551 Evaluation of the X Protein Truncate Expression Potential from the WPRE Element in Alipogene Tiparvovec, an AAV Based Gene Therapy Vector Molecular Therapy Volume 19, Supplement 1, May 2011 Copyri[.]
GENE REGULATION II 654 intron by modifying 5’ alternative splice site for tight regulation, and by reducing the size to comply with the size limitation of AAV genomes We found that the constructs containing the optimized intron had significant improvements in the level of induction of transgene expression In addition, we generated constructs that contain different sequences at 5’ alternative splice site of the intron We determined the expressions of these constructs are independently controlled by their corresponding ASOs We suggest that this system can be developed to independently regulate the expression of multiple transgenes, and can be applied to gene therapy of diseases that require treatment in a complex multi-step process for an extended period of time 549 Hypoxia-Specific Gene Expression System Using Oxygen Dependent Degradation Domain of Activating Transcription Factor for Cancer Specific Gene Therapy Su Hee Cho,1 Hyun Ah Kim,1 Ji Hwan Park,1 Min Hyung Lee.1 Bioengineering, Hanyang University, Seoul, Republic of Korea Solid tumor contains hypoxic region, which is an excellent target for tumor specific gene therapy In this study, the oxygen dependent degradation (ODD) domain from activating transcription factor (ATF4) was evaluated for hypoxia specific gene expression ATF4 is a basic leucine-zipper (bZip) transcription factor, which regulates amino acid metabolism, cellular redox state, and anti-stress responses ATF4 is over-expressed in hypoxic areas of tumors ATF4 is induced under hypoxia, which was mediated by the ODD domain To evaluate the ATF4 ODD domain for hypoxia specific gene expression, pSV-LucATF4 ODD was constructed For the construction of pSV-Luc-ATF4 ODD, the luciferase cDNA without the stop codon was inserted into pSV vector Then, the ATF4 ODD cDNA was chemically synthesized and inserted at the downstream of the luciferase cDNA In vitro transfection assay was performed and the cells were incubated under hypoxia or normoxia The results showed that luciferase expression was induced under hypoxia in the pSV-Luc-ATF4 ODD transfected cells RT-PCR showed that the luciferase mRNA levels were not different between normoxia and hypoxia, suggesting that the induction of luciferase expression was post-translational event For cancer therapy, pSV-TK-ATF4 ODD was constructed, in which the thymidine kinase (TK) expression was regulated by the ATF4 ODD To evaluate the TK expression under hypoxia, in vitro transfection assay was performed and the cells was treated with the ganciclovir (GCV) under hypoxia and normoxia condition The results showed that the cells transfected with pSV-TK-ATF4 ODD had lower cell viability under hypoxia than normoxia It suggests that the ATF4 ODD domain stabilized TK under hypoxia and promoted degradation of TK under normoxia However, the pSV-TK transfected cells did not show this effect In conclusion, a post-translational regulation system using the ATF4 ODD domain may be useful for the development of the cancer specific gene therapy system 550 DNA Methylation and Gene Expression in hESCs Grown in Defined and Xeno-Free Culture Systems Joshua D Tompkins,2 Christine A Hall,1 Vincent C Chen,1 Sylvana Couture,1 David Hsu,1 Larry A Couture,1 Arthur D Riggs.2 Center for Biomedicine and Genetics, City of Hope, Duarte, CA; Molecular and Cellular Biology, City of Hope, Duarte, CA Therapeutic applications for human embryonic stem cells (hESCs) will require the large-scale adaptation of cell lines to fully defined and xeno-free culture conditions Therefore, it is crucial that we first understand the interaction of hESCs and their culture environments, as well as the consequences of altering such interactions on a genome-wide scale In this study, we have comprehensibly analyzed Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy gene expression and DNA methylation profiles of hESCs during adaptation (and reverse adaptation) to multiple substrate/medium combinations: MEFs/DMEM-F12 KSR medium, Matrigel/mTeSR1, and CELLstart/STEMPRO These experimental conditions allow for the comparison of traditional hESC cultures to commercially available fully defined systems Results indicate that markers of pluripotency exhibit consistent DNA methylation and expression patterns appropriate for the stem cell state in all culture environments Yet, culture manipulation commensurately altered the expression of gene sets that appear fundamental to hESC adaptation In particular, differential expression of genes encoding membrane spanning and secreted cell adhesion factors suggest a substrate-specific membrane reconfiguration We further note, compensatory expression changes in genes controlling growth factor responses and metabolic pathways as a reaction to cellular signaling targeted by medium formulations We find that despite hundreds of differentially expressed genes between cultures, that DNA methylation remains remarkably stable, and the overwhelming majority of culture induced methylation events are highly specific and fully reversible However, DNA methylation changes were found to increase both with culture manipulation as well as with prolonged passage These sites of disparate DNA methylation were linked to expression levels of a small number of key genes involved in not only cellular adhesion, but in controlling G-protein coupled signaling, of which the latter may be responsible for the larger scale of microenvironment associated expression changes Genomewide, we identify correlations between DNA methylation and gene expression including a novel association of gene silencing and DNA hypomethylation at promoter distal sites These observations provide insight into the function of DNA methylation in hESCs Taken together, our results demonstrate that hESC acclimation to defined culture conditions is marked by a corresponding response at the gene regulation level and convey that epigenetic fidelity should be considered when maneuvering hESC cultures, especially when considering downstream therapeutic purpose 551 Evaluation of the X-Protein Truncate Expression Potential from the WPRE Element in Alipogene Tiparvovec, an AAV-Based Gene Therapy Vector Jacek Lubelski,1 Florence Salmon,1 Betty Au,1 Larbi Afia,1 Andrew Bakker,1 Harald Petry.1 Amsterdam Molecular Therapeutics, Amsterdam, Netherlands The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is a hepadnavirus sequence that is widely used as a cis-acting regulatory module in various types of plasmids and viral gene therapy vectors The WPRE is often essential to achieve sufficient levels of expression with integrative retroviral gene therapy vectors WPRE has been under scrutiny in the last years because parts of its sequence overlap with that of the woodchuck hepatitis virus X protein (WHx) which has been suspected of being implicated in the development of liver tumours that is thought to be linked with the expression of a truncated X-protein Here we have evaluated the potential of X-protein truncate expression upon transduction of two cell lines with GlyberaTM (Alipogene tiparvovec) GlyberaTM is an adeno-associated virus based vector encoding the Ser447X variant of the human lipoprotein lipase gene and containing a WPRE Thus far there are no reports of WHx truncated expression alone from WPRE in an AAV based vector in relation to the generation of tumours However, one report indicates the expression of an aberrant fusion protein comprising the WHx C-terminal truncate from a WPRE present in an AAV vector and the generation of tumours (Embury et al., 2008) We have adopted the same methodology by trying to detect the WHx-protein expression with MAb 8429 (Millipore), a monoclonal antibody directed against the human HBx protein, targeting amino acids 50-88 This antibody was claimed by Embury et al., to crossS211 OLIGONUCLEOTIDE & RNAI THERAPEUTICS I react with WHx In order to confirm this assumption, plasmids harbouring HBx (positive control), WHx and WHx 82aa truncate under the strong CMV promoter were synthesized These plasmids were used to express the corresponding proteins after transfection in HepG2 and C2C12 cells Additionally, in order to determine if WHx was properly expressed, if not recognized by MAb 8429, it was also his-tagged Here we show that the MAb 8429 directed against HBx failed to recognize either the full WHx protein or its truncate The lack of cross reactivity of MAb 8429 with the WHx-protein invalidates the approach to detect WHx in our experimental settings Therefore a polyclonal antibody was used, which adequately recognized the plasmid expressed WHx Only with this antibody it became possible to investigate the expression of the WHx C-terminal truncate successfully Subsequently, we demonstrated that no X-protein expression can be detected in the HepG2 and C2C12 cell lines using this appropriate tool after transduction with alipogene tiparvovec Oligonucleotide & RNAi Therapeutics I 552 Abstract Withdrawn 553 Efficient Delivery of siRNA to Brain Capillary Endothelial Cells with Endogenous Lipoprotein In Vivo Hiroya Kuwahara,1 Kazutaka Nishina,1 Kie Yoshida,1 Tomoko Nishina,1 Wenying Piao,1 Hidehiro Mizusawa,1 Takanori Yokota.1 Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan Brain capillary endothelial cell (BCEC) is a major functional component of blood-brain barrier and is associated with pathophysiologies of diseases including brain ischemia, multiple sclerosis, and neurodegenerative disorders We explored a genesilencing method in BCECs by introducing short-interfering RNA (siRNA) using endogenous lipoprotein in vivo Cholesterolconjugated 21/23-mer siRNA targeting organic anion transporter (OAT3) mRNA (Chol-siOAT3) was intravenously injected to mice after incorporation into extracted endogenous lipoproteins CholsiOAT3 was not delivered to neurons or glia, but was successfully delivered to BCECs with a significant reduction of OAT3 mRNA expression level when injected after incorporation into high density lipoprotein (HDL) An efficient delivery was not achieved, however, when CholsiOAT3 was injected without lipoproteins or after incorporation into low density lipoprotein (LDL) Investigations with apolipoprotein E (ApoE)-deficient and LDL receptor (LDLR)-deficient mice indicated that the uptake of HDL containing Chol-siOAT3 was mainly mediated by ApoE and LDLR in mice These findings indicate that siRNA can be delivered to BCECs using endogenous lipoprotein and thus, this strategy is applicable as a new gene-silencing therapy for the diseases related to BCECs 554 Chemically Modified Antisense Oligonucleotides Can Direct Transcriptional Regulation of Gene Expression Stuart E Knowling,1 Anne-Marie Turner,1 Kevin V Morris.1 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA Small non-coding RNAs have been shown to guide epigenetic silencing complexes to target loci in human cells When these targeted loci are located at a gene promoter the result of such targeting can lead to long-term stable epigenetic silencing of gene transcription To date, small RNAs have been shown to modulate transcriptional gene silencing of HIV-1 as well as several other genes but it has remained unknown as to what extent particular chemistries can be used to generate single stranded backbone modified oligonucleotides that are amenable to this form of gene targeting and regulation We present data here suggesting that specific combinations of backbone modified chemistires can be used to generate single stranded antisense oligonucleotides which can functionally direct transcriptional gene silencing of HIV-1 and the proto-oncogene PLK-1 These data suggest that backbone chemically-modified oligonucleotide based approaches could be implimented as a means to epigenetically regulate a specific genes transcription, in this case HIV-1 and PLK-1 expression Exapnding upon these observations one could surmise that virutally any actively transcribed gene in the human cell could be suscpetible to modulation by backbone chemically-modified oligonucleotides S212 Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... show that the MAb 8429 directed against HBx failed to recognize either the full WHx protein or its truncate The lack of cross reactivity of MAb 8429 with the WHx -protein invalidates the approach... expression of the WHx C-terminal truncate successfully Subsequently, we demonstrated that no X- protein expression can be detected in the HepG2 and C2C12 cell lines using this appropriate tool after transduction... WHx in our experimental settings Therefore a polyclonal antibody was used, which adequately recognized the plasmid expressed WHx Only with this antibody it became possible to investigate the expression