348 A Novel Jurkat LMO2 Assay System for Vector Safety Testing and Insulator Screening Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy S136[.]
RNA VIRAL VECTORS 346 Identification of a Domain Responsible for the Dose-Dependent Antiproliferative and Apoptotic Effects of Sleeping Beauty Transposase Melanie Galla,1 Zsuzsanna Izsvak,2 Christine S Falk,3 Kathrin Thomay,4 Gudrun Göhring,4 Zoltan Ivics,5 Axel Schambach,1 Christopher Baum.1 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2Division of Mobile DNA, Max Delbrueck Center, Berlin, Germany; 3Institute of Transplantation Immunology, Hannover Medical School, Hannover, Germany; Institute of Cell- and Molecular Pathology, Hannover Medical School, Hannover, Germany; 5Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany The Sleeping Beauty transposase (SB) and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy Potential off-target effects of transposases require careful assessment, as other DNA-modifying enzymes have been associated with dose-dependent cytotoxicity Furthermore, residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and result in uncontrollable transposition We compared retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, and found that the overexpression of the SB transposase may trigger premitotic cell cycle arrest followed by apoptosis Importantly, retrovirus particle-mediated mRNA transfer (RMT) prevented cytotoxicity by expressing the SB transposase in a transient and dose-controlled manner Cytotoxic effects induced by continued overexpression of the transposase were strictly dosedependent and occurred even in the absence of a co-transfected transposable element High and prolonged levels of SB were selflimiting due to the induction of caspase-mediated apoptosis within a few days after transduction We observed substantial induction of p53 and c-Jun as well as increased numbers of gH2AX foci, suggesting genotoxicity as the underlying mechanism (Galla et al., NAR 2011) However, inactivating mutations of the SB transposase DDE catalytic triad or the nuclear translocation signal could not abrogate cytotoxicity, indicating that the cytotoxicity cannot be associated with direct DNA damage inflicted by the transposase This promoted studies in which we introduced serial deletions of individual transposase (sub-)domains to determine their impact on intracellular distribution, cell cycle arrest, apoptosis, double strand breaks and stress signaling We were thus able to identify a small domain comprising 16 amino acids that is largely responsible for the cytotoxic effects, which will hopefully allow us to generate variants with a greater “therapeutic index” When using conventional SB or SB100X, we recommend reducing the level and duration of transposase expression via RMT or other transient expression methods to avoid the previously described overproduction inhibition and the newly discovered cytotoxicity 347 Addition of a Modified polyA Signal and Intron into a Lentiviral Platform for RNAi and Protein Over-Expression Provides Potent Efficacy While Maintaining High Vector Titers Ulrike Jung,1,2 Kellen C Fae,2 Juan P Patron,2 Stefan HE Kaufmann,2 John J Rossi.1 Molecular & Cell Biology, Beckman Research Institute of City of Hope, Duarte; 2Immunology, Max Planck Institute of Infection Biology, Berlin, Germany RNAi (RNA interference) as well as protein over-expression are widely used tools in research as well as gene therapy with viral vectors a common delivery vehicle When targeting primary cells effective expression of transgenes is a major problem due to cell S136 and tissue restrictions on promoter function This is especially true when targeting hematopoietic cells like CD34+ progenitors and descendants Another problem is the risk of perturbed expression of genes downstream of a strong promoter Previous publications using addition of polyA signals to prevent read-through interference have reported decreases in virus titers We have sought to address these issues by the use of a splicing based lentiviral platform that combines a pol II promoter for minimized toxicity and cell specific expression, shRNAs derived from intron based pre-microRNAs, exon based protein expression, a modified polyA signal for minimal downstream effects incorporated into a 3rd generation SIN lentiviral vector Thus this construct offers parallel expression of both mi/shRNAs as well as proteins to minimize the number of promoters required for transgene expression In this context the effects of several 3’ UTR modifications on gene expression as well as virus titer were tested Knockdown efficacy of different pre-miR30 variants was compared Our results show that a modified polyA signal does not decrease the vector titer When combined with the WPRE it significantly increases the exon expression and has a tendency to increase the titer We compared the effects of construct expressing human endogenous or artificial premiRNAs with control constructs lacking miRNAs In these studies we measured the processing efficacy and miRNA expression levels and found that while the addition of the intron decreases the titer somewhat it still remains high (average 3*107TU/ml with maximum 1*108TU/ml) This combinatorial platform was used for expression of shRNAs as well as endogenous miRNAs and target knockdowns of up to 80% were demonstrated in several cell lines The efficacy of the system in human CD34+ derived dendritic cells infected with M tuberculosis or the tuberculosis vaccine strain BCG has been demonstrated In conclusion our platform can be used for RNAi and protein based gene therapy as well as combinations of the two and should be useful for a variety of gene therapy applications 348 A Novel Jurkat-LMO2 Assay System for Vector Safety Testing and Insulator Screening Sheng Zhou,1 Taihe Lu,1 Zhijun Ma,1 John T Gray,1 Brian P Sorrentino.1 Department of Hematology, St Jude Children’s Research Hospital, Memphis, TN Insertional activation of the proto-oncogene LMO2 by the enhancers in gamma-retroviral vector LTRs have caused multiple cases of T-cell leukemia in gene therapy trials for selected immunodeficiencies One approach to avoid this complication is the use of cellular promoters that lack enhancer function; however, new vectors may require the use of viral or cellular enhancers to achieve adequate expression levels of the transgene Another approach is to incorporate chromatin insulator elements to block inadvertent interactions with adjacent cellular promoters However, relatively few insulator elements have been tested for this purpose, particularly in the context of T-cell gene therapy The much-studied cHS4 insulator only reduces, but does not eliminate insertional gene activation by LTR enhancers in human T cells The goal of this study is to develop an effective cellular assay to screen for and identify more effective insulators for use in T-cell disorders An AAV-vector was used to target a site upstream of both LMO2 promoters to recreate a previously described insertion site that was associated with leukemia in patient #5 (P5) in the Paris gene therapy study Homologous recombination with this vector inserts a retroviral LTR driving a mCherry cDNA in an antisense orientation to LMO2 Shuttle exchange of different constructs is possible due to the inclusion of non-homologous loxp sites (Figure1) After transducing Jurkat cells with this AAV vector, we isolated five out the 900 clones that contained the desired targeted insertion into the P5 locus All clones expressed high levels of mCherry, proving that the retroviral LTR is active in this position Quantitative RT-PCR showed that LMO2 mRNA was variably increased in all clones, at Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy RNA VIRAL VECTORS to 59% of that seen in K562 cells Western blot analysis of clones P5G27a, P5G27e and P5G27f showed significant LMO2 protein expression, verifying that LTR activation of the LMO2 allele had occurred (Figure1) We are now using these P5G27 clones to evaluate the activity of candidate insulator elements, such as the T-cell derived Bead element, using Cre-mediated homologous recombination to replace the Loxp-LTR-mCherry-Lox511 with Loxp-Insulator-LTRCerulean-Lox511 To adapt this system for high complexity screens of candidate insulators identified by chromatin immunoprecipitation, we are targeting the P5G27 clones to insert an IRES-GFP cassette into the 3’ untranslated region of the LMO2 gene This should provide a sensitive system for identifying clones with insulator activity and hence, loss of GFP expression Our plans are to pull down CTCFbinding sequences from human T cells, library clone these elements into a Cerulean exchange plasmid in a position between the LTR and LMO2 promoters (Figure1), and isolate Cerulean+, GFP- clones to clone out potential new insulators for further testing 349 A Cell-Based Assay To Detect Rare Psi-Gag Recombinants in Lentiviral Vector Preparations Seraphin Kuate,1 Michael P Marino,1 Jakob Reiser.1 Division of Cellular and Gene Therapies, FDA/CBER, Bethesda, MD For many of the advanced HIV-1-based lentivirus vectors, there is considerable overlap between sequences present in the packaging construct and sequences present in the vector genome Such regions of overlap have been shown to result in recombinant vectors (PsiGag recombinants) that can potentially lead to the generation of replication-competent lentivirus (RCL), an outcome that is clearly not desirable for clinical-grade vectors Psi-Gag recombinants are typically detected by PCR using genomic DNA from transduced cells A limitation of this method is that only a small fraction of the total genomic DNA can be sampled Our goal is to generate HIV-1based lentiviral vectors that are less prone to the formation of PsiGag recombinants Toward this goal we have designed a sensitive assay capable of detecting such recombinants The assay involves a packaging construct containing a blasticidin (BSD)-resistance gene inserted at the vpr locus In the event of a recombination event in the gag and RRE regions of the vector and the packaging construct, the BSD resistance gene is transferred to the vector genome Upon transduction of target cells with such vector particles, the BSDresistance gene confers BSD resistance to the transduced cells The occurrence of the predicted recombination events is verified by PCR using genomic DNA from BSD-resistant cell clones Our results indicate that one such recombinant in a total of 104 transduced cells can be detected In order to reduce the sequence overlaps between the packaging and the vector construct we have substituted the RRE Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy present in the vector construct using heterologous RRE elements The usefulness of this assay to detect Psi-Gag recombinants in lentiviral vectors attenuated in this way is currently being assessed 350 Vector Design-Dependent Modulation of Viral-Cellular Chimeric Fusion Transcripts Derived from Self-Inactivating (SIN) Lentiviral Vectors David Almarza,1 Fernando Larcher,1 Rodolfo Murillas.1 Epithelial Biomedicine, CIEMAT, Madrid, Spain The occurrence of chimeric viral-cellular fusion transcripts derived from integrated proviruses in keratinocytes transduced with self-inactivating (SIN) retroviral vectors is an emerging mechanism of genotoxicity that must be addressed in cutaneous gene therapy preclinical studies Readthrough-dependent fusion transcripts with different structures originate from K14-driven vector proviruses and their relative abundance depends on cellular RNA stability-regulating mechanisms We showed that transcripts containing non-coding sections of the genome fused to vector sequences are the predominant chimeric transcripts derived from SIN lentiviral vectors, while transcripts containing gene coding sequences are short-lived due to their downregulation by the Nonsense-Mediated Decay (NMD) system These vector-cellular-exons fusion transcripts are generated by splicing from a dominant, cryptic site located in a residual dNef fragment present in many HIV-1-derived lentiviral vectors Due to the structure of these transcripts and their reduced stability it is very unlikely that they can cause the inappropiate expression of cellular proteins or protein fragments, and the risk that they pose should therefore be small However, in the present study we show that mutations eliminating the dominant splice donor site in dNef change the pattern of fusion transcript generation from SIN vectors, resulting in the generation of transcripts encoding transgene-cellular fusion proteins These transcripts evade the NMD mechanism of transcript stability control and can therefore result in the expression of potentially troubling fusion proteins We have shown that very small changes in SIN vector design may cause substantial modifications in fusion-transcript related insertional mutagenesis, which should be considered during preclinical assessment of new gene therapy vectors 351 Directed Integration of Insulated Lentiviral Vectors to the Heterochromatin towards Safer Gene Transfer to Stem Cells Alexandre Artus,1 Caroline Duros,1 Yaïr Botbol,1,2 Simone Scholz,3 Emilie Bayart,1 Manfred Schmidt,3 Christof von Kalle,3 Marc Lavigne,2 Odile Y D Cohen-Haguenauer.1,4 Gene Therapy Programme, LBPA, Ecole Normale Superieure (ENSC), Cachan, France; 2Laboratory Joliot Curie, ENS-Lyon, Lyon, France; 3Laboratory of Translational Oncology, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany; 4Medical Oncology, Hôpital St-Louis and University Paris-Diderot, Paris, France We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing integration to heterochromatin Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, the insulated lentivector which shows efficient and stable expression into human primary cells: 4xIns2-DCaro4 (Duros et al, submitted), has been further tested and compared to its noninsulated counterpart, with chimeric HIV-1 derived integrases targeting heterochromatin through either histone H3 or methylated CpG islands (ML6 & ML10 chimeras, respectively) With DCaro4 and ML6 chimeras, a homogeneous expression is sustained over time With the control, GFP expression is just over background doublemutant in catalytic and ledgf binding-sites while expression can be induced with HDAC In CD34+ cells from cord-blood, these data S137 ... lentiviral vectors that are less prone to the formation of PsiGag recombinants Toward this goal we have designed a sensitive assay capable of detecting such recombinants The assay involves a packaging... Lentiviral Vector Preparations Seraphin Kuate,1 Michael P Marino,1 Jakob Reiser.1 Division of Cellular and Gene Therapies, FDA/CBER, Bethesda, MD For many of the advanced HIV-1-based lentivirus vectors,... the LTR and LMO2 promoters (Figure1), and isolate Cerulean+, GFP- clones to clone out potential new insulators for further testing 349 A Cell-Based Assay To Detect Rare Psi-Gag Recombinants in