339 Autoregulation of Transposition of Sleeping Beauty Transposons by Ubiquitous N Terminal, Dominant Negative Peptides DNA VECTORS FOR NON VIRAL GENE THERAPY 337 Mutational Derivatives of PhiC31 Inte[.]
DNA VECTORS FOR NON-VIRAL GENE THERAPY 337 Mutational Derivatives of PhiC31 Integrase with Increased Efficiency and Specificity Annahita Keravala,' Solomon Lee,' Bhaskar Thyagarajan.? Eric C Olivarcs.! Vanessa E Gabrovsky,' Lauren E Woodard,' Michele P Calos.' 'Genetics, Stanford University, Stanford, CA; lGenetics, Poetic Genetics LLC, Burlingame CA The phiC31 integrase system has emerged as a promising nonviral delivery strategy for gene therapy The level and duration of gene expression achievable with phiC31 integrase, as well as its safety observed in a wide variety of in vivo applications, makes it an attractive strategy for clin ical development of gene therapies for a number of diseases The phiC31 integrase is a phage integrase that functions in mammalian cells to provide chromosomal integration that is useful for gene therapy and other applications In its normal role , phiC31 intcgrasc pairs a short attll sequence with a sequence having 50% identity to attll, called attl', PhiC31 integrase can also promote integration of plasmids carrying attb at native mammalian sequences with partial identity to all? called pseudo all? sites, with integration frequencies in the range of - - 10% There may be as many as several hundred pseudo att]' sites in the human genome in cultured cells, though fewer sites arc probably used in vivo In order to increase the efficiency and specificity of integration, we mutated the phiC31 integrase gene and screened the mutants in human cells in a rapid assay for higher recombination frequency between attli and attl', We report the isolation ofa mutant, P2 that has twice the chromosomal integration frequency of wild-type phiC31 integrase, at both a chromosomal pre-integrated attl' site and at pseudo attl' sites in cultured human cells, with no apparent change in specificity We demonstrated the utility of this mutant also in mouse liver, where it provided twice the long-term serum levels of human factor IX, compared to wild-type phiC31 integrase, from a human factor IX gene delivered to hepatocytes by hydrodynamic injection We also describe a mutant, P3 that combines the higher efficiency mutant P2 with further changes and possesses higher specificity for integration at a chromosomally-placed attl' site With P3, 44% of integration occurred at one position in the genome, compared to - 5% specificity for this site with the wild-type enzyme These mutants are useful in gene therapy and genome engineering, and they demonstrate the potential ofmodifying phiC31 integrase toward more desirable properties 338 Terminal Inverted Repeats of the Sleeping Beauty DNA Transposon Possess Residual Promoter-Like Activities Brian Moldt, I Peter R Andersen,' Stephen R Yant,' Mark A Kay,2 Jacob G Mikkelsen.' 'Department ofHuman Genetics, University ofAarhus, Aarhus, Denmark; lStanford School ofMedicine, Stanford University; Stanford Sleeping Beauty (SB) DNA transposon-based vectors belong to a growing family of non-viral gene-integrating vectors that represent attractive alternatives to conventional virus-based integrating gene vehicles In early studies of gene transfer vectors based on the SB transposon we found indications that the terminal inverted repeat (IR)-sequences are able to drive gene expression of downstream genes To test the hypothesis that transposon IRs can act as promoters we carried out expression studies based on reporter constructs containing the Firefly luciferase (Iuc) gene In separate constructs, DNA sequences containing the left IR (LlR) and right IR (RIR) of SB were inserted in front of the reporter gene To detect potential orientation differences LlR and RIR were tested in both 'inwards' SI28 (pointing towards the transposon center) and ' outwards' (pointing away from the transposon body) orientations In initial studies carried out in HeLa cells we observed a 3-fold increase in expression compared to levels obtained with a promoter-deficient control construct The LlR-inwards and in particular the RIR-inwards sequences were consistently found to facilitate highest levels of activity in a number oftransfected cell lines The inverted repeats ofcommonly used SB-based vectors are flanked on the inner side by short sequences (about 150·bp long) derived from the transposase gene ofthe original Tanichthys albonubes element To investigate a potential importance ofthese regions we generated deletion mutants containing only the IR sequences (defined by the terminal direct repeats in each IR) To our surprise, these shorter promoters did not support transient luc expression To further characterize the role of this DNA segment we created a panel of 30-bp deletion mutants based on the RIR Luc expression assays revealed the existence of both transcriptional enhancer and repressor elements in this region of the SB vector In addition, IR-delctcd variants were inactive, indicating that promoter activity relied on sequences within the IR The impact of IRs with residual promoter activity on expression of transgenes from integrated SB vectors is currently not known We have demonstrated, however, that expression from SB-IRs is sufficient to drive expression of a selectable marker gene, located inside the transposon, indicating that the transposon termini ofa SB vector may influence expression of its genetic cargo We conclude that the SB invertedrepeatspossess promoter-like activities and that this activity is further enhanced by the sequences flanking the inner direct repeat of both LlR and RIR 339 Autoregulation of Transposition of Sleeping Beauty Transposons by Ubiquitous NTerminal, Dominant Negative Peptides Jeffrey M Schreifels,' Jason B Bell ,' Elena L Aronovich,' David C Erickson,' R Scott Mclvor,'? Perry B Hackett.'? 'GCD and Beckman Center for Transposon Research, Institute ofHuman Genetics, University ofMinnesota, Minneapolis, MN; 'Gene Therapy Program, Dept ofPediatrics, University ofMinnesota, Minneapolis AiN; JDiscovelY Genomics, lnc , Minneapolis, MN The Sleeping Beauty (SB) transposon system is being developed to direct the integration ofprecise DNA sequences into chromosomes for gene therapy and functional genomics studies SB transposase mediates transposition into TA sites in DNA, making it useful for gain-of-function and loss-of-function studies SB transposons leave footprints when they excise from a site in DNA, most often leaving a five base-pair sequence that would shift reading frames if it occurs in a translational open reading frame Thus, SB transposons can cause mutations not only by interrupting DNA sequences, but also by introducing frameshifts in translated sequences when they are excised From an evolutionary point of view, regulation of transposase activity is important to keep the mutational load as low as possible Moreover, Tc IImariner transposons, the family to which SB belongs, not have regul ated promoters driving their cognate transposase gene; rather, the transposase is expressed from a promoter proximal to the site of integration One way to ensure regulation of transposition to keep transposons from continually hopping is for the transposase to be auto-regulatory This can occur by over-expression inhibition Recently, we have identified a second potential mechanism for autoregulation oftransposition - production ofa N-terminal, dominant-negative cleav age product The protease responsible for site-specific e1eavage of SB transposase has been detected in many tissues of mice Western analyses of extracts of liver tissue from transgenic SB mice show a number of polypeptides of that are detected using monoelonal antibodies directed against SB transposase These antibodies also bind to the NI23 aminoMolecular Therapy Volume 15.Supplement Iã.\by 2007 Copyright â The Americ m Society {I t Gene Therapy terminal fragment of SB transposase, suggesting that the products include the amino-terminus of SB transposase We have modeled domina nt-negative inhib ition of transposition by both N 123 and ADDE, a related fragment of SB transposase Our results indicate that N-terminal cleavage products, even in low concentration, can repress transposit ion These findings suggest that the SB transposon system has an inherent ability to self-regu late and thereby reduce chances of continued hopping in cells into which an SB vector is used for gene therapy 340 Silencing of Episomal Transgene Expression in Liver by Plasmid Bacterial Backbone DNA Is Associated with Histone Modifications but Independent of CpG Methylation Efren Riu, Zhi- Ying Chen, Chen- Yi He, Hui Xu, Mark A Kay and Genetics, Stanford University, Stanford, CA I Pediatrics One ofthe major obstacles to non-viral gene therapy is transcriptional silencing ofthe DNA vector The mechanisms underlying gene silencing in mammalian cells are comp lex and remains unclear We previously demonstrated that transgene expression was transcriptionally silenced when the expression cassette was covalently linked to a plasmid DNA backbo ne Because alterations in CpG-DNA methylation and changes in chromatin structure including histo ne modificatio ns arc involved in transcriptional regulation of endogenous genes , we aimed to determine what role they may play in transgene expression from stable episomal DNA vectors in liver We used 17 antibodies against di fferent modifications present in histones in chromatin immunoprecip itation (Ch IP) analyses on liver Iysates prepared from mice transfected with different expression cassettes contained within a DNA minicirc le devoid of the bacterial plasmid backbone DNA (which showed marked persistence of trans gene expression) or their parental plasmid (which were silenced over time) Silencing of the transgenc from the parental vectors was accompanied by an increase in heterochromatin-associated histone modificat ions and a decrease in modificat ions typically associated with euchromatin Conversely, the pattern of histone modifications on the minicircle DNA was consistent with patterns observed in euchromatin This suggests that the transcr iptional state ofthe vector DNA is dependent on the type of chromatin that forms in vivo In addition, because of the high concentration of CpG dinucleotides present in plasmid DNA , we investigated the role of DNA methylation on transgcne silenci ng in vivo We compared transgene expression in vivo from plasmids containing their normal CpG content or devo id of CpG motifs in the bacterial backbone In addition, we were able to generate plasmids with CpG motifs that were unmethylated or fully methylated prior to infusion into mice Importantly, the methylation status ofthe injected plasm id DNA did not change aftertransfection into liver We observed that the silencing ofthe transgenc expression cassette was independent of the CpG content and/or methy lation status of CpG motifs in both the expression cassette and bacterial plasmi d DNA backbone We believe that episomal DNA vectors are quickly chrornatinized in vivo and that the transcrip tional fate ofthe transgene is determined by neighboring DNA sequences that result in specific histone-modification marks Interestingly, DNA methylation appears to play no role in this process Ident ification of additional chromatin proteins associated with episomal DNA vectors arc underway Ultimately, this work will be helpful in developing new tools and strategies to achieve persistent therapeutic gene expression in a clinical setting Molecular Therapy Volume t:;,Supplcmm t 1•.\1",.2007 O:lprrighf