Scholarly article on topic '558. Comparison of Gene Editing Strategies for Gene Correction of Wiskott-Aldrich Syndrome in Mouse Embryonic Stem Cells'

558. Comparison of Gene Editing Strategies for Gene Correction of Wiskott-Aldrich Syndrome in Mouse Embryonic Stem Cells Academic research paper on "Biological sciences"

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Academic research paper on topic "558. Comparison of Gene Editing Strategies for Gene Correction of Wiskott-Aldrich Syndrome in Mouse Embryonic Stem Cells"

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 ChlP-Seq data of histone modifications and DNasel 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, proto-oncogenes, 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 RNA-Guided 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. p-Globin Gene Editing in Human Cells Using TALENs and ssDNA Oligonucleotides: Towards a Gene Repair Approach for Sickle Cell Anemia and p-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 P-thalassemias and sickle cell anemia are congenital anemias caused by mutations in the P-globin gene, resulting in deficient or altered hemoglobin P-chain production. A current promising therapy for these diseases relies on the transplantation of autologous hematopoietic stem cells transduced with a retrovirally encoded wild-

type P-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-Crel-derived meganucleases, TALE nucleases (TALENs), and Zinc-Finger 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 P-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 P-globin gene. Using deep sequencing analysis, we determine that TALENs cleave the P-globin locus more efficiently than meganucleases in both 293 and sciPS cells. We also show specific targeting of the P-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 P-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 P-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 non-targeted 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 3 through 12) flanked by recombination arms homologous to the genomic sequence upstream and downstream of Was intron 2 and either a plasmid encoding a zinc-finger nuclease (ZFN) or a CRISPR/Cas9 constructs, both targeting specific Was intron 2 sequences. The "donor" construct also expressed puromycin N-acetyl-tranferase, which allowed for selection of transfected clones in puromycin (1ug/ml) for 6 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

occurrence of the "donor" cDNA sequence in the targeted Was locus. Southern Blot analysis was also consistent with successful targeting of the Was locus. Conclusion: Gene editing of the mouse Was locus in Was knockout ES cells can be achieved using ZFN and CRISPR/ Cas9 technologies with similar efficiencies. Our next step will be the creation of corrected mice by injecting targeted ES cells into Was knockout blastocysts, to provide proof-of-principle that in vitro gene editing can result in stable gene correction in living animals.

559. On- and Off-Target Cleavage of CRISPR Nickases Targeting Multiple Genes

Ciaran M. Lee,1 Thomas J. Cradick,1 Gang Bao.1 1 Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA.

The development of the clustered regulatory interspersed short palindromic repeats (CRISPR) systems for gene targeting has made targeted genomic modifications efficient, easily customizable, and can be multiplexed for genome-wide studies, compared with other technologies such as transcription activator like effectors and zinc finger nucleases. However, our lab and others have shown that CRISPRs have significant levels of off-target activity. The CRISPR system relies on an RNA guide strand for target site recognition and the CRISPR associated protein Cas9 for DNA cleavage, therefore, only one guide strand or DNA binding domain is required per target site. The Cas9 protein contains two DNA cleavage domains either of which can be inactivated by alanine substitutions, generating a Cas9 "nickase" capable of cutting only the sense or anti-sense strand. When two CRISPR nickases bind in close proximity they can induce two single-strand breaks on opposite strands to generate a DSB with either a 5' or 3' overhang, depending on target site orientation.

We chose to target four disease associated genes HBB, RYR2, CCR5, IL2R-Y, and the safe harbour site AAVS1. We report that the use of two CRISPR nickases targeted to opposite strands in close proximity can result in higher levels of DSB formation compared to unmodified CRISPRs (up to 80%) and that the level of DSBs can be influenced by both the spacing between the two guide RNA target sites and the type of overhang generated. When tested at previously identified CRISPR off-target sites, no detectable DSB formation was observed with the corresponding CRISPR nickase, demonstrating that the use of CRISPR nickase pairs may abrogate the off-target activity observed with unmodified CRIPSRs. DSBs induced by CRISPR nickase pairs are repaired by NHEJ resulting in a different indel profile when compared to unmodified CRISPRs, which may have an effect on homology directed repair.

560. "Getting Under the Skin": Peptide-Mediated Nucleic Acid Delivery

Manika Vij,1 Poornemaa Natarajan,1 Bijay R. Pattnaik,1 Shamshad Alam,2 Rajpal Sharma,1 Kausar M. Ansari,2 Rajesh S. Gokhale,1 Vivek Natarajan,1 Munia Ganguli.1

1Skin Biology, Institute of Genomics and Integrative Biology, New Delhi, India; 2Food and Toxicology Research, Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India.

Peptide mediated delivery of complex biomolecules in vitro and in vivo (as therapeutics) has gained widespread interest since the past few years. Till now multiple payloads have been delivered to various cell types and organs in order to target a plethora of diseases. Recently much attention has been directed towards delivery of macromolecules across skin. Owing to its favourable anatomical and biological features, skin holds immense potential to be explored for both systemic as well as localized delivery. A wide variety of debilitating and untreatable cutaneous disorders like psoriasis, atopic dermatitis, vitiligo, to name few, and also different conditions of

the skin like formation of wounds, make skin a possible therapeutic target. Skin mediated delivery not only overcomes the limitations of hepatic metabolism but also increases the patient compliance. Most of the non viral methods of nucleic acid delivery to the skin involve use of physical methods or chemical methods like electroporation, penetration enhancers or liposomes. However, issues with efficiency, toxicity, tissue damage, robustness and high production costs limit their universal use. Thus one of the key challenges in skin biology is to develop efficient methods of delivering biomolecules to (topically or intradermally) and through (transdermally) the skin. We have developed a peptide-based delivery system for efficient plasmid DNA delivery in skin upon topical application. The amphipathic nature and alpha-helicity of the peptide system as assessed by various biophysical techniques and its ability to retain in skin as seen by franz assay makes it a suitable vehicle to be used for biomolecule delivery in skin. We have also found that the application of bare peptide to skin cells and human foreskin tissue exhibits efficient cellular uptake as well as tissue penetration ability as assessed by confocal microscopy and flow cytometry analysis. The peptide was further explored for its ability to deliver plasmid DNA as cargo in both skin cells as well as human foreskin tissue using luciferase and fluorescence assays. We observed efficient transfection ability of the peptide in both keratinocyte and melanoma skin cells and human foreskin tissue without any additive physical or chemical methods. Also transfection efficiency observed was equivalent to the commercially known transfection agent. In in-vivo studies using SKH-1 hairless mice model we could observe similar activity for both bare peptide and peptide-DNA complex following topical application. The cytotoxicity analysis of bare peptide and peptide-DNA complex revealed minimal or no deleterious effect on skin cells. The studies to check specific localization of these peptide-DNA complexes in different skin layers are currently undergoing. Hence these novel peptides with dual ability to overcome cellular and tissue level transport barriers could facilitate delivery of a wide spectrum of therapeutic cargo in skin and increase the feasibility of treatment for various skin disorders.

561. Lessons Learned From TALEN Knockout of NANOG in Colorectal Carcinoma (CRC) Cells

Abid R. Mattoo,1 Snorri S. Thorgeirsson,1 J. M. Jessup.1 laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD.

NANOG is a key transcription factor maintaining pluripotency in embryonic stem cells and supporting stemness in human cancers. . NANOG gene family contains several pseudogenes that are associated with progression of leukemias, colorectal (CRC) and other carcinomas including NANOG2 and NANOGP8. Because NANOGP8 can replace NANOG to support stemness in CRC, we sought to knockout parental NANOG to clarify the role of NANOG and its pseudogenes in CRC. Knockout of NANOG in human cells is complicated by the presence of NANOG, NANOGP8, NANOGP4 and NANOGP7 transcripts. Here we tested whether a TALEN could knock out parental NANOG in the presence of these pseudogenes. A TALEN plasmid pair was designed by Cellectis to target a region 34 - 44 nucleotides from the ATG. Clone A, a subclone of the human DLD-1 CRC cell line, was tested because it has high transfection efficiency. Since inhibition of NANOG and NANOGP8 with a shRNA to the same target sequence inhibits proliferation in Clone A and other CRC lines with inhibition of WEE1, we screened for colonies whose proliferation was slower than the proliferation of control cells after transfection. Western blot analysis of cell lysates from the expanded slow growing clones revealed that NANOG protein levels in 3 of 20 clones were ~50% of that in control cells. Since NANOG and NANOGP8 have the same mass and react with anti-NANOG antibodies, cDNA from these 3 clones was then analyzed for NANOG and NANOGP8 transcripts were decreased by AlwN1 restriction enzyme digestion that

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy