1151 Effects of Steroid Drugs in Glioma Cell Lines Transduced with Ad egr 1 Luciferase Vector Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������®������������ �!����� ����"� �����[.]
GENE REGULATION: PROMOTER AND CONSTRUCT DESIGN subcutaneous space of nude mice and syngeneic, non-eGFP C57Bl/ mice The constructs were recovered at and weeks and assessed for GRP using direct fluorescence microscopy and immunohistochemistry GFP bearing cells were present but diminished in number in both the non-eGFP C57/BL6 mice and in the nude mice Loss of GFP bearing cells was time dependent in both model systems These experiments establish that there is turnover of cells in this dermal construct in both syngeneic and immunodeficient mice Thus, it would appear that the microenvironment of the nylon matrix and not species differences accounts for the observed loss of TGE and GMFb in vivo 1149 Optimization of Plasmid Based Gene Delivery to Murine Skeletal Muscle P Szymanski,1 F Jin,1 K Reiter,1 P Liu,2 H S Qian,2 K Kauser,3 R Harkins,1 G Rubanyi,1 T Hermiston,1 P Kretschmer.1 Gene Therapy Dept, Berlex Biosciences, Richmond, CA; Pharmacology Dept, Berlex Biosciences, Richmond, CA; Cardiovascular Dept, Berlex Biosciences, Richmond, CA Although electroporation of plasmid-injected muscle results in 100 to1000 fold increases in gene expression compared to muscle injected with plasmid alone, to date there are no reported clinical studies in which electroporation is used to deliver plasmids to skeletal muscle for gene therapy Consequently, the initial clinical applications of electroporation will involve considerable effort In order to maximize gene expression in human skeletal muscle without electroporation, we have identified optimal promoter constructs and polymer formulations for gene expression following injection of plasmid DNA We first evaluated the effect of a variety of poloxamers on gene expression in the mouse adductor muscle in vivo We found that the most effective poloxamers were F68, L44 and 25R2, which routinely increased gene expression up to ten-fold compared to injection of naked DNA To optimize the promoter driving transgene expression we compared a series of human CMV IE1 (hCMV) promoters to a variety of control promoters both in vitro and in vivo In addition to the commonly used pcDNA3.1 promoter, we constructed the fulllength human CMV promoter (fl hCMV containing the complete CMV enhancer, first exon and intron) and a hCMV promoter containing complete enhancer, 50% of the first exon and synthetic intron (sh hCMV) In vitro (SkMC, HAEC, EOMA and differentiated/ undifferentiated C2C12 cells), the fl and sh hCMV promoters were of similar strength and both ranged from – 10 fold the strength of the pcDNA3.1 promoter in different cell lines In vivo (mouse adductor muscle), the fl promoter was about two fold stronger than the sh hCMV promoter and approximately ten fold stronger than the pcDNA3.1 promoter Finally, we have formulated the optimal fl hCMV promoter plasmid with the poloxamers and demonstrated that the promoter and polymer formulation effects are additive in vivo 1150 Understanding Transcriptional Regulation of Exogenously Delivered DNA C L Bishop,1 M Ramalho,1 A A McBride,2 C F Higgins,1 N Krauzewicz.1 MRC Clinincal Sciences Centre, Imperial College London, London, United Kingdom; 2Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD A major challenge facing gene therapy is achieving long-term expression of desired genes in vivo Currently, no gene delivery system is ideally suited to this purpose Viral vectors are able to Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy provide long-term expression, but have raised safety concerns due to inherent toxicity and the possibility of generating recombinant virus Mouse polyoma capsid-like particles (CLPs), composed solely of the recombinant viral coat protein VP1, offer an alternative gene delivery vehicle CLPs can interact with plasmid DNA in cell free systems to form virus-like particles (VLPs), which deliver plasmid DNA to cell nuclei This delivery follows a ‘specific’ virus-like pathway, in contrast to ‘non-specific’ routes typical of non-viral systems Thus, while CLPs maintain a number of the advantages of viral based systems, they lack associated viral toxicity and are easy to generate Delivery of DNA to cells by VLPs has been established by PCR both in vitro and in vivo However, only low levels of reporter gene expression are detected (Krauzewicz et al., 2000 Gene Ther 7:1094-1102) Analysis of DNA from transfected cells suggests that plasmid DNA is silenced following chromatinisation Treatment of transfected cells with histone deacetylase inhibitors relieves repression, resulting in high levels of reporter gene expression Similar regulation is observed when these inhibitors are applied to cells treated with DNA alone, or as polyethylenimine complexes Thus, such regulation may also be exerted on DNA delivered by other methods This study aims to determine whether modifying plasmid design can relieve this repression of exogenous gene expression Replication of DNA results in a ‘loosening’ of chromatin conformation and may expose transcription start sites Thus, plasmid design was adapted to include the simian virus 40 (SV40) origin of replication (ori), and constructs were transfected into cells expressing SV40 viral replication protein, LT This resulted in reporter gene expression levels similar to those observed following calcium phosphate transfection and depended on replication of the input DNA These findings raise a number of questions For example, if chromatin conformation plays a role in exogenous DNA silencing, will using other nucleotide sequences that alter this conformation change the pattern of silencing of cells in vivo? Studies are underway to address this question Further, we asked if viral replication proteins could be exploited to specifically facilitate exogenous gene expression in cancerous cells To examine this, a cervical cancer cell line expressing papilloma virus replication proteins E1/E2 was transfected with a plasmid DNA contain the papilloma ori However this did not result in high levels of reporter gene expression Unlike the SV40 LT, the E1/E2 proteins are associated with cellular chromosomes Parallel characterisation of our system by fluorescence microscopy, suggests that VLPs deliver DNA to a specific nuclear location, distinct from the chromatin Thus, exogenous DNA entering cells may be subjected to several levels of transcriptional regulation: silencing may occur following chromatinisation, but further regulation of expression may be imposed by spatial positioning of the DNA once inside the nucleus 1151 Effects of Steroid Drugs in Glioma Cell Lines Transduced with Ad-egr-1 Luciferase Vector Francisco Martinez-F,1,2 Joanne T Douglas,3 Andres A Gutierrez-L.1 Cell Therapy Unit, Centro Nacional de Rehabilitacion, Mexico, DF, Mexico; 2Department of Pharmacology, School of Medicine, UNAM, Mexico, DF, Mexico; 3Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, United States Introduction Transcriptional control of genes delivered by adenoviral vectors in target cells has recovered importance in gene therapy research Several inductors have been reported to modulate transcriptional activity of egr-1 promoter Recently, receptors for steroids hormones on the cellular membrane surface of glioma cells have been identified Based on this approach, we explore the effect of several steroid derived drugs on glioma cell lines transduced with adenoviral vector containing luciferase reporter gene drived by –600 pb fragment of S443 GENE REGULATION: PROMOTER AND CONSTRUCT DESIGN egr-1 promoter Material and Methods Adenoviral vectors were generated based on the AdEasy system and homologous recombination in bacterials –600 pb fragment of egr-1 promoter was cloned upstream of the luciferase gene into the MCS of pShuttle plasmid Adenoviral plasmids resulting was screened, sequenced and packaged into HEK293 cells using effectene transfection system (Quiagen) Adenoviral large scale stock was screened, purified and titer for further experiments Glioma cell lines (CH235, D54 and U373) were grown in D-MEM media containing 10% of FBS and 1% Antibiotics in standard conditions 1x 105 cells were seeded and infected with 25 MOI for hours in low serum media After that, cells were washed and maintained in D-MEM media supplemented with % of FBS for 24 hrs before to start induction with 100 ng/ml of Betamethazone, 100 ng/ml of b-Estradiol and 50 ng/ml of Progesterone as previously reported Unstimulated and infected cells were maintained in % FBS and used as basal control Proteins were purified using Cell Culture Lysis (Promega) and stored at –20° C Luciferase Activity was quantified using Luciferase Assay System according to manufacturer instructions (Promega coorp.) in a Victor Wallac instrument All experiments were performed for triplicate Results and conclusions In CH235 cell line induction of activity reaches a maximum expression six hours after induction with BEstradiol and this level is maintained until 12 hrs These activities compared to basal expression means 2.7 holds of luciferase activity D54 cells not show significative differences of luciferase activity compared to basal with three inductors Finally, U373 cells show a slightly increase of luciferase activity with Betamethazone, bEstradiol and Progesterone at hours after induction 1152 Characterization of Copy Numbers of βActin and IFNγγ Genes in Mouse Tissues with Real Time Quantitative PCR Baohong Tian,1 Sarah S Rentz, Amanda P Cunningham, Tina Rogers, John G Page Safety Assessment Department, Southern Research Institute, Birmingham, AL, United States Characterization of copy numbers of some specific DNA sequences in animal tissues appeared to be impractical until the presence of real time quantitative PCR (Q-PCR) Since both βActin and IFNγ are highly interesting genes with a substantially different amount of gene products, we designed Molecular Beacon probes and their corresponding primers for both sequences to characterize their copy numbers in mouse tissues Tissues from both male and female mice, including cerebrum, heart, lung, kidney, liver, spleen, stomach, small intestine, ovary and testes, were collected and lysed through homogenization in GITC solution DNA was isolated with QIAGEN DNeasy Tissue Kit (Valencia, CA) and quantitated with PicoGreen (Molecular Probes, Eugene, OR) The copy numbers of both standards, a commercial DECAtemplate (Ambion, Austin, TX) containing mouse β-Actin cDNA sequence and a plasmid containing mouse IFNγ sequence, were calculated on the basis of the size and concentration and validated by limiting dilution assay (LDA) The copy numbers of β-Actin and IFNγ in a variety of tissues, based on their standards, were detected with real time Q-PCR (LightCycler, Roche Molecular Biochemicals, Mannheim, Germany), and then normalized on the basis of the DNA amount In addition, two DNA samples from cerebrum and ovary were tested with LDA to validate the copy numbers of both β-Actin and IFNγ The copy numbers of the standards validated by LDA with QPCR were consistent with the calculated copy numbers Based on the standard curve, the copy numbers of β-Actin and IFNγ detected in a variety of tissues from four mice and normalized with DNA amount were constant among different tissues for both β-Actin and IFNγ Surprisingly, the copy numbers of mouse β-Actin detected in S444 all these tissues was over tenfold higher than the expected two copies per 6.6 pg DNA (DNA amount per cell) In contrast, the copy numbers of mouse IFNγ had an average of copies per 6.6 pg DNA, close to the expected two copies Furthermore, LDA for both β-Actin and IFNγ in cerebrum and ovary supported the result that the same amount of DNA had tenfold different copy numbers between β-Actin and IFNγ The following table shows the copy numbers of both β-Actin and IFNγ per 6.6 pg DNA in ten tissues from four mice (Mean±SE) Tissues Cerebrum Heart Lung Kidney Liver Copies / 6.6 pg DNA β-Actin IFNγ 38.4±3.0 3.1±0.8 39.0±2.7 3.1±0.8 39.6±4.3 3.5±0.7 36.6±2.7 3.3±0.8 34.2±3.4 3.2±0.7 Tissues Spleen Stomach Small Intestine Ovary Testes Copies / 6.6 pg DNA β-Actin IFNγ 31.2±5.0 3.2±0.6 35.4±2.9 3.3±0.9 36.2±2.7 3.4±0.8 29.9±4.9 2.0±0.1 34.6±2.3 3.6±0.8 This result successfully demonstrates that multiple gene sets of mouse β-Actin related sequences, possibly including many pseudogenes, were detected in mouse genome with Q-PCR Additionally, a single set of mouse IFNγ was also quantitated as predicted Altogether, this study validates the sensitivity, specificity, and accuracy of Q-PCR technology and supports its utility for its extensive applications 1153 Strand Bias in Gene Repair Using SingleStranded Oligonucleotides Charlotte B Sorensen,1 Anne-Margrethe Krogsdam,2 Karsten Kristiansen,2 Lars Bolund,1 Thomas G Jensen.1 Department of Human Genetics, University of Aarhus, Aarhus, Denmark; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Targeted gene conversion is a method developed for site-specific correction of dysfunctional target genes The method is based on the interaction of either chimeric RNA/DNA or modified single-stranded oligonucleotides with their homologous target genes thereby generating a single mismatched nucleotide base pair This mismatch is believed to be recognized by the endogenous repair system resulting in excision and substitution of the mismatched base using the oligonucleotide sequence as template Although the potential of this technique is enormous, it still faces many obstacles such as the relatively low correction efficiencies obtained as well as low reproducibility among and even within laboratories In order to optimize the efficiency, we are investigating the effect of substituting deoxyribonucleotides in single-stranded oligonucleotides with synthetic DNA-analogues with increased affinity and specificity As a model system for testing designs of oligonucleotides, we use an episomally expressed beta-galactosidase gene Oligonuclotides of varying lengths and polarity have been investigated with regard to correction efficiency Based on the most efficient design, new oligo designs with synthetic DNA-analogues have been made and are being tested It has previously been shown that antisense oriented singlestranded oligonucleotides results in a higher conversion frequency compared to its sense oriented counterpart Using standard DNAcontaining oligonucleotides, we have found that this holds true for only one of the beta-galactosidase mutations studied For the other mutation, the highest efficiency is obtained using the sense oriented oligonucleotide suggesting that limited accessibility due to presence of transcription complexes is not the only cause of the observed strand bias Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy ... Glioma cell lines (CH235, D54 and U373) were grown in D-MEM media containing 10 % of FBS and 1% Antibiotics in standard conditions 1x 10 5 cells were seeded and infected with 25 MOI for hours in. .. that, cells were washed and maintained in D-MEM media supplemented with % of FBS for 24 hrs before to start induction with 10 0 ng/ml of Betamethazone, 10 0 ng/ml of b-Estradiol and 50 ng/ml of Progesterone... and infected cells were maintained in % FBS and used as basal control Proteins were purified using Cell Culture Lysis (Promega) and stored at –20° C Luciferase Activity was quantified using Luciferase