128 no recombinant vector sequences detected in tumor tissues from two research subjects received adenovirus ornithine transcarbamylase gene therapy

128 No Recombinant Vector Sequences Detected in Tumor Tissues from Two Research Subjects Received Adenovirus Ornithine Transcarbamylase Gene Therapy Molecular Therapy Volume 21, Supplement 1, May 2013[.]

ADENOVIRUS AND OTHER DNA VIRUS VECTORS I virion DNA to double-strand In recombinant AAV (rAAV) genedelivery vectors, they are targets for DNA repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration We investigated the effect of the TR HP on recombination by comparing specific DNA substrates transfected into WT and DNA-repair deficient cells DNA molecules with the TR sequences constrained in the cruciform hairpin conformation at one or both ends were subject to loss of gene expression, which was partially relieved in ATM-/- (ataxia telangiectasia mutated) cells The ATMdependent effect was mediated by transcriptional silencing of a subset of HP-containing molecules in cis, and was dependent on the specific T-shaped structure of the HP and not the primary sequence DNA molecules with simple U-shaped HP ends were unaffected by ATMdependent silencing DNA molecules with the TR sequence in the open duplex conformation, or without TR sequences, were unaffected by ATM mutation In vivo testing of the ATM effect on vector expression in mouse liver demonstrated a more uniform distribution of transduced cells throughout the liver, in contrast to the pericentral dominant pattern normally observed These results suggest that the presence of the cruciform HP structure on rAAV vector genomes subjects them to specific, and sometimes unproductive, DNA repair/ recombination pathways Adenovirus and Other DNA Virus Vectors I 126 Shielding the Hexon Hyper Variable Region of Ad5-Based Gene Transfer Vectors by PEGylation Decreases Sequestration by Erythrocytes and Increases Blood Coagulation Factor X -Mediated Hepatocyte Transduction Lea Krutzke,1 Jan-Michael Prill,1 Tatjana Engler,1 Stefan Kochanek,1 Florian Kreppel.1 Gene Therapy, University of Ulm, Ulm, Germany The use of adenovirus serotype -based gene transfer vectors in gene therapy is limited by multiple non-target vector-host interactions, in particular with human blood components Recently it was shown that human, but not murine erythrocytes express CAR as well as complement receptor (CR1) on their surface This leads to sequestration of administered Ad vector particles via binding of fiber knob to CAR and complement-mediated binding to CR1 However, in order to generate retargeted Ad vectors suitable for in vivo delievery into the bloodstream it is mendatory to decrease this sequestration by erythrocytes Here we analyzed the role of hexon hyper variable region (HVR1) in this context We generated Ad vector mutants with CAR-binding ablation and a genetically inserted cysteine residue in HVR1 of hexon, that allows for a site-specific attachment of shielding polymers Using human whole blood we could show that ablation of CAR binding in absence of complement-activating antibodies is sufficient to completely prevent erythrocyte binding If anti-Ad IgGs (provided in form of human IVIG) were present virtually 100% of the Ad vector particles were sequestered due to binding to CR1 Of note, PEGylation of HVR1 prevented vector sequestration by erythrocytes in the presence of IVIG by more than 20% Comparative analysis of HVR1 and HVR5 shielding revealed HVR1 as a favorable site to prevent erythrocyte binding Furthermore, our study provides evidence that modification of HVR1 with PEG moieties  5kDa was sufficient to shield from blood coagulation factor X (FX) -mediated cell transduction However and interestingly, it was also shown that modification of HVR1 with PEG moieties  2kDa significantly increased FX dependent cell transduction levels We assume that this is due to a shielding of the negatively charged amino acid stretch within HVR1, which is primarily responsible for the overall negative charge of the vector particles This neutralization probably favors the bridging effect of FX between vector and cell The influence of the S52 surface charge on interactions has not been investigated before and is of great interest to understand the natural biodistribution of Ad5 vectors Finally in vivo data will be presented 127 Therapeutic Efficacy in a Breast Cancer Model of Novel Conditionally Replicative Adenovirus (CRAd) Based upon the Deletion of Minor Core Protein Gene V Hideyo Ugai,1,4 George C Dobbins,2 Minghui Wang,3 David T Curiel.1,4,5 Radiation Oncology, Washington University in St Louis, St Louis, MO; 2Surgery, University of Alabama at Birmingham, Birmingham, AL; 3Genetics, University of Alabama at Birmingham, Birmingham, AL; 4Cancer Biology Division; Biologic Therapeutic Center Malignant tumors are aggressive and often metastasize from the primary site to other organs Therefore, more effective treatments are urgently required for human cancer therapy Conditionally replicative adenoviruses represent a promising new direction for human cancer treatment in order to effectively kill target cells However, recent evidence shows that conditionally replicative adenoviruses which are incapable of antagonizing tumor suppressor proteins p53 and RB replicate and induce cytocidal effect in normal cells Therefore, novel approaches to develop conditionally replicative adenoviruses that are distinct from current approaches are needed to achieve stringent replicative specificity In this regard, our previous investigation found that an adenoviral pV-deletion mutant, Ad5-dV/TSB, replication is restricted in normal cells due to a defect in induction of subcellular relocalization of nucleolar nucleophosmin 1/NPM1/B23.1 which is required for adenoviral replication In contrast to normal cells, Ad5-dV/TSB effectively replicated in various types of cancer cells which express nucleoplasmic NPM1 Therefore, we evaluated therapeutic efficacy of Ad5-dV/TSB by using in vitro and in vivo models of human breast cancer Ad5-dV/TSB infection resulted in viral replication and tumoricidal effect in human breast cancer cells, but Ad5-dV/TSB replication was restricted in a primary normal cell line of human mammary epithelial normal cells Additionally, Ad5dV/TSB infection, as well as wild type Ad5 infection, effectively blocked tumor growth of MDA-MB-231 stably expressing EGFP in a subcutaneous xenograft in nude mice Thus, our present study shows that Ad5-dV/TSB is a promising agent for cancer treatment, and we propose that pV-deletion is a novel strategy for achieving stringent replicative specificity of conditionally replicative adenovirus-based virotherapy agents 128 No Recombinant Vector Sequences Detected in Tumor Tissues from Two Research Subjects Received Adenovirus Ornithine Transcarbamylase Gene Therapy Li Zhong,1,2 Shaoyong Li,1,3 Mengxin Li,1,3 Yu Zhang,1,3 Brendan Lee,4 Mark L Batshaw,5 James M Wilson,6 Guangping Gao.1,3 Gene Therapy Center, UMass Med School, Worcester, MA; Pediatrics, UMass Med School, Worcester, MA; 3Microbio and Physio Sys, UMass Med School, Worcester, MA; 4Mol and Hum Genetics, Baylor College of Med, Houston, TX; 5Pediatrics, Children’s Nat Medical Center, George Washington Univ School of Medicine and Health Sci, Washington, DC; 6Gene Therapy & Pathology, Lab Medicine, UPENN School of Med, Philadelphia, PA Although wild-type (wt) Adenovirus (Ad) serotype could transform cells in vitro via E1a gene, natural infections of Ad in human not associate with cancer Like its wild type counterpart, E1-deleted replication defective recombinant adenoviral (rAd) vector does not undergo integration in chromosome and persistence Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy 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 ... 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... 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. .. 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