WO2017100683A1 - Criblage différentiel de médicaments utilisant des cellules souches pluripotentes induites avec des nanoparticules fonctionnalisées - Google Patents

Criblage différentiel de médicaments utilisant des cellules souches pluripotentes induites avec des nanoparticules fonctionnalisées Download PDF

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WO2017100683A1
WO2017100683A1 PCT/US2016/065987 US2016065987W WO2017100683A1 WO 2017100683 A1 WO2017100683 A1 WO 2017100683A1 US 2016065987 W US2016065987 W US 2016065987W WO 2017100683 A1 WO2017100683 A1 WO 2017100683A1
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cell
cells
induced
stem cells
pluripotency
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PCT/US2016/065987
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Frederick Stamner Hagen
Andranik Andrew APRIKYAN
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Stemgenics, Inc.
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells

Definitions

  • This disclosure relates to methods and compositions for generating pluripotent stem cells from multiple sources and cells differentiated therefrom to screen molecules for functional activity.
  • CF cystic fibrosis
  • CFTR cystic fibrosis transmembrane regulator
  • PSCs and airway epithelial cells may be produced from a homozygous F508del patient.
  • in vitro cellular assays of CFTR activity are limited to biopsy tissue. Due to the low prevalence of CF, biopsy tissue from CF patients is limited and competition for this tissue is high. Even laboratories and companies with priority access are forced to curtail tests. Due to limited availability of tissue, researchers typically attempt to expand the number of cells from biopsies with cell culture. However, biopsy tissue from patient airways is usually accompanied by infectious agents that further thwart culturing attempts. Such setbacks can delay drug development progress for months. Even when tissue is forthcoming, researchers have little control over the genotype of the samples provided and must contend with lack of continuity in their assays.
  • induced pluripotent stem cell produced from cells of a patient's skin patch or from blood cells and differentiation into airway epithelial cells has many advantages over the use of biopsy tissue including: 1. defined genotype of starting material, 2. choice of genotypes of starting material, 3. essentially unlimited supply of airway epithelial assay cells, 4. continuity and consistency of assay cells, and 5. consistent sterility of assay cells.
  • a 4 millimeter patch of skin is obtained from a CF patient with known genotype.
  • the cells of the skin patch are disrupted with collagenase, placed in tissue culture conditions, and incubated for outgrowth of fibroblast cells.
  • the fibroblast or blood cells are exposed to functionalized nanoparticles. Beginning about day 7 pluripotent stem cell colonies appear in culture. When colonies reach 50 microns in size they are picked, trypsinized, and grown on non-adherent plates into embryoid bodies or on adherent plates into monolayers.
  • the iPSC monolayer cells are differentiated in a series of steps into airway epithelial cells.
  • the last step in maturation of airway epithelial cells can include plating on SnapWells to induce tight junctions between cells.
  • the SnapWells are placed in the Ussing chamber (Physiological Instruments, San Diego, CA) and used according to standard protocols [30] for drug screening.
  • iPSCS induced pluripotent stem cells
  • Germline variants are inherited variations and are often associated with the pharmacokinetic behavior of a drug, including drug disposition and ultimately drug efficacy and/or toxicity, whereas somatic mutations are often useful in predicting the pharmacodynamic response to drugs.
  • Pharmacoethnicity, or ethnic diversity in drug response or toxicity is an increasingly recognized factor accounting for interindividual variations of drug response. Pharmacoethnicity is often determined by germline pharmacogenomic factors and the distribution of single nucleotide polymorphisms across various populations [34].
  • a pharmaceutical screen that utilizes a pluripotent stem cell, and/or a differentiated mature cell derived therefrom, that was originally derived from a single individual will reflect biases that are due to the individual's unique reaction to the pharmaceutical drugs. It may be that initial drug screens can be performed with cells from one source or individual but to broaden the applicability of a drug to the general population; a much wider selection of cells from different individuals is needed. The larger the number of source individuals the greater the probability the drug is going to have uniform response in the general population. Without this wider screening effort the drug may be effective for only a percentage of the population, for example 50, 40, or 20 %, with this percentage reducing the profitability of a drug. The larger the number of source individuals for generation of PSCs and mature cells used in drug screening, the greater the percentage of people being effectively treated with a given drug.
  • participants in clinical trials can be pre-qualified for a clinical trial with a cellular assay with mature cells produced from iPSCs of the candidate participant. If the cells respond well to the drug being assessed in the clinical trial the individual would be included in the clinical trial. If the cells did not respond well, the individual can be excluded from the trial. With pre-validation of the participants better outcomes of the clinical trial may be assured.
  • compositions and techniques to implement comprehensive pharmaceutical screening such that the results more accurately reflect the entire target population as a whole and avoids individual response bias and to prequalify participants in clinical trials.
  • present disclosure seeks to fulfill these needs and provides further related advantages.
  • FIGURES 1A and IB illustrate the entry of fluorescent labeled functionalized nanoparticles into the cytoplasm of fibroblast cells.
  • Two different human fibroblast cell types MHF2 (FIGURE 1A) and HI SF (FIGURE IB) were incubated for 30 minutes with FITC-conjugated functionalized nanoparticles, washed, fixed, and exposed to DAPI.
  • Blue fluorescence (indicated in dark grey; representative signals indicated with solid arrows) indicates cell nuclei; green fluorescence (light grey; representative signals indicated with dashed arrows) indicates FITC labeled nanoparticles.
  • FIGURES 2A and 2B illustrate entry of fluorescent labeled functionalized nanoparticles into the cytoplasm of human hematopoietic cells.
  • Human HL60 hematopoietic cells were incubated for 60 minutes in the presence (FIGURE 2A) or absence (FIGURE 2B) of FITC-conjugated functionalized nanoparticle, washed, fixed, and exposed to DAPI.
  • Blue fluorescence (indicated in dark grey) indicates cell nuclei; green fluorescence (light grey; representative signals indicated with solid arrows) - FITC labeled nanoparticles.
  • FIGURE 3 illustrates the nuclear localization of functionalized nanoparticles in human fibroblasts.
  • Cells were treated with FITC-conjugated functionalized nanoparticles and the images of live cells were acquired approximately 42 h later using fluorescent microscopy.
  • Green fluorescence (indicated in light grey; representative signals indicated with dashed arrows) indicates localization of the nanoparticles to nuclei.
  • FIGURE 4 illustrates the induction of target pluripotency gene expression by functionalized nanoparticles in human primary fibroblasts upon 72h treatment. Quantitative PCR data are presented as fold difference in the expression level of target pluripotency genes.
  • FIGURES 5A-5D illustrate the reprogramming human fibroblasts (FIGURE 5A) with cell penetrant nanoparticles functionalized with bioactive reprogramming factors Nano-OS L (scale bar (sb) ⁇ ). Representative images of newly formed small niPSC colonies (FIGURE 5B, sb 50 ⁇ ) and expanding larger colonies (FIGURE 5C, sb 200 ⁇ ; FIGURE 5D, sb 500 ⁇ ). Live staining of niPSC colony with alkaline phosphatase substrate is shown (FIGURE 5D, sb 500 ⁇ ).
  • FIGURES 6A-6E illustrate embryoid bodies (A-C) generated from functionalized nanoparticle-induced pluripotent stem cells.
  • Embryoid bodies (FIGURE 6D) and niPSCs at higher magnification are positive for human Tra-1-60 expression as evidenced by red staining in each respective right panel (illustrated in light grey; representative signals indicated with solid arrows).
  • Tra-1-60 expression is a characteristic pluripotency marker specific to human embryonic stem and induced pluripotent stem cells. DETAILED DESCRIPTION
  • Pluripotent stem cells differentiated into mature cells are frequently used to screen for pharmaceutical drugs.
  • the power and utility of this approach is that the PSCs, and the mature cells differentiated therefrom, retain the particular phenotypic characteristics from the original source somatic cell.
  • using only one source or individual as the source of the PSCs and mature cells for drug screening diminishes the utility of such a screen because it may not reflect the response of a broader patient population.
  • Using a larger number of sources or individuals to generate the PSCs and mature cells for screening increases the probability that the drug or combination of drugs will effectively treat a larger percentage of patients.
  • PSCs common methods for producing the PSCs involve inducing pluripotency from somatic cells using viruses, DNA, oncogene RNA, or any form of nucleic acid, have the danger of mutating or changing the genome of the generated cells and providing unintended alterations in cell phenotype, that may further skew screening results.
  • the disclosure addresses the above issues of cell-based drug screens.
  • the disclosure describes the use of multiple sets of induced pluripotent stem cells (iPSCs) and/or mature cells to screen for pharmaceutical drugs to provide more uniform and accurate indications of the pharmaceutical effect across the target population of potential subjects. This allows for identification and optimization of drug candidates that have an equivalent effect on a broad spectrum of people.
  • sets of induced pluripotent stem cells and mature cells of specific clinical trial subjects may be used to pre-validate the individual subject for inclusion and/or progress in a given clinical trial.
  • the present invention in some embodiments is directed to functionalization methods of linking proteins and/or peptides to biocompatible nanoparticles for modulating cellular functions. In some embodiments, the present invention is directed to the functionalized biocompatible nanoparticles themselves.
  • a functionalized biocompatible nanoparticle capable of penetrating through a mammalian cell membrane and delivering intracellularly a plurality of bioactive molecules for modulating a cellular function comprises: a central nanoparticle ranging in size from 1 to 50 nm or more and having a polymer coating thereon, a plurality of functional groups covalently attached to the polymer coating, wherein the plurality of bioactive molecules are attached to the plurality of the functional groups, and wherein the plurality of bioactive molecules can include at least a peptide and a protein, and wherein the peptide is capable of penetrating through the mammalian cell membrane and entering into the cell, and wherein the protein is capable of providing a new functionality within the cell.
  • a nucleic acid such as a DNA or RNA molecule can be used, which will provide a new functionality within or outside the cell.
  • the central nanoparticle can comprise iron and be magnetic.
  • the peptides of the present invention can be attached to the protein, DNA, or RNA (as opposed to being attached to the nanoparticle).
  • the peptides and proteins can each be attached to the nanoparticle by way of one or more interposing linker molecules.
  • the peptide can include five to nine basic amino acids in some embodiments, whereas in other embodiments the peptide includes nine or more basic amino acids.
  • the protein can be a transcription factor such as, for example, a transcription factor selected from the group consisting of Oct4, Sox2, Nanog, Lin28, cMyc, and Klf4.
  • the DNA or RNA molecules referred to above can encode or represent a bioactive molecule that can add new functionality within or outside the cells. By encoding a bioactive molecule, it is the ultimate product (e.g., translated polypeptide product) that is bioactive.
  • the encoded bioactive molecule can be, e.g., any transcription factor that affects induction of pluripotency or development of the cells or other bioactive protein. Such induced cells can be useful for screening drugs, as described herein, or even for other applications, such as in regenerative medicine, and the like.
  • the phrase "represent a bioactive molecule" indicates that the DNA or RNA itself is bioactive.
  • an RNA molecule can influence the endogenous transcription activities of the cell not by encoding a protein, but rather by way of RNA interference.
  • the RNA can be an inhibitory or microRNA, and the like, that can influence the abundance of endogenous transcription factors that are not desired for an intended functionality.
  • RNA-functionalized nanoparticles can deliver the RNA into the cytoplasm where it can be translated into a bioactive protein, or function to influence endogenous mRNA abundance.
  • the use of nanoparticles functionalized with RNA is integration-free and, thus, can implement a desired change (e.g., functional, developmental, or other phenotypic change) in the cell, while simultaneously avoiding interruption of endogenous genomic information.
  • DNA-functionalized nanoparticle can deliver the attached DNA molecule(s) into the cytoplasm and nucleus, where it can be transcribed first into RNA and then into a bioactive protein of interest.
  • DNA- functionalized nanoparticles ensure that the extent of any DNA integration into the cell genome is relatively minimized as compared with typical viral-based DNA integration, and is therefore safer.
  • bioactive molecule e.g., an encoded factor of interest
  • This new functionality can contribute to, e.g., a more precise and accurate platform for drug screening or to achieve certain desired cellular characteristics.
  • the present invention is directed to a method of changing a cellular functionality within or outside of a mammalian cell.
  • the novel method comprises administering an effective amount of functionalized biocompatible nanoparticles, as described herein, to the cell and changing the cellular functionality within or outside of the cell.
  • the changing of the cellular functionality can involve a change in a physico-chemical property of the cell, a change in proliferative and/or differentiation properties of the cell, a change in surviving ability of the cell, a change in the nature and level of molecules secreted from the cell, or a change in morphological phenotypical property of the cell.
  • the changing of the cellular functionality can involve an acquired ability of the cell to make a new cell type including a stem cell or a more specialized cell type.
  • compositions can be screened against PSCs or mature cells.
  • the mature cells are often generated (i.e., "derived” or “differentiated") from a stem cell, such as embryonic stem cells, umbilical cord blood stem cells, mesenchymal stem cells, or naturally occurring stem cells in the tissues of the patient.
  • the mature cells can also be generated from pluripotent stem cells (PSCs) including induced PSCs (iPSCs).
  • PSCs pluripotent stem cells
  • iPSCs induced PSCs
  • the mature cells used in the present disclosure can be generated from iPSCs or other cell types, where the iPSCs are induced by any known means, such as with nucleic acid-based technologies.
  • the iPSCs are generated using the functionalized nanoparticles, as described herein.
  • the stem cells can be directed to differentiate into preferred and/or intended mature cell types according to any known means acceptable for this purpose.
  • the functionalized nanoparticle reprogramming of mature cells into niPSCs has demonstrated production of niPSCs with naive mouse ESC-like morphology that effectively form monolayer of single cells or uniform embryoid bodies (EB), both of which have been shown in the literature to be good starting points for differentiation in mature cell types [20-23].
  • EB uniform embryoid bodies
  • CF effective drugs can be derived by differentiation of niPSCs through definitive endoderm cells, anterior foregut endoderm cells, and lung progenitors to multiciliated airway epithelial cells [17-21].
  • the present disclosure provides mature cells differentiated from PSCs, where the PSC population contains PSCs from different sources (e.g., different initial somatic cells types, different individuals, etc.).
  • sources e.g., different initial somatic cells types, different individuals, etc.
  • the advantage of screening pharmaceutical compositions using cells that were obtained, ultimately, from a diverse population of sources is that the ultimate results are not skewed toward the potentially unique phenotype of any one source, but rather more accurately predict the functional response of an entire population or subpopulation.
  • transcription factors and “reprogramming factors” and “maturation factors” encompass any molecule demonstrated to elicit and/or facilitate a desired reprogramming of a stem cell and/or the differentiation of a mature cell from a pluripotent cell.
  • results of future screenings for more effective modulators of cellular programming, functions, and morphology in stem or other cell types to convert them into more or less primitive stem cells are envisioned to be useful in the application of the present drug screening method.
  • the following Examples are intended to illustrate, not limit, the application of the present disclosure.
  • Oct4, Sox2, Nano, and Lin28 (OSNL) transcription factors were produced in bacteria as his-tag proteins with a free sulfhydryl.
  • the proteins were purified by affinity chromatography and, through a linker chain (LC) linked to paramagnetic nanoparticles using sulfhydryl linking chemistry previously described in WO 2013/059831, incorporated herein by reference in its entirety.
  • the same transcription factors and other bioactive factors of interest can be generated employing nanoparticles functionalized with RNA or DNA, which encode these bioactive factors, using corresponding DNA or RNA conjugation chemistry methods known in the art.
  • a poly arginine peptide was added to the nanoparticles using the same chemistry.
  • Nano-OS L pluripotent stem cells
  • HDAC histone deacetylase
  • iPSC small induced pluripotent stem cell
  • the colonies are mechanically transferred and then trypsinized and plated onto irradiated mouse embryonic fibroblasts (iMEFs) or matrigel for long term culturing with FGF or 2iLIF. Efficiencies of reprogramming are typically 8 to 25 %.
  • niPSCs Nano-OS L generated colonies
  • niPSCs Upon further replating and culturing in the presence of 2iL continuous formation of a large number of small round dome-shaped niPSCs approximately 30 to 100D in size is observed. These niPSCs, passaged through trypsinization, resemble mouse-like naive human embryonic stem cell colonies as recently reported [11-14]. Live staining of the niPSCs with alkaline phosphatase substrate and monoclonal antibody against human Tra-1-60 pluripotency marker using commercially available kits (Life Technologies and Stem Cell technologies, respectively) demonstrate that the niPSCs are positive for alkaline phosphatase and Tra-1-60. See FIGURES 5D-5F.
  • niPSC colonies spontaneously differentiate to three-dimensional aggregates of cells called embryoid bodies [15, 16].
  • Subsequent culture of niPSC-embryoid bodies in non-adherent plates reveal their further expansion and formation of a monolayer of embryoid bodies as depicted in FIGURES 6A-6E.
  • these embryoid bodies represent the starting material for generation of various more specialized cell types and tissues, and in adherent plates, these embryoid bodies can be further differentiated into different cell types of interest, including airway epithelial cells [17-21].
  • an individual set of nanoparticles can also be functionalized with RNA or DNA encoding each one of the transcription factors included Oct4, Sox2, Klf4, cMyc, Nanog, Lin28. Other factors can also be used such as Klf4, cMyc, Glisl, etc.
  • RNA RNA
  • RNA end-labeling kits which will make it suitable for subsequent covalent binding to the maleimide group of a LC linker on the nanoparticle.
  • the unbound is removed using a magnetic column (such as one from Myltenyi Biotech) yielding nanoparticles functionalized with covalently bound RNA molecule.
  • a poly arginine peptide is added to the nanoparticles using the same sulfhydryl chemistry.
  • the resultant RNA-functionalized nanoparticle is used to reprogram somatic cell into pluripotent stem cell or other cell type using cell reprogramming protocol and the setting similar to that described in Example 2 thereby resulting in a similar outcome.
  • niPSCs nanoparticle induced pluripotent stem cells
  • the patients' and healthy donor skin samples obtained from the National Disease Resource Interchange (NDRI, Philadelphia, PA).
  • the fibroblast cells will be treated twice with Nano-OSNL and transferred onto irradiated mouse embryonic fibroblasts (iMEFs).
  • iMEFs irradiated mouse embryonic fibroblasts
  • the newly generated niPSC colonies will be hand-picked and transferred for expansion for a week onto fresh iMEFs followed by trypsinization and further passage on iMEFs in the presence of FGF or GSK-3 D, MEK inhibitors, and human Leukemia Inhibitory Factor LIF (2iL).
  • FGF or GSK-3 D irradiated mouse embryonic fibroblasts
  • MEK inhibitors irradiated mouse embryonic fibroblasts
  • human Leukemia Inhibitory Factor LIF (2iL) human Leukemia Inhibitory Factor LIF
  • skin fibroblasts from CF and normal individuals will be treated with functionalized nanoparticles and will generate CF-specific and normal pluripotent stem cells with intact genome, which will be further used for differentiation into airway epithelial cells based on recently established and published protocols suitable for a monolayer of single cells as well as for embryoid bodies [17-21].
  • patient-specific functional respiratory epithelial cells will be generated from niPSCs generated by reprogramming fibroblasts of CF patients and control donors and these cells will be evaluated in functional assays and drug screening tests described below.
  • Ussing chamber analysis is used to assess electrophysiological phenotypes of engineered normal and CF airway epithelial cells and establish the suitability of the cultures for comparing CF drug candidates. This includes assessment of CFTR, Ca ++ - activated CI " channels' (CaCC) and Na + channels' (ENaC) functionality and subsequently testing the effects of various drug candidates and/or their combination including VX-770, VX-809, and INO-4995 on these parameters.
  • D15 AFE cells generated are seeded 10 5 cells/cm 2 on Corning Costar R - SnapWellTM cell culture inserts (0.4 micron pore size) coated with a combination of fibronectin (5 ⁇ g/mL; BD Biosciences), laminin (5 ⁇ g/mL; Sigma-Aldrich), and collagen IV (60 ⁇ g/mL; Sigma- Aldrich), 1 g/cm 2 Vitrogen and differentiated as described [18].
  • the transepithelial PD will be recorded, and subsequently the cells will be voltage clamped at 0 mV, and the resulting short circuit current (I sc ) continuously recorded.
  • I sc short circuit current
  • a periodic bipolar voltage pulse will monitor resistance calculated using Ohm's law.
  • baseline values will be recorded and amiloride (20miol/L) will be added to apical bath solutions to assess basal amiloride-inhibitable I sc. This will assess epithelial Na + channel (ENaC) status while excluding a potential interference by ENaC on subsequent recordings.
  • CFTR function will be assessed by the sequential addition of the cAMP agonist, forskolin (Sigma Aldrich, 20 miol/L), the CFTR potentiator, genistein (50 miol/L), and the CFTR channel blocker Inhm (Tocris, 30 miol/L).
  • ATP 100 ⁇
  • ATP will be added to the apical compartment prior to CFTR activation to trigger transient CaCC current, the presence of which is another validation of a differentiated airway epithelial phenotype.
  • a CaCC-inhibitor, 4,4 - diisothiocyanostilbene-2,2 -disiilfonic acid (DIDS) added prior to forskolin to check whether the CaCC inhibitor does not inhibit forskolin-stimulated I sc but glibenclamide or Inhi-2 added post-forskolin abolishes it.
  • DIDS 4,4 - diisothiocyanostilbene-2,2 -disiilfonic acid
  • cultures will be pretreated with CFTR potentiator VX-770 (Ivacaftor), CFTR corrector VX-809 (lumacaftor), or the combination VX-770+VX-809 (Orkambi) all available from Selleck Chem, Kunststoff Germany following previously described protocols [74].
  • Cultures will be pretreated by adding 3-5 ⁇ compound (or DMSO control) to the basolateral compartment 48 hours prior to Ussing analysis.
  • cultures will be pretreated with INO-4995 [75] alone or in combination by adding 3 ⁇ M/day basolaterally for 3 days prior to Ussing analysis. While all compounds are expected to suppress amiloride-inhibitable I sc in CF epithelia, only VX-770, VX-809 or the combination will be expected to restore CFTR function.
  • Example 6 Using multiple airway epithelial cell and stem cell sets to validate drug candidates for a broad patient population To assure that drug candidate compounds identified with airway epithelial cellular assays developed in Example above affect the broadest range of homozygous F508del CF patients, the drug candidate compounds will be screened with airway epithelial cells developed from 20 different or more homozygous F508del CF patients. These airway epithelial cells will be developed according to the protocols described above. The candidate drugs will be independently screened with airway epithelial cells from each homozygous F508del CF patient.
  • the lead compounds from Example 5 can be assessed in animal model systems and the best drug candidates chosen.
  • the clinical trial subjects contribute a patch of skin, niPSCs are developed from fibroblast outgrowth from the skin patch, airway epithelial cells are differentiated from the niPSCs, and used for screening against the drug candidate(s) being tested in the clinical trial.
  • niPSCs are developed from fibroblast outgrowth from the skin patch
  • airway epithelial cells are differentiated from the niPSCs, and used for screening against the drug candidate(s) being tested in the clinical trial.
  • Those clinical trial subjects whose cells react as expected from the drug screening as per Example above will be included in the clinical trial.
  • Firth AL Dargitza CT, Quallsa SJ, Menona T, Wright R, Singera O, Gage FH, Khanna A, Verma FM. Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells. PNAS 2014, 111 :E1723-30. Firth AL,Tushar Menon T, Parker GS, Quails SJ, Lewis BM, Ke E, Dargitz CT, Wright R, Khanna A, Gage FH, Verma FM. Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs. Cell Reports, 2015, 12: 1385-1390.
  • SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Molecular Pharmacology . 2002;62:65-74.
  • ALK transforming growth factor-beta superfamily type I activin receptor-like kinase

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Abstract

L'invention concerne des méthodes, des compositions et des systèmes utiles pour le criblage de médicaments et des applications associées qui impliquent la reprogrammation et/ou l'induction de la différentiation de cellules obtenues à partir d'un ou plusieurs sujets pour former un type de cellule d'intérêt. L'induction de la reprogrammation et/ou de la différenciation peut être réalisée par l'application d'agents, tels que des agents incorporés dans des nanoparticules fonctionnalisées, pour améliorer l'efficacité de la reprogrammation et/ou de la différenciation.
PCT/US2016/065987 2015-12-09 2016-12-09 Criblage différentiel de médicaments utilisant des cellules souches pluripotentes induites avec des nanoparticules fonctionnalisées WO2017100683A1 (fr)

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US11821890B2 (en) 2019-04-12 2023-11-21 Pinpoint Science Inc. Nanosensor chip with compound nanopores and methods of use thereof

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WO2009045063A2 (fr) * 2007-10-02 2009-04-09 Seoul National University Industry Foundation Complexe constitué d'un peptide de translocation et de nanoparticules magnétiques, et utilisation d'un tel complexe
US8257941B2 (en) * 2007-06-15 2012-09-04 Kyoto University Methods and platforms for drug discovery using induced pluripotent stem cells
US20140051085A1 (en) * 2011-03-01 2014-02-20 The Scripps Research Institute Direct reprogramming of human fibroblasts to functional neurons under defined conditions

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US20060160066A1 (en) * 2005-01-20 2006-07-20 The Regents Of The University Of California Cellular microarrays for screening differentiation factors
US20080019949A1 (en) * 2006-06-28 2008-01-24 University Of Kansas Differentiation of stem cells from umbilical cord matrix into hepatocyte lineage cells
US8257941B2 (en) * 2007-06-15 2012-09-04 Kyoto University Methods and platforms for drug discovery using induced pluripotent stem cells
WO2009045063A2 (fr) * 2007-10-02 2009-04-09 Seoul National University Industry Foundation Complexe constitué d'un peptide de translocation et de nanoparticules magnétiques, et utilisation d'un tel complexe
US20140051085A1 (en) * 2011-03-01 2014-02-20 The Scripps Research Institute Direct reprogramming of human fibroblasts to functional neurons under defined conditions

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Publication number Priority date Publication date Assignee Title
US11821890B2 (en) 2019-04-12 2023-11-21 Pinpoint Science Inc. Nanosensor chip with compound nanopores and methods of use thereof

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