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1. WO2018026723 - HUMAN INDUCED PLURIPOTENT STEM CELLS FOR HIGH EFFICIENCY GENETIC ENGINEERING

Publication Number WO/2018/026723
Publication Date 08.02.2018
International Application No. PCT/US2017/044719
International Filing Date 31.07.2017
IPC
C12N 15/113 2010.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
11DNA or RNA fragments; Modified forms thereof
113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides
C12N 5/10 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
10Cells modified by introduction of foreign genetic material, e.g. virus-transformed cells
CPC
C12N 15/11
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
11DNA or RNA fragments; Modified forms thereof
C12N 15/111
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
11DNA or RNA fragments; Modified forms thereof
111General methods applicable to biologically active non-coding nucleic acids
C12N 2310/20
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
2310Structure or type of the nucleic acid
10Type of nucleic acid
20involving clustered regularly interspaced short palindromic repeats [CRISPRs]
C12N 2320/12
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
2320Applications; Uses
10in screening processes
12in functional genomics, i.e. for the determination of gene function
C12N 2330/51
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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2330Production
50Biochemical production, i.e. in a transformed host cell
51Specially adapted vectors
C12N 2501/602
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
2501Active agents used in cell culture processes, e.g. differentation
60Transcription factors
602Sox-2
Applicants
  • UNIVERSITY OF PITTSBURGH - OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION [US]/[US]
Inventors
  • SOTO-GUTIERREZ, Alejandro
  • COLLIN DE I'HORTET, Alexandra, Sylvie
  • HANDA, Kan
  • LEPE, Jorge, Guzman
  • TAKEISHI, Kazuki
  • FOX, Ira, Jacob
  • WANG, Yang
Agents
  • SIEGEL, Susan, Alpert
Priority Data
62/369,69801.08.2016US
Publication Language English (EN)
Filing Language English (EN)
Designated States
Title
(EN) HUMAN INDUCED PLURIPOTENT STEM CELLS FOR HIGH EFFICIENCY GENETIC ENGINEERING
(FR) CELLULES SOUCHES PLURIPOTENTES INDUITES HUMAINES POUR UN GÉNIE GENETIQUE À HAUT RENDEMENT
Abstract
(EN)
Methods are disclosed herein for efficiently generating human induced pluripotent stem cells (iPSC) containing a nucleic acid including a doxycycline promoter operably linked to a nucleic acid encoding Cas9. These methods include transfecting a human somatic cell with a nucleic acid molecule comprising a doxycycline promoter operably linked to a nucleic acid encoding a Cas9, and constitutive promoter operably linked to a tetracycline responsive element and inducing the somatic cell to form an iPSC, thereby producing an iPSC that can undergo CRISPR/Cas9-mediated recombination at a high efficiency. The human iPSC, or a cell differentiated therefrom, is cultured in the presence of doxycycline to induce expression of the Cas9. These cells can then be used to target in any gene of interest by introducing nucleic acids encoding sgRNAs. Induced pluripotent stem cells produced by these methods are also disclosed.
(FR)
L'invention concerne des procédés pour générer d'une manière efficace des cellules souches pluripotentes induites (iPSC) humaines contenant un acide nucléique comprenant un promoteur de doxycycline fonctionnellement lié à un acide nucléique codant pour une Cas9. Ces procédés comprennent la transfection d'une cellule somatique humaine avec une molécule d'acide nucléique comprenant un promoteur de doxycycline fonctionnellement lié à un acide nucléique codant pour une Cas9, et un promoteur constitutif fonctionnellement lié à un élément sensible à la tétracycline, et induisant une cellule somatique pour former une iPSC, en produisant de ce fait une iPSC qui peut subir une recombinaison, médiée par CRISPR/Cas9, avec un rendement élevé. L'iPSC humaine, ou une cellule différenciée obtenue à partir de cette dernière, est cultivée en présence de doxycycline pour induire l'expression de la Cas9. Ces cellules peuvent ensuite être utilisées pour cibler tout gène d'intérêt, par introduction d'acides nucléiques codant pour des petits ARN guides (sgRNA). La présente invention concerne en outre les cellules souches pluripotentes induites produites par ces procédés.
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