555 Genome Wide Mapping of Genomic Instability and NHEJ Repair Activity In Vivo Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S215 GENE TA[.]
GENE TARGETING AND GENE CORRECTION III level after mRNA transcripts have already been expressed In contrast, dCas9/dCas9-KRAB is an orthogonal engineered system adapted from prokaryotes that is not known to interfere with natural mammalian pathways and controls gene expression at the genomic level, expanding potential targets beyond expressed transcripts This increased flexibility, along with the potential to multiplex gRNAs with a single Cas9 effector to repress multiple genes, makes CRISPR an exciting new method to manipulate gene expression for therapeutic applications, such as treatment of dominant-negative diseases, protection against viral infection, and inhibition of oncogenes In order to further characterize the potential of this system, we targeted the HS2 enhancer, a well-characterized distal enhancer necessary for activation of the globin locus, in K562 cells, a human leukemia cell line We stably expressed dCas9 or dCas9-KRAB using a lentiviral vector and transfected individual gRNAs targeted to different sites along the enhancer region Multiple gRNAs effected potent repression of e-, γ-, and β-globin genes, as measured by quantitative RT-PCR Delivering dCas9-KRAB generally resulted in more potent silencing than dCas9, suggesting an important role of this repression domain that actively induces heterochromatin formation These model studies demonstrate the potential of RNA-guided CRISPR/Cas9-based gene regulation to disrupt distal regulatory elements and thus influence the expression of multiple genes with a single gRNA Ongoing studies include elucidating the effects of dCas9 and dCas-KRAB on endogenous transcription factor binding in regulatory regions and evaluating the long-term effects of KRAB-based repression in the HS2 enhancer and other therapeutically relevant targets These findings establish the unique capability of CRISPR/dCas9 to disrupt expression at the genomic level and contribute to the characterization of this new technology for future application 553 AAV-Mediated Delivery of HSV-Specific Homing Endonucleases To Neurons of the Trigeminal Ganglia for HSV-1 Inhibition Martine Aubert,1 Chung Dang,1 Roman Galetto,2 Daniel Stone,1 Keith R Jerome.1,3,4 Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Cellectis Therapeutics, Paris, France; 3Department of Laboratory Medicine, University of Washington, Seattle, WA; 4Department of Microbiology, University of Washington, Seattle, WA Herpes simplex virus type (HSV-1) is the most common etiology of infectious blindness (keratitis) and oral ulceration HSV-1 can cause viral encephalitis, and severe and potentially fatal infections in newborns and immunocompromised hosts After primary infection HSV-1 establishes lifelong latency in neurons of the trigeminal ganglia (TG) and can later reactivate, causing recurrent ulcers While antiviral therapy with acyclovir or related drugs diminishes the severity of primary or recurrent disease, it does not eliminate latent infection Despite much effort, an effective vaccine for HSV remains elusive Therefore, there is a need for a new therapeutic approach to cure these infections As a novel therapeutic approach to HSV-1 treatment we are using homing endonucleases (HE) to perform targeted mutagenesis of latent HSV genomes We have two HSV-specific HEs that target sequences in the essential HSV-1 genes UL19 and UL30 After HE-mediated cleavage these genes become disrupted upon error-prone repair by non-homologous end joining, and HSV-1 subsequently becomes replication incompetent Using scAAV2 as a delivery vector and an in vitro model of HSV-1 latency that uses primary human fibroblasts we were able to show that expression of HSV-1 targeted-HEs in combination with the 3’ to 5’ exonuclease Trex can disrupt HSV-1 genomes and inhibit reactivation of HSV-1 from latency HSV-1 mutation frequencies could be significantly increased by pre-treatment of cells with HDAC inhibitors Targeted HEs could also mutate HSV-1 genomes after AAV Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy delivery to the neural cell line U373MG To determine the optimal method of HE delivery to the TG we first screened cultures of primary mouse TG neurons for transduction with AAV serotypes 1, 2, 4, 5, 6, & High levels of transduction were seen in TG neurons upon incubation with scAAV1 and scAAV8 vectors Mice in which latent HSV-1 infection can be established, were then inoculated with up to 1x1012 genomes of mCherry expressing scAAV8 reporter virus in the snout to determine whether TG neurons that project to the whisker pad could be transduced in vivo TG were explanted between and 30 days post AAV inoculation and placed in culture to assess the levels of neuron transduction By day post-explant more than 50% of TG neurons were positive for mCherry The ability of HSV-targeted HEs to inhibit HSV-1 replication and reactivation is currently being assessed in primary cultures of TG neurons and HSV-1 infected mice 554 CRISPR/Cas9-Based Transcriptional Activation of Endogenous Myod1 to Reprogram Murine Fibroblasts into Skeletal Myocytes Syandan Chakraborty,1 HaYeun Ji,1 Ami M Kabadi,1 Charles A Gersbach,1,2,3 Nicolas Christoforou,1,4 Kam W Leong.1 Department of Biomedical Engineering, Duke University, Durham, NC; 2Institute for Genome Sciences and Policy, Duke University, Durham, NC; 3Department of Orthopaedic Surgery, Duke University, Durham, NC; 4Department of Biomedical Engineering, Khalifa University, Abu Dhabi, United Arab Emirates RNA-guided, catalytically-inactive Cas9 (dCas9) fused to a VP64 transactivation domain has been recently used to activate expression of endogenous genes However, the potency of this system to achieve cellular reprogramming is yet to be demonstrated In this study, we describe the direct conversion of mouse embryonic fibroblasts to skeletal myocytes by activating the endogenous Myod1 gene by the lentiviral delivery of a single guide RNA in conjunction with a VP64fused dCas9 designer activator protein The expression of endogenous Myod1 was stable and continued even after the termination of activator protein expression Myod1 activation was independent of the genomic DNA strand being targeted by the gRNA Cross-striated organization of the sarcomeric markers, α-actinin and F-actin, marked the maturity of the reprogrammed cells In addition, upregulation of other skeletal muscle markers was detected by immunocytochemistry and RT-PCR Spontaneous twitching of the multi-nucleated myotubes after 14 days in culture further demonstrated the structural and functional integrity of the cells reprogrammed by this new method 555 Genome-Wide Mapping of Genomic Instability and NHEJ Repair Activity In Vivo Philipp K Zimmermann,1 Sara Chiblak,2 Christian Weber,1 Raffaele Fronza,1 Jürgen Debus,2 Amir Abdollahi,2 Christof von Kalle,1 Manfred Schmidt,1 Ali Nowrouzi.1 Translational Oncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany; 2Molecular Radiooncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany Detecting genomic instable regions in vivo is becoming increasingly important to evaluate secondary genomic side effects induced by therapeutic agents, radiation, cancer drugs or genome editing The detection of double strand breaks (DSB) by specific antibodies binding cellular repair proteins has genomic limitations, since upon repair these proteins engage from chromatin and not enable the mapping of DSB signatures in clonally expanded cells over time We have recently introduced DNA coded baits that are delivered by IDLV-based viral vectors into cells (Gabriel R, Lombardo A et al 2011) These DNA baits become stably incorporated at S215 GENE TARGETING AND GENE CORRECTION III induced DSB via the cellular non homologous end joining (NHEJ) pathway Here, we have applied these genetic tags that serve as templates for amplification and sequencing of the in vivo captured DSB to map radiation induced damage in clonally expanded cell populations Accordingly, we have sequenced and mapped >10.000 unique repaired NHEJ sites in the genome of three tumor cell lines (A549, PC3, U87) and primary human fibroblasts exposed to ionizing radiation Analysis of the gene expression status in the cells did not reveal evidence for a strong correlation of DSB induction and repair with transcriptional activity of the genome The correlation of captured DSB with ChIP-Seq data of histone modifications and DNaseI hypersensitive sites revealed that the probability for DSB in irradiated and clonally expanded cell populations is increased in open chromatin and in genomic regions containing both eu- and heterochromatin marks Furthermore, the probability for DSB is reduced in heterochromatin More interestingly, we identified many narrow genomic regions harboring up to 75 DSB in genomic intervals not spanning more than 10kb, representing genomic areas vulnerable or fragile to radiation induced genomic instability These hot spots are enriched in regions coding for known tumor suppressor genes, protooncogenes, and increasingly show copy number alterations in various cancers and genetic disorders We conclude that radiation induced DSB in clonally expanded cancer cell lines and human primary fibroblasts show a non-random distribution, potentially enabling the identification of genomic locations and structures likely to influence resistance to therapy We show that this genome-wide detection of induced DSB is applicable to identify genomic factors and intervals influencing the frequency of DSB and DNA repair activity in vivo 556 High Precision Genome Editing by RNAGuided CRISPR Cas9 Patrick D Hsu,1 Fei Ann Ran,1 David A Scott,1 Chie-yu Lin,1 Jonathan S Gootenberg,1 Silvana Konermann,1 Feng Zhang.1 Broad Institute of MIT and Harvard, Cambridge, MA Targeted genome editing technologies have enabled a broad range of research and medical applications The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20-nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis Here, we describe an approach that combines a Cas9 nickase mutant with pairs of guide RNAs to introduce targeted double-strand breaks Given that individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs effectively extends the number of specifically recognized bases in the target site We demonstrate that paired nicking can be used to reduce off-target activity by 50-1,000 fold in cell lines and facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency This versatile strategy thus enables a wide variety of genome editing applications with higher levels of specificity 557 β-Globin Gene Editing in Human Cells Using TALENs and ssDNA Oligonucleotides: Towards a Gene Repair Approach for Sickle Cell Anemia and β-Thalassemias Jorge Mansilla-Soto,1 Nicholas Socci,2 Yan Leifman,1 Michel Sadelain.1 Center for Cell Engineering, Memorial Sloan-Kettering Cancer Center, New York, NY; 2Bioinformatics Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY The β-thalassemias and sickle cell anemia are congenital anemias caused by mutations in the β-globin gene, resulting in deficient or altered hemoglobin β-chain production A current promising therapy for these diseases relies on the transplantation of autologous hematopoietic stem cells transduced with a retrovirally encoded wildS216 type β-globin gene However, one of the main concerns associated with the use of recombinant retroviruses to deliver therapeutic genes in stem cells is the risk of insertional mutagenesis following semirandom retroviral DNA integration Alternatively, gene repair by homologous recombination (HR) is recognized as the ideal approach to repair mutations Although HR is intrinsically inefficient in human cells, enzymes that create specific DNA double-strand breaks, such as I-CreI-derived meganucleases, TALE nucleases (TALENs), and ZincFinger nucleases (ZFNs), as well as the CRISPR/Cas9 system, can efficiently increase HR frequency Our long-term goal is to develop an efficient gene repair approach to repair sickle cell anemia and β-thalassemia mutations in stem cells Here, we show the generation of human induced pluripotent stem (iPS) cells carrying a homozygous sickle cell mutation Using this iPS line (sciPS) and human 293 cells, we investigate the activity of a series of single-chain meganucleases and TALENs that cleave specific sequences in the β-globin gene Using deep sequencing analysis, we determine that TALENs cleave the β-globin locus more efficiently than meganucleases in both 293 and sciPS cells We also show specific targeting of the β-globin gene in sciPS1 cells by using TALENs and a targeting construct carrying a selection cassette Finally, using oligonucleotides as donor DNA and TALENs we introduce short sequences in the β-globin gene of sickle iPS cells In the short term, we expect to translate this latter system to repair point mutations in sickle cell anemia and β-thalassemias patient-specific cells, such as hematopoietic stem cells and iPS cells, an approach that hold promise for future autologous stem cell therapies 558 Comparison of Gene Editing Strategies for Gene Correction of Wiskott-Aldrich Syndrome in Mouse Embryonic Stem Cells Lisa Peterson,1,2 J Douglas Burke,2 G Jaya Jagadeesh,2 Sangho Myung,2 Lisa Garrett,3 Rasoul Pourebrahim,4 Brian R Davis,4 Fabio Candotti.2 Dept of Neonatology, Walter Reed National Military Medical Center, Bethesda, MD; 2Disorders of Immunity, NIH, Bethesda, MD; 3Transgenic Stem Cell Core, NIH, Bethesda, MD; 4Stem Cell & Regenerat Med, Inst Mol Med, Univ Texas Hlth Sci Ctr, Houston, TX Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency which causes severe platelet defects, defective cellular and humoral immunity, and leads to recurrent infections and development of autoimmune diseases and cancer Clinical trials using gene addition approaches with gammaretroviral and lentiviral vectors have demonstrated the feasibility of gene therapy for WAS, but also stressed the potential for insertional oncogenesis of nontargeted gene delivery methods Our objective is to develop strategies for gene editing at the Was mouse locus as a model for targeted gene correction of the human disease Methods: Was knockout mouse ES and iPS cells were co-transfected with a “donor” construct containing the Was mouse cDNA sequence (exons through 12) flanked by recombination arms homologous to the genomic sequence upstream and downstream of Was intron and either a plasmid encoding a zinc-finger nuclease (ZFN) or a CRISPR/Cas9 constructs, both targeting specific Was intron sequences The “donor” construct also expressed puromycin N-acetyl-tranferase, which allowed for selection of transfected clones in puromycin (1ug/ml) for days Selected clones were screened with PCR to identify correct insertion of the targeted sequence Results: 60 and 96 ES cell clones transfected with the ZFN or CRISPR/Cas9 systems, respectively, were isolated Of these, 11 and 37, respectively, showed PCR positive fragments of the size expected by the homologous recombination of the “donor” cDNA sequence into the targeted region Of 60 IPS selected cell clones, 31 were PCR positive for “donor” cDNA recombination Sequence analysis of DNA extracted from CRISPR/Cas9 clones confirmed the Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... sequencing of the in vivo captured DSB to map radiation induced damage in clonally expanded cell populations Accordingly, we have sequenced and mapped >10.000 unique repaired NHEJ sites in the genome. .. the probability for DSB in irradiated and clonally expanded cell populations is increased in open chromatin and in genomic regions containing both eu- and heterochromatin marks Furthermore, the... to influence resistance to therapy We show that this genome- wide detection of induced DSB is applicable to identify genomic factors and intervals influencing the frequency of DSB and DNA repair