563 Generation and Genetic Correction of Patient Derived Disease Specific Human Induced Pluripotent Stem Cells Using Gene Editing Nucleases Molecular Therapy Volume 22, Supplement 1, May 2014 Copyrigh[.]
Trang 1Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
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selectively degrades NANOGP8 Both NANOG and NANOGP8 were
present in cDNA NANOG has introns but the pseudogenes do not
PCR amplifi cation of the intron1-exon1 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 fi rst 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
Adeno-Sleeping Beauty Transposon in COL7A1-Defi cient
Keratinocytes From Epidermolysis Bullosa
Patients
Maria Carmela Latella,1 Fabienne Cocchiarella,1 Giandomenico
Turchiano,1 Manuel Gonçalves,2 Fernando Larcher,3 Zsuzsanna
Izsvak,4 Zoltan Ivics,5 Alessandra Recchia.1
1 Center for Regenerative Medicine, Department of Life Sciences,
University of Modena and Reggio Emilia, Modena, Italy;
2 Department of Molecular Cell Biology, Leiden University Medical
Center, Leiden, Netherlands; 3 Cutaneous Regenerative Medicine
Unit, Epithelial Biomedicine Division, CIEMAT, Madrid, Spain;
4 Max Delbruck Center for Molecular Medicine, Berlin, Germany;
5 Paul 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 effi ciency 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 fi
rst-generation (Ad) adenoviral vectors, to combine the major advantages
of each system
Furthermore, since the transposition from the linear adenoviral
genome requires circularization of the 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 effi cient 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
Patient-Derived Disease-Specifi c Human Induced Pluripotent Stem Cells Using Gene Editing Nucleases
Sivaprakash Ramalingam,1 Chandrasegaran Srinivasan.1
1 Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD.
Introduction:
Generation and precise genetic correction of disease-specifi c 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-defi ned cystic fi brosis (CF) and Gaucher disease (GD) human induced pluripotent stem cells (hiPSCs) respectively from human CF
fi broblasts homozygous for CFTRΔF508 mutation and GD fi broblasts homozygous for GBA 1448T>C mutation through non-viral approach, using CCR5-specifi c TALENs We also demonstrate successful in-situ genetic correction of the sickle cell disease mutation in patient-derived hiPSCs using HBB-specifi c TALENs
Results and Discussion:
Site-specifi c addition of fi ve stem cell factor genes fl anked by loxP sites at the endogenous CCR5 safe harbor locus of human disease-specifi c fi broblasts using CCR5-specifi cTALENs induced reprogramming, giving rise to both single allele (heterozygous) CCR5-modifi ed hiPSCs as well as biallele CCR5-modifi ed hiPSCs (including homozygous hiPSCs) Subsequent Cre recombinase treatment of the CCR5-modifi ed hiPSCs resulted in the removal of the stem cell factor transgenes
TALEN-mediated somatic cell reprogramming is safe and simpler than the viral approaches Furthermore, it is more effi cient than most nonintegrating approaches
We also demonstrate site-specifi c correction of sickle cell disease (SCD) mutation at the endogenous HBB locus of patient-specifi c TNC1 hiPSC line that are homozygous for mutated β-globin alleles (βS/ βS), using HBB-specifi c TALENs SCD-corrected hiPSC lines
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Copyright © The American Society of Gene & Cell Therapy S219
showed gene conversion of the mutated βS to the wild-type βA in
one of the HBB alleles, while the other allele remained a mutant
phenotype After Cre recombinase-mediated excision of the
loxP-fl anked selection cassette from SCD-corrected hiPSC lines, we
obtained secondary βS/ βA heterozygous hiPSCs The level of HBB
expression in gene corrected wild type βA allele in red blood cells is
>100 fold higher than that in mutant cells, when differentiated from
corrected hiPSCs
Furthermore, we show that the TALEN mediated generation and
genetic correction did not induce any off-target mutations at closely
related sites
Conclusion:
TALEN-mediated gene targeting could be used not only to generate
precisely targeted genetically well-defi ned disease-specifi c hiPSCs,
but also for site-specifi c repair of disease-causing mutations in
patient-specifi c hiPSCs
Systems Using Cas9 Nickase Variants
Fuqiang Chen,1 Qiaohua Kang,1 Yuping Huang,1 Gregory D
Davis.1
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 signifi cantly
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 signifi cantly negative impact for genome editing applications
requiring high specifi city (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 effi ciency of paired nickase
design and application Interestingly, we observed that effi cient 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 fi nd that off-target double strand breaks are rendered undetectable
as measured by gel-based mismatch detection assays Thus, paired
nickases represent a signifi cant improvement in CRISPR specifi city,
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
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
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; 2 Institute 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 specifi c 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) specifi c 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 confi rmed the high specifi city of the 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 effi ciency Because
of the low transfection effi ciencies 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 artifi cial 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