339 GENIS: a bioinformatics tool for reliable and automated genome insertion site analysis

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[.]

Molecular Therapy Volume 23, Supplement 1, May 2015

Copyright © The American Society of Gene & Cell Therapy S135

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

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 1 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 2 kb donor DNAs and

a single sgRNA to promote gene correction by homology-mediated

repair Our experimental data revealed 8 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

1 Microbiology, Immunology, and Molecular Genetics, University

of California, Los Angeles, Los Angeles, CA; 2 Division of Allergy

and Immunology, Department of Pediatrics, David Geffen School

of Medicine, University of California, Los Angeles, Los Angeles,

CA; 3 Neurology, David Geffen School of Medicine, University of

California, Los Angeles, Los Angeles, CA; 4 Biology Department,

California State University, Northridge, Northridge, CA; 5 Eli

& 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

4 distinct TALEN pairs generated, only 2 demonstrated targeted

cleavage at rates nearing 10% of alleles In addition to on-target

cleavage at globin, nuclease off-target cleavage at other

beta-globin family genes was evaluated for each technology by Cel-1 of

nucleofected K562 cells Here the two TALEN pairs demonstrate

cleavage in the highly-homologous delta-globin gene with the optimal TALEN pair cleaving 11% of alleles In this assay, of the 6 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

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,

Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

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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

1 Biology and Biochemistry Department, University of Houston,

Houston, TX; 2 Center 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

1 Neurology, University of Massachusetts Medical School,

Worcester, MA; 2 Gene 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

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 AAV9-miR, 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 Muñoz,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

1 Genomic Medicine, Centre for Genomics and Oncological Research: Pfizer-University of Granada-Andalusian Regional Goverment), Granada, Spain; 2 Regulación Génica, Células Madre

y Desarrollo, Centre for Genomics and Oncological Research: Pfizer-University of Granada-Andalusian Regional Goverment), Granada, Spain; 3 Sangamo 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 1 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 WAS-specific 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