods: Various wild-type human adenovirus serotypes were incubated with freshly-collected serum from naive C57BL/6 mice, then diluted and assayed for infectivity on 293 cells In some cases FX was blocked using X-bp (an FX-binding protein) Results: As expected, Ad5 retained full infectivity in naive mouse serum, and S54 Alexandra Vetter,1 Kulpreet S Virdi,2 Sigrid Espenlaub,3 Wolfgang Rödl,1 Ernst Wagner,1,4 Florian Kreppel,3 Christine Spitzweg,5 Manfred Ogris.1,4 Center for System Based Drug Research, Department of Pharmacy, LMU, Munich, Germany; 2Department of Chemistry, Physical Chemistry, LMU, Munich, Germany; 3Department of Gene Therapy, University of Ulm, Ulm, Germany; 4Center for NanoScience (CeNS), LMU, Munich, Germany; 5Medical Clinic II, Department of Molecular Endocrinology, LMU, Munich, Germany Adenovirus type (Ad) has a high transduction efficiency in many cell types It is able to infect dividing as well as non-dividing cells, but exhibits the limitation of being dependent on the presence of viral receptors on target cells for sufficient infection The Coxsackie- and Adenovirus Receptor (CAR), which is stated to be the key player for efficient cell infection is often down regulated on tumour cells, correlating with the aggressiveness of a tumour Therefore there is a need in seeking for beneficial coating agents enabling infection of these Ad-refractory cancers Here we show a hybrid system of viral and non-viral vectors, by coating Ad with PAMAM dendrimers allowing for CAR independent infection of CAR negative cancer cell lines, which not only increases and specifies transduction but also protects the Ad from neutralizing antibodies We coated luciferase encoding Adenovirus (Ad-Luc) with PAMAM dendrimers generation 2, 3, or with linear polyethylenimine (LPEI, 22 kDa) a gold standard for non-viral gene delivery Luciferase and metabolic activity after transduction of U87MG human glioma (low CAR) or HuH7 human hepatoma (high CAR) cells was measured Uptake studies with coated versus naked Ad-Alexa488 were carried out by laser scanning microscopy (LSM) To analyse if PAMAM dendrimers protect the Ad from neutralizing antibodies, naked or PAMAM coated Ad-GFP was preincubated with anti Ad IgG (Privigen®) before transducing A549 cells Furthermore, retargeting studies using GE11 peptide as an EGFR targeting ligand coupled to PAMAM via a kDa PEG spacer were carried out EGF preincubation was performed to proove the selectivity of the targeting benefit achieved with PAMAM G2-PEGGE11 when compared with its untargeted control construct PAMAM G2-PEG-Cystein PAMAM G5 coating improved transduction efficiency especially on CAR negative SKOV-3 cells; on the low CAR cell line U87MG transgene expression was still increased up to 38-fold compared to naked Ad, whereas LPEI showed only a 5-fold increase Reasons are the toxicity of LPEI starting from 50 ng/well onwards as well as the considerable particle aggregation causing a slower and less efficient cellular uptake compared to PAMAM coated Ad PAMAM coating protected Ad from neutralizing antibodies leading to 65 % of GFP positive A549 cells, while naked Ad was inactivated at the same IgG concentration A significant retargeting effect to the EGFR of 2.3-fold was seen for the GE11 construct compared to the cystein control, which was demonstrated as specific after EGFR downregulation by EGF preincubation This study clearly proves noncovalent, charge-based coating of Ad vectors with ligand-equipped dendrimers as a viable strategy for efficient and specific transduction of cells otherwise refractory to Ad infection Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... extracellular binding of versican results in JAK activation, facilitating enhanced transcription of the vector transgene Both CD44 -dependent and CD44-independent pathways appear to be regulated... efficient cellular uptake compared to PAMAM coated Ad PAMAM coating protected Ad from neutralizing antibodies leading to 65 % of GFP positive A5 49 cells, while naked Ad was inactivated at the same IgG... cells In vivo, hAd6 infects, replicates its genome, and amplifies luciferase expression in BALB/c mice after intravenous and intranasal delivery These data suggest that non-human animal models can