127 PROGNOSE: an online tool for predicting and analyzing the off target site effects of designer nucleases

127 PROGNOSE An Online Tool for Predicting and Analyzing the Off Target Site Effects of Designer Nucleases Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene[.]

DNA VECTOR DEVELOPMENT & GENE TARGETING I attributed to expression of HAC-encoded genes This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications 125 Novel Antitumor Strategy by Transfection of Plasmids Encoding Pathogenic Antigen- and Cytokine-Genes Yoshiyuki Koyama,1 Chieko Yoshihara,1 Tomoko Ito,1,2 Masazumi Eriguchi.3 Otsuma Women’s University, Tokyo, Japan; 2Musashino University, Nishitokyo City, Japan; 3Shin-yamanote Hospital, Higashi-Murayama City, Japan Although several viral vectors harboring cytokines have been successfully employed to induce tumor-regression, non-viral vectors having the same cytokine genes showed lower therapeutic efficacy One possible reason for this limited efficacy would be low transfection efficiency of the non-viral systems However, it may be difficult to induce immune response to tumor cells with low immunogenicity only by cytokine-production Expression of the viral antigenic proteins on the infected tumor cell surface would be essential to stimulate APCc Not only the virus infection, but transfection of the virus- or bacteria-specific antigenic protein genes could also induce the pathogenic antigen presentation on the tumor cell surfaces Simultaneous transfection with plasmids expressing cytokine and pathogenic antigen into tumor cells should, thus, be expected to lead to highly effective anti-tumor immune-enhancement Here, we employed two pathogenic proteins, adenovirus death protein (ADP) and mycobacterium tuberculosis early secretory antigenic target-6 protein (ESAT-6) as an immune response-inducing antigen As a carrier of these gene, we have developed a highly effective gene transfection systems comprising very small (< 70 nm) plasmid complexes with negative surface charge (Biomaterials 31 (2010) 2912–2918) In this study, the small complexes were made of the plasmids harboring the pathogenic protein genes Those having GM-CSF or IL-2 genes were also prepared Co-transfection of the cytokine-gene with the pathogenic antigen-gene showed very high anti-tumor therapeutic effect in tumor-bearing mice Animal clinical study on primary tumor-bearing dogs was also carried out, and evident suppression of the tumor growth was observed 126 Zinc Finger Nuclease-Mediated Transgenesis in Human Cord-Derived Cells: En Route to Cell Therapy for Hemophilia A Jaichandran Sivalingam,1,2 Toan Thang Phan,3,4 Oi Lian Kon.1,2 Laboratory of Applied Human Genetics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Singapore; 2Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore The inability of current gene therapy vectors to integrate transgenes precisely into safe genomic sites has been associated with serious adverse events in clinical trials Zinc finger nucleases (ZFNs) have emerged as a potentially safer technique for targeted gene integration ZFNs are chimeras of sequence-specific DNA-binding zinc finger peptides fused to the catalytic domain of FokI endonuclease They induce highly site-specific genomic incisions that enhance homologous recombination-mediated integration of an exogenously provided transgene (donor DNA) We have used ZFNs to integrate a hybrid FVIII cDNA into the human AAVS1 locus Our goal is to achieve durable constitutive secretion of FVIII from primary human umbilical cord-lining epithelial cells (CLECs) with the intent of developing them as autologous bioimplants for hemophilia A therapy A pair of codon-optimised four-finger obligate heterodimeric ZFNs Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy (Miller JC, et al Nat Biotechnol 2007; 25:778) targeting the AAVS1 locus was assembled and expressed from a dual expression cassette plasmid vector N496D and H537R mutations were introduced into the FokI domains of left and right ZFNs, respectively, to restore catalytic activity of obligate heterodimeric ZFNs (Doyon Y, et al Nat Methods 2011; 8:74) S418P and K441E mutations were introduced into both ZFNs to enhance cleavage activity (Guo J, et al J Mol Biol 2010; 400:96) Expression of optimized ZFNs in concert with ZFNstabilising effects of mild hypothermia resulted in a 4.6-fold increase in targeted integration of a 50-bp donor DNA in K562 cells (Student’s t-test, p=0.001) Under optimized conditions of ZFN expression, we have achieved integration of a 9-kb hybrid FVIII DNA cassette into the AAVS1 locus of K562 cells and primary CLECs Junctional PCR and sequencing showed precise insertion without evidence of indels at both transgene-genome junctions We have incorporated a codon-optimized HSV TK in the donor DNA as a supplementary mechanism to minimize random transgene integrations A B-domain deleted human-porcine hybrid FVIII cDNA comprising porcine A1 and A3 domains was assembled in our lab with 93% identity to human FVIII cDNA Compared to human FVIII cDNA, hybrid FVIII cDNA increased in vitro FVIII secretion by transfected CLECs by 5-fold (Student’s t-test, p=0.003) The biosafety of ZFNmodified FVIII-expressing CLECs will be evaluated for alterations in the transcriptome, genome copy number, chromosomal structures and for acquired tumorigenic potential in immunocompromised mice The presence of unintended off-target genome modifications will be investigated by whole genome sequencing of clonal cells Comprehensive assessment of low genotoxic risk coupled with therapeutically meaningful and durable FVIII secretion in vivo could justify developing transgenic CLECs as autologous cell therapy for hemophilia A 127 PROGNOSE: An Online Tool for Predicting and Analyzing the Off-Target Site Effects of Designer Nucleases Eli J Fine,1 Charles L Zhao,1 Thomas J Cradick,1 Gang Bao.1 Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA Designer nucleases are engineered restriction enzymes created by fusing a DNA binding domain to a non-specific DNA cleavage domain When designer nucleases cleave a target locus, cellular DNA repair can be used to knockout or edit a gene, thereby facilitating therapeutic gene correction or the creation of genetically modified model organisms Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) are major classes of engineered nucleases that are deployed in pairs, greatly increasing their specificity Although each pair of nucleases is designed to target a unique genomic sequence, there have been a number of reports of ZFNs and TALENs cleaving additional genomic sites Off-target cleavage may significantly impact the efficacy and safety of biomedical applications of designer nucleases We have developed, PROGNOSE (Predicted Report Of Genome-wide Nuclease OffSite Effects), an online tool that performs exhaustive searches of genomic DNA sequences for potential nuclease off-target cleavage sites Different genomes can be searched based on homology to the nuclease pair’s target DNA binding sequences and user-supplied criteria, including the allowed spacings between nuclease binding sites, and the number of mismatches allowed in each binding domain PROGNOSE provides a list of possible off-site cleavage locations and annotates each site as being in an exon, promoter, intron, or intergenic region This information can be used while designing nuclease pairs to help identify target sequences without significant homology to other sites in the genome For each potential off-target site identified, PROGNOSE also automatically generates flanking PCR primers allowing amplification for quantification of non-specific S51 DNA VECTOR DEVELOPMENT & GENE TARGETING I cleavage and misrepair events at these genomic loci using nuclease assays or deep sequencing To validate the tool, we used PROGNOSE to analyze off-target site cleavage events after transfecting pairs of TALENs and ZFNs that target the same gene segment We performed PCR amplification and mutation detection assays to quantify the level of misrepair from cleavage at the intended nuclease target sites and putative off-target sites We compared these results to the predicted off-target sites for both TALENs and ZFNs In summary, PROGNOSE is a user-friendly online tool that makes the analysis of nuclease off-target cleavage easily accessible, without the need for bioinformatics and biochemical skill sets Presently off-target effects of ZFNs and TALENs are poorly understood since only a limited number of nuclease pairs have been tested As more offtarget site cleavage events are analyzed, the underlying trends that dictate nuclease specificity may become apparent This will greatly aid the design and application of engineered nucleases in biological and medical studies 128 Implementation of a Nuclear Factor Kappa B DNA Nuclear Targeting Sequence Improves In Vitro Suicide Gene Therapy Efficacy in Both Smalland Non-Small Cell Lung Cancer Cell Lines Frederik Cramer,1 Camilla L Christensen,2 Melissa Badding,3 David A Dean,3 Hans S Poulsen.1 Department of Radiation Biology, Copenhagen University Hospital, Copenhagen, Denmark; 2Dana-Farber Cancer Institute, Boston; 3Department of Pediatrics, University of Rochester The year survival rate of small cell lung cancer (SCLC) is currently 6% and new treatments are in high demand Gene therapy could be a promising candidate for a novel treatment of SCLC To target primary and metastatic tumor tissue systemic administered treatment is required However non-viral systemic delivery of therapeutic DNA is currently insufficent We therefore investigated if gene therapy delivery could be improved by implementing a DNA Nuclear Targeting Sequence (DTS) strategy To test this, multiple Nuclear Factor kappa B (NFkB) binding sequences were cloned into a transcriptional targeting plasmid encoding the yeast cytosine deaminase (YCD) and yeast uracil phosphoribosyl transferase (YUPRT) genes The expression of the suicide genes was regulated by the SCLC-specific promoter Insulinoma-associated (INSM1) The therapeutic effects of the different plasmid constructs were evaluated by a viability assay in vitro and the nuclear translocation effect of plasmids by microscopy A positive correlation between the number of inserted NFkB binding sites and the therapeutic effect were observed in several SCLC cell lines The effect was most likely due to elevated nuclear translocation of the suicide gene encoding plasmids as we observed that 68% of SCLC cells microinjected with plasmids containing NFkB binding sites, had nuclear localized plasmids hours post cytoplasmic microinjection When the experiment was repeated with plasmids lacking the NFkB binding sites, the number of cells with nuclear localized plasmids had dropped to 10 % The NFkB inserts did not compromise the specificity of the INSM1-promoter Furthermore, the NFkB-inserts also resulted in a similar nuclear translocation and cytotoxic effect in a non-small cell lung cancer (NSCLC) cell line, indicating that therapeutic efficiencies in other types of lung diseases than SCLC could be obtained by the implementation of a DTS strategy To our knowledge this is the first time a DTS strategy has been implemented in a suicide gene therapy system The results show that a significant improvement of gene therapeutic efficiency can be obtained by increasing the nuclear translocation of therapeutic DNA in vitro We are currently testing the system in vivo S52 129 Stimulation of Homologous Recombination-Mediated Genome Editing through Donor DNA Processing Maarten Holkers,1 Manuel A F V Gonỗalves.1 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Zuid-Holland, Netherlands Current approaches to bring about efficient homologous recombination (HR)-mediated genome editing of human somatic cells are based on artificial hybrid nucleases designed to introduce double-stranded DNA breaks (DSBs) at predefined sequences of choice Recent research from our laboratory and those of others indicates that site-specific nicks or single-stranded DNA breaks (SSBs) provide for an alternative trigger for HR during gene targeting or gene repair protocols (van Nierop et al [2009] Nucleic Acids Res 37: 5725-5736; McConnell-Smith et al [2009] Proc Natl Acad Sci USA 106: 5099-5104; Metzger et al [2011] Nucleic Acids Res 39: 926-935) These SSB-assisted genetic modification strategies have exploited either natural or engineered sequence- and strand-specific enzymes in the form of the adeno-associated virus (AAV) Rep78/68 proteins or a mutant I-AniI homing endonuclease, respectively Despite their differences in what the tools and the experimental set-ups are concerned, DSB- and SSB-mediated genome editing approaches have in common the fact that, hitherto, they have relied exclusively on the endonucleolytic cleavage of the target chromosomal template Recently, using a cellular model based on AAV Rep78/68 proteins and the human AAVS1 locus at position 19q13.42, we asked whether introduction of SSBs in both donor and chromosomal target DNA molecules would enhance the rate of HR in human cells as posited by initial nick-based HR models We found that donor DNA processing via AAV Rep78/68-induced nicking greatly fosters HR-dependent AAVS1 gene targeting (Gonỗalves et al [2011] Nucleic Acids Res Epub ahead of print) Here we hypothesized that recognition and processing of inverted repeats with the capacity to acquire highorder conformations in vivo, by cellular structure-specific nucleases, stimulate the use of donor DNA as DSB-repairing template To test this new gene targeting principle based on donor DNA processing, we generated and characterized cell lines containing a reporter expression unit whose open reading frame is disrupted by a premature stop codon plus the 18-bp I-SceI homing endonuclease target site Restoration of reporter gene expression was shown to be strictly dependent on the presence of I-SceI and homologous gene-correcting exogenous DNA sequences Importantly, cell cultures exposed to wild-type I-SceI and homology-bearing templates equipped with secondary structure-forming sequences displayed a modest but nonetheless highly significantly increase in the frequency of gene-corrected cells when compared to those incubated with I-SceI and conventional donor DNA (143%, p