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WO2025062355A1 - Lignées de cellules souches contenant des protéines marquées endogènes avec expression différentielle, procédés de production et leur utilisation pour l'identification de cycle cellulaire - Google Patents

Lignées de cellules souches contenant des protéines marquées endogènes avec expression différentielle, procédés de production et leur utilisation pour l'identification de cycle cellulaire Download PDF

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Publication number
WO2025062355A1
WO2025062355A1 PCT/IB2024/059158 IB2024059158W WO2025062355A1 WO 2025062355 A1 WO2025062355 A1 WO 2025062355A1 IB 2024059158 W IB2024059158 W IB 2024059158W WO 2025062355 A1 WO2025062355 A1 WO 2025062355A1
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WIPO (PCT)
Prior art keywords
cell
gene
marker
cells
expression product
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PCT/IB2024/059158
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English (en)
Inventor
Moises DI SANTE
Francesco PASQUALINI
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Universita degli Studi di Pavia
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Universita degli Studi di Pavia
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Filing date
Publication date
Priority claimed from IT102023000019377A external-priority patent/IT202300019377A1/it
Application filed by Universita degli Studi di Pavia filed Critical Universita degli Studi di Pavia
Publication of WO2025062355A1 publication Critical patent/WO2025062355A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates to a stem cell line containing endogenous marked proteins with differential expression, production methods and use thereof Background Art
  • HTS high- throughput screening
  • fewer than five compounds are used for human clinical trials and the vast majority of these fails. This is dramatic not only for the company but also for society. Indeed, once on the market, the new drug will be offered at a price capable of covering not only the research costs associated with its development, but also the development of the many failed assays.
  • hPSCs human pluripotent stem cells
  • HTS assay generally the typical HTS assay is offered at ⁇ 0.1 €/datapoint but culture procedures for hPSCs have much higher costs because they comprise a maintenance phase in pluripotency and a differentiation phase towards the cellular phenotype, e.g., hPSC-derived cardiac muscle cells (hPSC-CM).
  • these costs levitate the market price for HTS with hPSC at least 10- 100 times higher.
  • potentially safe and effective compounds in humans can be discarded because they show signs of unsafety or ineffectiveness in pre-clinical models that we know (after the FDA modernization act) to be poorly predictive.
  • a drug to prevent heart attack (1 in 2 men, 1 in 3 women) may already have been discarded simply because it did not give the right signals when tested on rodent or human cells of other tissue, since the only human heart muscle cells can be derived from hPSCs.
  • hPSCs human heart muscle cells
  • the main aim of the present invention is to make stem cell lines containing endogenous marked proteins with differential expression.
  • Another object of the invention is to use computational models to interpret potential errors in HTS.
  • the invention in accordance with the present invention allows HTS to be made more predictive by introducing hPSCs in a convenient maimer.
  • a further object of the present invention is to genetically engineer a line of hPSCs so that they express fluorescent proteins which are sensitive to a variety of pathophysiological signals. In this way, the cells would independently manufacture the reagent and the fixed component of the marginal cost for imHTS with hPSCs would disappear.
  • TEMPO drug screening and regenerative medicine
  • FIG. 1 is a schematic representation of the FUCCIplex sensor in accordance with the present invention.
  • FIGS. 2 and 3 are schematic representations of the gene construct in accordance with the present invention.
  • Figure 4 is representative of a functional analysis of the FUCCIplex gene construct in accordance with the present invention.
  • Figure 5 is representative of a computational analysis aimed at eliminating the overlap in the emission spectra between the green and cyan channels
  • Figure 6 is representative of a segmentation analysis of cell compartments using the gene construct in accordance with the present invention.
  • FIGS 7 and 8 are representative of expression analysis of the gene construct in accordance with the present invention.
  • the invention according to the invention relates to a gene construct, comprising:
  • the first expression product is at least one of: a cyan fluorescent protein (CFP) or an infrared fluorescent protein (iRFP).
  • CFP cyan fluorescent protein
  • iRFP infrared fluorescent protein
  • the second expression product is at least one of: a fluorescent protein (RFP-LifeAct) the quantity of which in a cell varies depending on the presence of actin, a fluorescent protein the quantity of which in a cell varies depending on the presence of tubulin.
  • a fluorescent protein RFP-LifeAct
  • the third expression product is a fluorescent protein (GCaMP6f) the quantity of which in a cell varies depending on the presence of intracellular calcium.
  • construct in accordance with the present invention comprises at least one fluorescent marker (FLUO-4) adapted to detect the presence of intracellular calcium.
  • FLUO-4 fluorescent marker
  • the invention relates to a method for performing the phase identification of a cell cycle, the method comprising the phases of: visualizing, using at least a first marker, at least one or more gene expression products the quantity of which in a cell vary in a cell cycle-dependent manner; and detecting at least the first marker so as to distinguish a proliferation phase of the cell cycle from a resting phase of the cell cycle.
  • the method according to the invention comprises visualizing, using at least a second marker, at least one or more gene expression products the quantity of which in a cell vary in a cell structure-dependent maimer; and detecting at least the second marker so as to distinguish actin microfilaments.
  • the method according to the invention comprises visualizing, by using at least a third marker, at least one or more gene expression products the quantity of which in a cell vary in a cell function-dependent manner; and detecting at least the third marker so as to distinguish intracellular calcium.
  • the invention relates to a cell, comprising an exogenous polynucleotide integrated into a target genomic locus, the exogenous polynucleotide comprising polynucleotide sequences encoding:
  • the target genomic locus is a locus of a gene that encodes for a differentially expressed protein.
  • the invention relates to a lentiviral expression vector comprising the previously described gene construct.
  • such a vector consists of the sequence SEQ. ID. No: 1.
  • Such a vector is a lentiviral vector having the following nucleotide sequence:
  • AAAGATACCT AAAGGATCAA CAGCTCCTGG GGATTTGGGG
  • GCAACTACAA GACCCGCGCC GAGGTGAAGT TCGAGGGCGA
  • AGAACACCCC CATCGGCGAC GGCCCCGTGC TGCTGCCCGA
  • CTGACGCAAC CCCCACTGGT TGGGGCATTG CCACCACCTG
  • CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT
  • GAAGAGCGCC CAATACGCAA ACCGCCTCTC CCCGCGCGTT
  • the lines hiPSC WTC-11 (GM25256) and GCaMP6 (WTC11-AAV-CAG- GCaMP6) were kindly provided by Dr. Bruce Conklin (Gladstone. Institute of Cardiovascular Research). Cells were maintained in complete Essential 8 Flex Medium (catalog no. A2858501, ThermoFisher Scientific) supplemented with Penicillin/Streptomycin (P/S) (catalog no. A001, HiMedia) on Matrigel Growth Factor Reduced (GFR) 1: 100 (7-10 mg/ml, depending on the batch) without phenol red (catalog no. 356231, Coming).
  • the cells were detached using 0.5 mM EDTA (ethylenediaminetetraacetic acid, Life Technologies) in Dulbecco’s PBS (DPBS) without Ca2+ or Mg2+ (catalog No. C-40232, PromoCell) for 5 min at 37 degrees 5%, CO2 environment.
  • DPBS Dulbecco
  • Ca2+ or Mg2+ Catalog No. C-40232, PromoCell
  • cells were expanded in 6-well plates by means of 1: 10 passage in the presence of RevitaCell (catalog No. A26445-01, LifeTechnologies). Twenty-four hours after plating, the medium was replaced with complete Essential 8 Flex Medium (without RevitaCell) and changed every other day.
  • the hiPS cells were differentiated into cardiomyocytes (CMs) using a direct differentiation method adapted from the small molecule protocol described in Lian et al (PMID: 23257984). More specifically, hiPSCs were plated at a density of 8 x 104/well in 12-well plates which were pre-coated with Matrigel (day -3/4) in the presence of RevitaCell. The next day, the medium was changed to complete Essential 8 Flex Medium (without RevitaCell). After 3-4 days, or when the cells reached 90% confluence (day 0), the medium was replaced with pre-warmed cardio differentiation medium (RPMI 1640+ Glutamax Medium (lx) (catalog no.
  • RPMI 1640+ Glutamax Medium (lx) catalog no.
  • the strategy for the generation of the triple reporter and 4-color hiPS TEMPO clonal cell line followed the same scheme adopted for the creation of the HaCaT FUCCIplex prototype clonal cell line with some adaptations (see below).
  • the FUCCIplex cell cycle indicator and the genetically encoded actin sensor RFP- LifeAct were introduced into the WTCl l-AAV-CAG-GCaMP6 cell line.
  • antibiotic selection Hygromycin B (50 mg/ml in PBS) 50 ul/ml for FUCCIplex- positive cells and 2 pl/ml Puromycin (1 pg/ pl) for LifeAct and nd-positive cells
  • TEMPO hiPSc were sorted and positive clones were expanded.
  • hiPS WTC-11 (GM25256) cells were genomically edited to specifically insert the FUCCIplex cassette into the ROSA26 THUMPD3-AS1 locus (PMID: 18037879) by means of CRISPR/Cas9n-based gene-trap strategy. Unless otherwise indicated, genomic editing was performed as described in Bertero et al (PMID: 27899508).
  • the FUCCIplex cDNA fragment flanked by ROSA26 homology arms was amplified by PCR starting from the original FUCCIplex plasmid (pLV-Hygro- EFla-FUCCIplex) using primers fwd: CATCATTTTGGCAAAGAATTAATTCGGATCCgccaccatggtgagcaagg and rev: CGAGGCTGATCAGCGAGCTACGCGTttacagggccttccgccg.
  • the plasmid pR26-Bsd_CAG-EGFPd2 (kindly provided by Professor Bertero, Department of Molecular Biotechnology and Health Sciences, Turin, Italy) was digested with BamHI and Mlul to remove the EGFPd2 fragment and ligated by NEBuilder HiFi DNA assembly (catalog No. E5520S, New England Biolabs) with the FUCCIplex DNA fragment to generate the pR26-Bsd_C AG- FUCCIplex construct.
  • hiPSc were transfected using the Lipofectamine stem cell reagent (catalog No.
  • the present invention relates to a method for the production of a cell comprising at least one endogenous marked protein, expressed in a differential maimer, the method comprising:
  • a donor plasmid comprising: a first polynucleotide encoding for a selection cassette, where the selection cassette comprises a first selectable marker; a second polynucleotide encoding for a second selectable marker that is different from the first selectable marker;

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La construction génique comprend : (i) au moins un premier gène (FUCCIplex) ou un fragment partiel d'un gène pour au moins un premier produit d'expression dont la quantité dans une cellule varie d'une manière dépendant du cycle cellulaire ; et au moins l'un parmi : (ii) au moins un deuxième gène ou un fragment partiel d'un gène pour au moins un deuxième produit d'expression dont la quantité dans une cellule varie d'une manière dépendant de la structure cellulaire ; (iii) au moins un troisième gène ou un fragment partiel d'un gène pour au moins un troisième produit d'expression dont la quantité dans une cellule varie d'une manière dépendant de la fonction cellulaire.
PCT/IB2024/059158 2023-09-20 2024-09-20 Lignées de cellules souches contenant des protéines marquées endogènes avec expression différentielle, procédés de production et leur utilisation pour l'identification de cycle cellulaire Pending WO2025062355A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
IT102023000019377 2023-09-20
IT102023000019377A IT202300019377A1 (it) 2023-09-20 2023-09-20 Linee di cellule staminali contenenti proteine endogene marcate con espressione differenziale, metodi di produzione e loro utilizzo per l’identificazione del ciclo cellulare
IT202400021063 2024-09-20
IT102024000021063 2024-09-20

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WO2025062355A1 true WO2025062355A1 (fr) 2025-03-27

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010042333A1 (fr) * 2008-10-06 2010-04-15 Merck Sharp & Dohme Corp. Génération d'un rapporteur en phase mitotique pour l'imagerie par bioluminescence in vivo chez l'animal vivant
US20190365818A1 (en) * 2017-02-09 2019-12-05 Allen Institute Genetically-tagged stem cell lines and methods of use
WO2019236893A2 (fr) * 2018-06-07 2019-12-12 Allen Institute Lignées de cellules souches contenant des protéines marquées endogènes étiquetées de manière différentielle, leurs procédés de production et leur utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010042333A1 (fr) * 2008-10-06 2010-04-15 Merck Sharp & Dohme Corp. Génération d'un rapporteur en phase mitotique pour l'imagerie par bioluminescence in vivo chez l'animal vivant
US20190365818A1 (en) * 2017-02-09 2019-12-05 Allen Institute Genetically-tagged stem cell lines and methods of use
WO2019236893A2 (fr) * 2018-06-07 2019-12-12 Allen Institute Lignées de cellules souches contenant des protéines marquées endogènes étiquetées de manière différentielle, leurs procédés de production et leur utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAMEL MARGRIT ET AL: "Catching new targets in metabolic disease with a zebrafish", CURRENT OPINION IN PHARMACOLOGY, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 37, 6 September 2017 (2017-09-06), pages 41 - 50, XP085302347, ISSN: 1471-4892, DOI: 10.1016/J.COPH.2017.08.007 *
LAETITIA KURZAWA ET AL: "Cell-Cycle Markers and Biosensors", CHEMBIOCHEM, vol. 11, no. 8, 15 April 2010 (2010-04-15), Hoboken, USA, pages 1037 - 1047, XP055528490, ISSN: 1439-4227, DOI: 10.1002/cbic.200900729 *

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