339 GENIS A Bioinformatics Tool for Reliable and Automated Genome Insertion Site Analysis Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S1[.]
Gene Targeting and Gene Correction II 337 CRISPR/Cas9 Mediated Highly Efficient Genome Engineering in Mouse Embryos Khurshida Begum,1 Bert W O’Malley,1 Francesco J DeMayo,1 Paul Overbeek.1 Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX CRISPR/Cas9 is an RNA-based adaptive immune system used by bacteria and archaea to cleave foreign nucleic acids (e.g bacteriophage DNA) This system has been adapted as a tool to edit mammalian genes with high efficiency and specificity Here, we describe our use of the CRISPR/Cas9 system to achieve targeted gene knock outs and targeted gene repair in one cell stage mouse embryos We have efficiently mutated tyrosinase (a coat color gene), p53 (a tumor suppressor gene), and Cacna1a (a voltage-gated ion channel gene) in mouse embryos by injection of Cas9 mRNA plus either a single or paired guide RNAs (sgRNAs) Single sgRNAs typically induced deletions of bp to several hundred bp at their target site whereas paired sgRNAs generated localized deletions at high efficiency For gene repair, we used 700 bp to kb donor DNAs and a single sgRNA to promote gene correction by homology-mediated repair Our experimental data revealed to 18% efficiency of gene correction When we used two sgRNAs to target a single exon, all of the newborn mice from one experiment showed targeted therapy for an inherited genetic disorder, albinism No one has previously published a protocol that has achieved gene repair with this level of effectiveness Our results confirm that the CRISPR/Cas9 system can be used for efficient gene targeting and gene repair in vivo 338 Evaluation of TALENs and the CRISPR/ Cas9 Nuclease System To Correct the Sickle Cell Disease Mutation Megan D Hoban,1 Dianne Lumaquin,1 Caroline Kuo,2 Zulema Romero,1 Courtney Young,3 Michelle Ho,1 Joseph Long,1,4 Nathan Coss,1 Carmen Bjurstrom,1 Michelle Mojadidi,1 Roger P Hollis,1 Donald B Kohn.1,4 Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA; 2Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA; 3Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA; 4Biology Department, California State University, Northridge, Northridge, CA; 5Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, Los Angeles, CA Targeted genome editing technology can correct the sickle cell disease mutation of the beta-globin gene in hematopoietic stem and progenitor cells (HSCs) The correction induces production of red blood cells that synthesize normal hemoglobin proteins Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease systems have been developed to target the sickle mutation in the beta-globin gene for site-specific cleavage to facilitate precise correction of the sickle mutation by a co-delivered homologous donor template K562 cells were electroporated with TALEN and CRISPR/Cas9 expression plasmids and, using the Surveyor Nuclease Assay (Cel-1), cleavage rates were quantified and compared between TALEN- and CRISPR/Cas9-treated cells Of the six CRISPR/Cas9 guides tested, each of them led to target disruption of the beta-globin locus with the highest cleavage rates upwards of 35% of alleles Of distinct TALEN pairs generated, only demonstrated targeted cleavage at rates nearing 10% of alleles In addition to on-target cleavage at beta-globin, nuclease off-target cleavage at other betaglobin family genes was evaluated for each technology by Cel-1 of nucleofected K562 cells Here the two TALEN pairs demonstrate Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy cleavage in the highly-homologous delta-globin gene with the optimal TALEN pair cleaving 11% of alleles In this assay, of the CRISPR guides tested, none showed off-target disruption of delta-globin or any of the other beta-globin cluster genes Of note, in each of the tested guides, at least one base differed from the target site in beta-globin to the respective sequence in delta-globin in the 10bp PAM proximal region Further experiments are being conducted to determine the genome-wide off-target effects of each of these nucleases Upon co-delivery of a plasmid donor template containing the corrective base at the sickle site as well as a restriction fragment length polymorphism (RFLP) for rapid assessment of targeted gene modification, both nuclease technologies led to gene modification Gene modification rates were assayed by qPCR with primers specific to the modified base TALENs drove gene modification rates of 18%, while the optimal CRISPR guides resulted in 37% modification in K562 cells without sorting for transfected cells These results provide the basis for pursuing the use of the CRISPR/Cas9 nuclease system for targeted correction of the sickle mutation in human HSCs 339 GENIS: A Bioinformatics Tool for Reliable and Automated Genome Insertion Site Analysis Saira Afzal,1 Raffaele Fronza,1 Stefan Wilkening,1 Cynthia Bartholomä,1 Christof von Kalle,1 Manfred Schmidt.1 German Cancer Research Center (DKFZ), National Center for Tumor Diseases (NCT), Heidelberg, Germany Over the last two decades, gene therapy has shown rapid advancements as a promising approach to treat genetic diseases by introducing corrected genes into patient cells Viruses are the most common carriers in the vector-mediated gene therapy However, integration of viral vectors at undesirable genomic locations can lead to deleterious effects, e.g insertional mutagenesis Therefore, an efficient, stable and safe vector system is the major prerequisite for a successful gene therapy Long term monitoring of the distribution pattern of vector integration sites (IS) is the most feasible strategy to address vector safety and stability concerns Recent advancements in next generation sequencing technologies have dramatically increased the possibility to generate substantial amount of vector-genome sequencing data for comprehensive IS analysis An efficient downstream analysis of this data requires automated and fast computational methods Here, we present Genome Insertion Site (GENIS) pipeline, a suite for time-efficient and reliable analysis of vector-genome junctions GENIS has been designed to analyze the sequencing data generated from traditional linear amplification mediated PCR (LAM-PCR) based methods and also from new targeted DNA single and paired end sequencing technologies (e.g., Agilent SureSelect) Our suite consists of six basic modules including barcode sorting, quality filtering and adapter trimming, mapping of sequence reads to the reference genome, extraction of soft-clip reads and clustering of IS for subsequent annotation GENIS is implemented on Linux platform with minimum external software dependencies Users can adjust the required parameters in the provided configuration file It takes about 30 minutes for complete processing, starting from raw reads till annotation, of 10 million paired end reads generated by targeted sequencing In case of LAM-PCR data, 30 million reads are sorted in about 30 minutes (50 different PCR) and time required for rest of processing to obtain annotated IS is also approximately 30 minutes for 15 million reads Three final files present the conclusion of the analysis process and contain: 1) the information about read ID, chromosome position (genomic IS), vector position (vector IS), sequence, genomic and vector orientation and sequence span; 2) all the clustered IS with their respective sequence count and 3) the annotated IS with respect to nearby genomic features, including gene identifier and gene name, S135 Gene Targeting and Gene Correction II transcription start site, coding region start and end sites etc Our tool is highly appropriate for in-depth quantitative analysis of biosafety and transduction efficiency of viral vectors 340 Development of a Nuclease Screen to Improve Cas9 Targeting Specificity Jeffrey M Spencer,1,2 Xiaoliu Zhang.1,2 Biology and Biochemistry Department, University of Houston, Houston, TX; 2Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) bacterial immunity targets invading nucleic acid sequences using an RNA guide (gRNA) The well-defined Cas9 from Streptococcus pyogenes’ type II CRISPR system has been utilized as a gene editing tool in a vast assortment of organisms However, an inherent lack of specificity limits the potential of the current system and presents challenges to interpreting experimental results A plasmid cleavage based screening platform has been developed with the intent to increase the fidelity of the Cas9 system Utilizing both positive and negative selection, the screen can be used to identify variants of Cas9 or its gRNA with improved specificity Positive selection of variants with activity towards the appropriate target site proceeds through the removal of a plasmid containing an inducible suicide gene Conversely, negative selection of variants with reduced activity on off-target substrates involves the retention of a plasmid with an antibiotic resistance gene Preliminary results suggest that a gRNA with an insertion in the scaffold region of the RNA can decrease the level of off-target cleavage These results suggest that the screening platform has the potential to identify novel protein or RNA variants with greater targeting specificity compared to the naturally occurring components A Cas9 system with enhanced fidelity will expand the potential applications of the technology and accelerate its ability to interrogate biological systems 341 Therapeutic Approach for SOD1-ALS Using AAV9 Delivered Artificial microRNAs Lorelei Stoica,1,2 Johnny Salameh,1 Christian Mueller,2 Robert Brown,1 Miguel Sena-Esteves.1,2 Neurology, University of Massachusetts Medical School, Worcester, MA; 2Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons This results in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis About 10% of cases are inherited typically in a dominant manner - of which twenty percent are due to mutations in the superoxide dismutase gene (SOD1) Experiments in transgenic ALS mouse models that overexpress human SOD1 have shown that decreasing levels of mutant SOD1 protein alters, and in some cases eliminates, disease progression We postulated that silencing SOD1 expression with a micro RNA (miR) would be therapeutic in ALS We developed a single stranded AAV9 vector encoding GFP and a miR against human SOD1, driven by a CBA promoter, which we injected into the SOD1G93A ALS mouse at postnatal day one Each mouse received 2ml into each lateral ventricle, for a total vector dose of 4e10 vector genomes At four weeks post injection, hSOD1 mRNA was reduced by almost 50% at all three levels of the spinal cord Transduction was visible in both motor neurons and astrocytes in the spinal cord as well as neurons in layer V of the motor cortex Transduced cells, assessed by GFP RNA FISH, had a decrease in hSOD1 mRNA This translated into a 50% extension in median survival of treated mice (206 days) compared to untreated (135 days) mice S136 To assess neuromuscular health during the duration of the experiment, we performed motor unit number estimates (MUNEs) and needle electromyography (EMG) Treated mice had mild or no muscle denervation as opposed to the severe denervation seen in untreated SOD1 In fact, the treated mice did not develop paralysis but instead had to be euthanized due to weight loss and a hunched posture We also assessed neuropathology in the spinal cord and nerves in untreated and treated animals, at their respective endpoints The treated mice showed no spinal cord motor neuron loss, while the untreated mice lost the majority of the motor neurons The axonal integrity of the lumbar ventral roots was also improved in treated animals Furthermore, there was no axonal degeneration in the sciatic nerves of the treated animals when analyzed at 120 days, the endpoint of untreated mice Lastly, treated animals show delayed onset of astrogliosis and microgliosis, as observed by IHC for inflammatory markers GFAP and Iba1, and confirmed by RT-qPRC for genes upregulated in inflammation In conclusion, we were successful at extending the lifespan of the SOD1G93A mouse by 50% with our high dose neonatal AAV9miR, and our treated animals remain ambulatory and active until the humane endpoint with minimal or no signs of paralysis 342 Gene Edition for Wiskott-Aldrich Syndrome Gene Therapy Francisco Martin,1 Pilar Moz,1 Marién Cobo,1 Almudena Sanchez-Gilabert,1 Pedro Real,2 Verónica Ramos,2 Philip D Gregory,3 Michael C Holmes,3 Miguel G Toscano.1 Genomic Medicine, Centre for Genomics and Oncological Research: Pfizer-University of Granada-Andalusian Regional Goverment), Granada, Spain; 2Regulación Génica, Células Madre y Desarrollo, Centre for Genomics and Oncological Research: Pfizer-University of Granada-Andalusian Regional Goverment), Granada, Spain; 3Sangamo Biosciences, Inc.Pt Richmond Tech Center, Richmond, CA Site Specific nucleases (SSN) are powerful tools for genome editing that are revolutionizing basic and applied science However, for most therapeutic applications their efficiency and/or specificity must be still improved before translation into clinic Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency (PID) caused by mutations in the WAS gene An interesting characteristic of this disease is that, due the growth selective advantage of the WAS-expressing cells, a small number of corrected hematopoietic stem cells (HSCs) could be enough to achieve a complete cure of the patients In this context, our final aim is to develop gene edition tools for WAS gene therapy with the idea to achieve safe genetic correction in a small numbers of patients’ HSCs To study efficiency and safety of our tools, we first developed a WAS-specific GFP-reporter cellular model harboring GFP coding sequences disrupted by the intron of the WAS gene This reporter cell allowed us to have a direct measurement of the efficiency and specificity of WAS-specific nucleases to achieve genetic restoration We constructed different CRISPR-based and ZFNs-based WASspecific nucleases expressed by Lentiviral vectors (LV) With these set of plasmids we performed a side by side comparison of two different nucleases (ZFNs and CRISPR) and two different delivery systems (nucleofection versus Integrative deficient lentiviral vectors (IDLV)) to edit the WAS locus We showed that CRISPR and ZFNs were equally efficient for WAS gene edition (over 60% gene disruption and 4% gene restoration) when delivered by plasmid nucleofection However, IDLV-ZFNs were more efficient than IDLV-CRISPR, probably due to the low expression levels of Cas9 in the IDLV backbone Our data indicates that CRISPR based systems can be as efficient as other SSN using standard delivery systems, however, their efficiency using IDLV is limited Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy ... Granada, Spain; 2Regulación Génica, Células Madre y Desarrollo, Centre for Genomics and Oncological Research: Pfizer-University of Granada-Andalusian Regional Goverment), Granada, Spain; 3Sangamo...Gene Targeting and Gene Correction II transcription start site, coding region start and end sites etc Our tool is highly appropriate for in-depth quantitative analysis of biosafety and transduction... untreated mice Lastly, treated animals show delayed onset of astrogliosis and microgliosis, as observed by IHC for inflammatory markers GFAP and Iba1, and confirmed by RT-qPRC for genes upregulated