Scholarly article on topic '563. Generation and Genetic Correction of Patient-Derived Disease-Specific Human Induced Pluripotent Stem Cells Using Gene Editing Nucleases'

563. Generation and Genetic Correction of Patient-Derived Disease-Specific Human Induced Pluripotent Stem Cells Using Gene Editing Nucleases Academic research paper on "Biological sciences"

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Academic research paper on topic "563. Generation and Genetic Correction of Patient-Derived Disease-Specific Human Induced Pluripotent Stem Cells Using Gene Editing Nucleases"

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.


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

showed gene conversion of the mutated PS to the wild-type PA in one of the HBB alleles, while the other allele remained a mutant phenotype. After Cre recombinase-mediated excision of the loxP-flanked selection cassette from SCD-corrected hiPSC lines, we obtained secondary PS/ PA heterozygous hiPSCs. The level of HBB expression in gene corrected wild type PA allele in red blood cells is >100 fold higher than that in mutant cells, when differentiated from corrected hiPSCs.

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Furthermore, we show that the TALEN mediated generation and genetic correction did not induce any off-target mutations at closely related sites.


TALEN-mediated gene targeting could be used not only to generate precisely targeted genetically well-defined disease-specific hiPSCs, but also for site-specific repair of disease-causing mutations in patient-specific hiPSCs.

564. Comparison of High Fidelity CRISPR Systems Using Cas9 Nickase Variants

Fuqiang Chen,1 Qiaohua Kang,1 Yuping Huang,1 Gregory D. Davis.1

'ZFN Group, Sigma-Aldrich, St. Louis, MO.

Recently, CRISPR systems have been rapidly and broadly adopted in the research community for genome editing applications. Initial bioinformatic and biochemical analysis of the native bacterial Type II CRISPR system from Streptococcus pyogenes indicated that the DNA binding requirements (20 bp or less) were significantly smaller than that implemented by established ZFN and TALEN systems (30 bp or greater). In the context of larger and more complex mammalian genomes, this difference in DNA binding extent could have a significantly negative impact for genome editing applications requiring high specificity (i.e. gene and cell therapy). To expand the target site length required for CRISPR systems, we evaluated Cas9 nickase mutants for their ability to produce double strand breaks when two separate Cas9-gRNA nickase complexes are designed in close proximity. Through a systematic evaluation of mutants and nicking positions, we have collected data which suggests that gRNA orientation is a primary factor in the efficiency of paired nickase design and application. Interestingly, we observed that efficient double strand breaks could be produced in the context of putative 5' or 3' overhangs, dependent upon nick position. Upon applying the paired nickase systems to previously characterized CRISPR off-target loci, we find that off-target double strand breaks are rendered undetectable as measured by gel-based mismatch detection assays. Thus, paired nickases represent a significant improvement in CRISPR specificity, and increase CRISPR nuclease design density to aid genome editing applications with site restricted requirements near disease SNPs.

Lastly, we present data which explores the performance of CRISPR nucleases using one and two-plasmid systems, RNA, and lentiviral delivery formats for cell culture applications.

565. TALEN Mediated Gene Correction of Coagulation Factor IX Via Homologous Recombination in Context of a Canine Hemophilia B Model

Thorsten Bergmann,1 Eric Schulz,1 Maren Gebbing,1 Verena Schildgen,2 Oliver Schildgen,2 Anja Ehrhardt.1 'Institute of Virology and Microbiology, Center of Biomedical Education and Research, Department of Human Medicine, Faculty of Health, Private University of Witten/Herdecke, Witten, Germany; 2Institute of Pathology, Clinics of the City of Cologne gGmbH, Hospital of the Private University Witten/Herdecke (Cologne), Private University Witten/Herdecke, Cologne, Germany.

Gene correction at specific target loci is an emerging strategy to overcome diseases caused by genetic disorders like hemophilia B. This disease is provoked by different mutations in the coagulation factor IX gene leading to a dysfunction of the protein. In an existing canine model there is only one point mutation responsible for causing the disease, which makes it ideal for testing gene correction approaches.

In this study we aim at correcting the canine factor IX gene (cFIX) via homologous recombination (HR) replacing the mutation by the homologous wild type sequence. To do so we designed transcription-activator-like-effector nucleases (TALENs) specific for the mutated locus in the cFIX gene, to introduce a DNA double strand break. This leads to several DNA repair pathways including HR. With several detection methods we confirmed the high specificity ofthe TALENs to this locus in plasmid based transfection experiments, including hetero duplex based mutation detection assay (T7E1) and we are establishing a pyrosequencing protocol to measure TALEN efficiency. Because of the low transfection efficiencies for canine cells, we furthermore established a human HEK293 cell line in which a 2kb fragment of the genomic cFIX gene including the mutated target locus is stably integrated.

As constitutive expression would also lead to a continuous cutting of the target sequence, even after HR, as well as potential genotoxic side effects, short-time expression of TALENs would be advantageous. Thus, we created an inducible expression system based on the TET-ON 3G system (Clontech), in which the expression of the TALENs is induced by the addition of doxycycline and an artificial transactivator protein. We show that this system results in functional TALENs after induction, whereas without induction no functionality of the TALENs in transfected cells was detected.

In current experiments we are testing several approaches for providing the homologous target sequence after TALEN treatment. For this propose we designed different cassettes for integration of target sequences via HR.

Next we plan to produce high capacity adenoviral vectors (HC-AdV) containing the TALEN expression cassettes as well as a vector containing the HR-cassettes in order to do infection experiments in vitro and in vivo in murine and canine liver. In summary we established functional TALENs for the cFIX locus which in the future will allow treatment of hemophilia in a canine model for hemophilia B.

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