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0occurrence 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
selectively degrades NANOGP8. Both NANOG and NANOGP8 were present in cDNA. NANOG has introns but the pseudogenes do not. PCR amplification of the intronl-exonl junction of NANOG failed to identify the presence of NANOG in TALEN-treated cells but did reveal NANOG in the parental cells. The 3 clones with lower NANOG levels initially showed slower proliferation compared to control but all clones increased proliferation to control levels within 2 weeks -in the absence of increased in NANOG protein levels. Attempts to re-transfect the original clones did not further decrease proliferation or NANOG protein expression. This study was undertaken with the knowledge that the target site also targeted NANOGP4, P7 and P8. Since our first active shRNA targeted the same site, we felt this was an appropriate risk. Our data suggest that NANOG was in fact partially disrupted, perhaps on an allelic basis. However, compensatory mechanism(s) prevented the use of growth inhibition as a selection phenotype. Thus, gene knockout may be enhanced by 1) a stable but more facile phenotype for selection and 2) a single gene target without essentially identical sequences in pseudogenes.
562. Collagen VII Gene Delivery Via an Adeno-Sleeping Beauty Transposon in COL7A1-Deficient Keratinocytes From Epidermolysis Bullosa Patients
Maria Carmela Latella,1 Fabienne Cocchiarella,1 Giandomenico Turchiano,1 Manuel Gonjalves,2 Fernando Larcher,3 Zsuzsanna Izsvak,4 Zoltan Ivics,5 Alessandra Recchia.1 'Center for Regenerative Medicine, Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Netherlands; 3Cutaneous Regenerative Medicine Unit, Epithelial Biomedicine Division, CIEMAT, Madrid, Spain; 4Max Delbruck Center for Molecular Medicine, Berlin, Germany; 5Paul Ehrlich Institute, Langen, Germany.
EB is a family of severe skin adhesion defects due to disruption of the dermal-epidermal junction.
In particular the autosomal recessive epidermolysis bullosa (RDEB) is caused by mutations in the type VII collagen gene (COL7A1). The COL7A1 gene has a large coding sequence (9kb) full of repetitive elements and therefore is not easily suitable for a retroviral delivery that could cause rearrangements.
The Sleeping Beauty (SB) transposon-based integration system can potentially overcome these issues by taking advantage of the hyperactive SB100X transposase in combination with the pT2 transposon. We constructed the pT2 transposon plasmid carrying the COL7A1 cDNA driven by the PGK promoter.
Despite its enormous potential, the low efficiency of plasmid transfection procedure of the transposon/transposase integrating system in primary keratinocytes from RDEB patients remains an obstacle to its practical application in gene therapy. To overcome this limitation we incorporated the T2.Col7 transposon/ SB100X transposase system respectively into helper-dependent (HD) and firstgeneration (Ad) adenoviral vectors, to combine the major advantages of each system.
Furthermore, since the transposition from the linear adenoviral genome requires circularization ofthe vector genome, we incorporated the FRT sites into HD.T2.Col7 vector providing the Flp recombinase into integration defective lentiviral vector.
Employing the established three vectors platform we observed 25% transposition of T2.Col7 in immortalized RDEB keratinocytes. Genomic analysis of isolated single clones showed that the transposition events occurred with an average copy number of 1.5, in the absence of collagen VII cassette rearrangements, and through a genuine cut and paste transposition events as demonstrated by
sequencing of the transposon-genome junctions. Restoration of cytoplasmic and secreted full-length collagen VII protein was demonstrated by Western blot analysis on transposed clones.
Transposition experiments on primary RDEB keratinocytes showed an efficient and non-toxic infection by the 3 vectors platform, resulting in a considerable collagen VII expression in the transposed cells respect to the untreated population.
To demonstrate the safety and the transposition in long term repopulating epithelial stem cells we will investigate the proliferation and clonogenicity of primary RDEB keratinocytes infected with the viral-mediated transposon system.
563. Generation and Genetic Correction of Patient-Derived Disease-Specific Human Induced Pluripotent Stem Cells Using Gene Editing Nucleases
Sivaprakash Ramalingam,1 Chandrasegaran Srinivasan.1 'Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD.
Introduction:
Generation and precise genetic correction of disease-specific hiPSCs has great potential in regenerative medicine. Such genetic manipulations can be achieved by gene-editing nucleases. Here, we report the generation of precisely targeted genetically well-defined cystic fibrosis (CF) and Gaucher disease (GD) human induced pluripotent stem cells (hiPSCs) respectively from human CF fibroblasts homozygous for CFTRAF508 mutation and GD fibroblasts homozygous for GBA 1448T>C mutation through non-viral approach, using CCR5-specific TALENs. We also demonstrate successful in-situ genetic correction of the sickle cell disease mutation in patient-derived hiPSCs using HBB-specific TALENs.
Results and Discussion:
Site-specific addition of five stem cell factor genes flanked by loxP sites at the endogenous CCR5 safe harbor locus of human disease-specific fibroblasts using CCR5-specificTALENs induced reprogramming, giving rise to both single allele (heterozygous) CCR5-modified hiPSCs as well as biallele CCR5-modified hiPSCs (including homozygous hiPSCs). Subsequent Cre recombinase treatment of the CCR5-modified hiPSCs resulted in the removal of the stem cell factor transgenes.
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TALEN-mediated somatic cell reprogramming is safe and simpler than the viral approaches. Furthermore, it is more efficient than most nonintegrating approaches.
We also demonstrate site-specific correction of sickle cell disease (SCD) mutation at the endogenous HBB locus of patient-specific TNC1 hiPSC line that are homozygous for mutated P-globin alleles (PS/ PS), using HBB-specific TALENs. SCD-corrected hiPSC lines
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
