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WO2018057782A1 - Méthodes de prévention de la différenciation de cellules souches à l'aide d'un inhibiteur de pask - Google Patents

Méthodes de prévention de la différenciation de cellules souches à l'aide d'un inhibiteur de pask Download PDF

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WO2018057782A1
WO2018057782A1 PCT/US2017/052781 US2017052781W WO2018057782A1 WO 2018057782 A1 WO2018057782 A1 WO 2018057782A1 US 2017052781 W US2017052781 W US 2017052781W WO 2018057782 A1 WO2018057782 A1 WO 2018057782A1
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pask
cells
differentiation
cell
inhibitor
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Jared Rutter
Chintan K. KIKANI
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University of Utah Research Foundation Inc
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • PAS domain containing protein kinase is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species.
  • the identification of a role for PASK in differentiation in different cell types would be useful as a cell therapy for the treatment of various diseases and disorders.
  • the present disclosure provides a mechanism for PASK in promoting the differentiation of progenitor and stem cells.
  • inhibition of PASK can be useful in preventing cell differentiation.
  • Described herein are methods of preventing differentiation of stem cells, the methods comprising contacting stem cells with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • Described herein are methods of reprogramming mouse embryonic fibroblast cells, the methods comprising contacting mouse embryonic fibroblast cells with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • Described herein are methods of inhibiting differentiation of pluripotent embryonic stem cells into neurons, the methods comprising contacting pluripotent embryonic stem cells with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • Described herein are methods of inhibiting differentiation of C3H10T1/2 mesenchymal stem cells into adipocytes, the methods comprising contacting C3H10T1/2 mesenchymal stem cells with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • Described herein are methods of inhibiting differentiation of mouse C2C12 muscle progenitor cells into muscle fibers, the methods comprising contacting mouse C2C12 muscle progenitor cells with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • Described herein are methods of increasing the number of preadipocytes present in sample comprising administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having preadipocytes and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress preadipocytes cell differentiation into adipocytes; and suppressing PASK activity in the sample; thereby increasing the number of preadipocytes present in the sample.
  • PASK protein kinase
  • Described herein are methods of increasing the number of neural stem cells present in a sample comprising administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having neural stem cells and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress neural stem cells differentiation into neurons; and suppressing PASK activity in the sample; thereby increasing the number of neural stem cells present in the sample.
  • PASK protein kinase
  • Described herein are methods of increasing the number of myoblasts present in a sample comprising administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having myoblasts and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress myoblasts cell differentiation into myoctyes; and suppressing PASK activity in the sample; thereby increasing the number of myoblasts present in the sample.
  • PASK protein kinase
  • Described herein are methods of producing a culture of undifferentiated cells the method comprising providing a population of preadipocytes having with a PAS domain containing protein kinase (PASK); and contacting the population of cells in a) with a PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells; thereby producing a culture of undifferentiated cells.
  • PASK protein kinase
  • Described herein are methods of producing a culture of undifferentiated cells comprising providing a population of myoblasts having a PAS domain containing protein kinase (PASK); and contacting the population of cells in a) with PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells;
  • PASK protein kinase
  • RNA short-interfering ribonucleic acid
  • Described herein are methods of inhibiting expression of a PAS domain containing protein kinase (PASK) in a subject comprising administering to a subject an effective amount of a pharmaceutical composition comprising a short- interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK), thereby inhibiting the expression of PASK in the subject.
  • a pharmaceutical composition comprising a short- interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK)
  • an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein and one or more guide RNAs that target the gene encoding the PASK, whereby the one or more guide RNAs target the genomic loci of the gene encoding the PASK and
  • FIGs. 1 A-K shows that PASK is required for skeletal muscle regeneration after acute muscle injury.
  • FIG. IB shows the comparison of fusion index between control and siRNA (Si_PASK) or inhibitor (BioE-1197)-treated C2C12 cells from FIG. 1A
  • FIG. IE shows the results of qRT-PCR analysis of C2C12 cells showing abundance of Mylpf and Actal mRNAs in PASK-silenced cells expressing GFP, WT or KD PASK. 18S rRNA was used as normalizer. Error bars ⁇ S.D. *P ⁇ 0.05, #P ⁇ 0.05 WT vs KD PASK in control samples, # P ⁇ 0.05 WT vs KD hPASK in Si PASK samples.
  • FIG. 1G shows the quantification of fusion index from FIG. IF at Day 4.
  • FIG. 1H shows qRT-PCR analysis of fold change in the expression of Pask and Myh3 mRNA following BaC12 induced muscle injury to TA muscle relative to uninjured (DPI 0). 18S rRNA was used as normalizer.
  • FIG. II is a Western blot analysis of isolated TA muscle following BaC12 induced muscle injury from WT and Pask-/- mice.
  • FIGs. 2A-L shows that Pask is required for transcriptional activation of MyoG in response to differentiation cues.
  • FIG. 2A is a schematic of myogenesis from satellite cells that depicts the progression of transcription factors during myogenesis.
  • FIG. 2B shows qRT-PCR analysis of WT and Pask '1' satellite cells prior to (day 0) or at the indicated time after initiation of differentiation with ⁇ insulin in serum free DMEM. 18S rRNA was used as normalizer. Transcript levels of WT cells at Day 0 were set at 1 to calculate fold changes during differentiation. Error bars ⁇ S.D. *P ⁇ 0.05, **P ⁇ 0.005,
  • FIG. 2C is a Western blot analysis of the indicated proteins at 5 days post- injury from isolated TA muscles of WT and Pask ⁇ mice.
  • FIG. 2D shows the
  • FIG. 2F is an immunofluorescence microscopy showing MyoG and Pask in control, Pask-siRNA or 25 ⁇ BioE-119- treated cells at Day 1 of differentiation.
  • FIG. 2G shows the percent MyoG + cells in Control, Pas ⁇ -siRNA or 25 ⁇ BioE-1197-treated cells during differentiation as in FIG. 2F.
  • FIG. 2K shows that an empty GFP vector or GFP vector containing hPask were retrovirally introduced to proliferating C2C12 cells at a sub-confluent density in growth media.
  • 2L shows qRT-PCR analysis of GFP or hPask-expressing C2C12 myoblasts after puromycin selection from FIG. 21.
  • Cells were collected from growth media (GM) or 24 h after addition of differentiation media (DM).
  • GM growth media
  • DM differentiation media
  • FIGs. 3A-F show that Pask is required for myogenic conversion of C3H10T1/2 cells by MyoD.
  • FIG. 3A is a schematic depiction of the mechanism by which MyoD- induces myogenic conversion of adipogenic C3H10T1/2 cells.
  • FIG. 3C shows MyoD-expressing C3H10T1/2 cells were allowed to differentiate in the presence of DMSO or 25 ⁇ BioE-1197 and processed for immunofluorescence microscopy using anti-MyoG antibody.
  • FIGs. 4A-I shows that Pask directly interacts with and phosphorylates Wdr5 at Ser49.
  • FIG. 4A shows that endogenous Pask was immunoprecipitated from C2C12 cells either before (day -1, 0) or after induction of differentiation (Day 1).
  • FIG. 4B shows V5-tagged LacZ, WT Pask or KD (K1028R) Pask was co-expressed with Flag-YFP or Flag-Wdr5 in 293T cells.
  • FIG. 4C shows V5-hPask was expressed in HEK293T cells with control or Flag-Wdr5 vector.
  • FIG. 4D shows in vitro phosphorylation of purified His-Wdr5 was performed using WT or KD Pask and analyzed by autoradiogram of the reaction mixture after western blotting, with total protein visualized by Ponceau S staining. pPask indicates autophosphorylation of WT-Pask during kinase reaction.
  • FIG. 4E shows that the Pask-Wdr5 complex was immunoprecipitated from cells incubated with 2P in the presence of DMSO or 25 ⁇ BioE-1197.
  • FIG. 4F shows endogenous Pask was immunoprecipitated from C2C12 cells growing in growth media or 12 h after replacement with differentiation media containing ⁇ Insulin and was incubated with purified Flag- Wdr5 and [ ⁇ - 2 ⁇ ] ATP. Autoradiogram shows incorporation of 2 P into Pask (p-Pask) and Wdr5 (p-Wdr5).
  • FIG. 4G is a schematic showing Ser49 and upstream sequence in Wdr5, compared to the site of Pask phosphorylation in Ugpl, a bona fide substrate of S.
  • FIG. 4H shows GST-tagged WT, S49A or S49E Wdr5 was incubated with Pask and [ ⁇ - 2 ⁇ ] ATP and phosphorylation was detected by autoradiography after SDS-PAGE.
  • [I] WT or S49A Wdr5 was co-immunoprecipitated with Pask from cells incubated with 2 P-phosphate and analyzed as in FIG. 4E.
  • FIGs. 5A-H show that the phospho-mimetic S49E Wdr5 mutant rescues myogenesis in Pask-silenced cells.
  • FIG. 5C shows the quantification of percent Pax7+ cells from FIG.
  • FIG. 5F shows the same as in FIG. 5B, except cells were stained for MHC on Day 3 of differentiation.
  • FIG. 5H shows C2C12 myoblasts were infected with retrovirus expressing GFP, Flag tagged WT or KD Pask or WT, S49A or S49E Wdr5 and infected cells were selected with puromycin in growth media for 48 hrs.
  • FIGs. 6A-G shows that Pask is required for recruitment of Wdr5 and MyoD to the Myog promoter during differentiation.
  • FIG. 6A is a depiction of the Myog genomic locus depicting MyoD and RNAPolII occupancy as well as H3K4me2 and H3K4me3 abundance at 60 h of differentiation from the ENCODE dataset for the C2C12 cell line.
  • TSS Transcription Start Site. Colored horizontal bars indicate the positions of ChIP amplicons a, b or c.
  • FIG. 6E shows that endogenous Wdr5 ChIP was performed from control or Pask-siRNA C2C12 cells at Day 0 or Day 1 of differentiation and fold enrichment on the Myog promoter was determined by qRT-PCR. Error bars ⁇ S.D. * P ⁇ 0.05.
  • FIGs. 7A-F shows that differentiation induced H3K4mel to H3K4me3 conversion is dependent upon Pask phosphorylation of Wdr5.
  • H3K4mel (FIG. 7A) and total H3 ChIP (FIG. 7B) were performed from control or Pask-siRNA C2C12 cells at the indicated day of differentiation and fold enrichment on the Myog promoter was determined by qRT-PCR using primer set b. Error bars ⁇ S.D. * P ⁇ 0.05, **P ⁇ 0.005.
  • FIG. 7C shows that the differentiation potential of C2C12 myoblasts subjected to Pask or M113 siRNA treatments was assessed by qRT-PCR using primers specific for Myog, Mylpf, M113 and Pask.
  • FIG. 7F is a model depicting the role of Pask and Wdr5 phosphorylation in regulating MyoD recruitment to the Myog promoter during differentiation.
  • FIG. 8A-E shows that Pask is enriched in stem cells and is required for terminal differentiation of neuronal, adipogenic and myogenic cell types.
  • FIG. 8A shows a comparison of Pask mRNA expression profile from GeneAtlas MOE430 dataset obtained from BioGPS.
  • FIG. 8B shows induction of Pask mRNA expression during iPSC formation from indicated differentiated cell types plotted as percentile rank across all genes.
  • FIG. 8C shows Pask mRNA expression during differentiation of hESCs into cardiomyocytes and in comparison with adult heart expressed as a percentile rank across all genes.
  • FIG. 8D is a depiction of the mouse Pask locus showing positions of Oct4 and Nanog transcription factor binding and corresponding chromatin marks indicative of active transcription in ESCs. ChlP-Seq data was obtained from ENCODE browser, see Examples for citation.
  • FIG. 8E shows the relative mRNA after shRNA treatments.
  • FIGs. 9A-C shows that Pask is required for differentiation of ES cells but not for iPS reprograming.
  • FIG. 9A shows mouse embryonic fibroblasts (MEFs) containing an IRES-GFP cassette expressed from the Oct4 locus were induced for reprogramming by mSTEMCCA factors as described in material and methods in the presence of DMSO or 50 ⁇ BioE-1197. Representative images of iPS colonies at day 8 post-initiation of reprogramming showing expression of GFP and its corresponding brightfield image are shown.
  • FIG. 9B shows the quantification of GFP+ colonies 8 days after initiation of reprograming from experiment in FIG. 9A.
  • FIG. 9C shows mouse ES cells were treated with either DMSO or 25 ⁇ BioE-1197 24 h prior to induction of neuronal
  • FIGs. 10A-D shows that Pask is required for adipogenesis of mesenchymal stem cell.
  • FIG. 10A shows C3H10T1/2 cells that were treated with either DMSO or 25 ⁇ BioE-1197 48 h prior to induction of adipocyte differentiation.
  • FIG. 10D shows the proliferation rate of C3H10T1/2 cells that were measured by seeding cells at 10,000 cells/well and counting cells using a hemocytometer over the ensuing three days.
  • FIGs. 11-F shows that genetic and pharmacological inhibition of Pask suppresses myogenesis of mouse and human myoblasts.
  • FIG. 11A shows MHC staining of C2C12 cells with Cas9 guided deletion of Pask during differentiation.
  • FIG. 11B shows the fusion index calculation from FIG. 11A.
  • FIG. 11C is a Western blot analysis of MHC expression during differentiation of C2C12 cells with Cas9 guided Pask deletion.
  • FIG. 11D shows human primary myoblasts that were subjected to differentiation by either 2% horse serum or ⁇ insulin in presence of DMSO or BioE-1197.
  • FIG. 11A shows MHC staining of C2C12 cells with Cas9 guided deletion of Pask during differentiation.
  • FIG. 11B shows the fusion index calculation from FIG. 11A.
  • FIG. 11C is a Western blot analysis of MHC expression during differentiation of C2C12 cells with Cas9 guided Pask deletion.
  • FIG. 11D shows human primary myoblasts
  • FIG. 1 IF shows the proliferation rate of mouse primary myoblasts derived from WT or Pask-/- mice treated with DMSO or BioE-1197.
  • FIGs. 12A-C show that Pask is required for transcriptional activation of MyoG in response to differentiation cues.
  • FIG. 12A is a Western blot analysis of the abundance of the indicated proteins during myoblast differentiation in control or Pask-silenced C2C12 cells using 2% Horse Serum media.
  • FIGs. 13A-B shows that Pask does not regulate MyoD+ cell population.
  • FIG. 13A is an immunofluorescence microscopy showing MyoD expression and localization in control, Pask-siRNA or 25 ⁇ BioE-1197 treated cells on Day 0 of differentiation.
  • FIGs. 14A-C shows a Pask promoter occupied by MyoG and MyoD during differentiation.
  • FIG. 14A shows ChlP-Seq data from the ENCODE dataset for H3K4me3 levels, MyoG, MyoD and POL II binding at the indicated time-points at the Pask promoter during C2C12 differentiation.
  • Vertical turquois line represents the peak of MyoG/MyoD binding at the predicted E-Box motif (sequence in brown).
  • the transcription start site is indicated by an arrow.
  • the green and red horizontal bars represent Pask a and Pask _b ChIP primer sets used.
  • FIG. 14A shows ChlP-Seq data from the ENCODE dataset for H3K4me3 levels, MyoG, MyoD and POL II binding at the indicated time-points at the Pask promoter during C2C12 differentiation.
  • Vertical turquois line represents the peak of MyoG/MyoD binding at the predicted E-Box motif (se
  • FIG. 14B shows MyoG and MyoD ChIP was performed from proliferating (Day 0) or differentiating (Day 1 and Day 2) C2C12 cells and analyzed by Pask a and Pask b primer sets.
  • Myh3 primers spanning - lOObp from TSS were used as a positive control for MyoG binding.
  • n 3, Error bars ⁇ S.D.
  • FIG. 14C shows a region containing ⁇ 300bp upstream of the predicted Pask transcriptional start site, which contains a putative E-box element, was cloned upstream of firefly luciferase.
  • RLU Relative Light Units.
  • FIGs. 15A-B show that Pask is required for myogenic conversion of mesenchymal stem cells by MyoD.
  • FIG. 15A shows the immunofluorescence of MyoD expression in DMSO or BioE-1197 treated samples at Day 1 and Day 2.
  • FIG. 15B shows the immunofluorescence of MyoG expression in DMSO or BioE-1197 treated samples at Day l and Day 2.
  • FIGs. 16A-F show that Pask directly interacts with and phosphorylates Wdr5 at Ser49.
  • FIG. 16A shows flag-tagged YFP or indicated members of various protein complexes of which Wdr5 is a member were expressed in 293T cells.
  • FIG. 16B shows bacterially purified GST-Wdr5 or GST control was attached to glutathione sepharose and purified His-tagged Pask was passed over and the beads were washed extensively.
  • FIG. 16C shows the domain truncation of Pask to determine Wdr5 binding region.
  • FIG. 16D shows the in vitro phosphorylation of Wdr5 by Pask in the presence of DMSO or 25 ⁇ 1197.
  • FIG. 16E shows that bacterially purified GST-(Flag)-WT, S49A or S49E Wdr5 were used for GST pull-down assays of Pask as in FIG. 16B.
  • FIG. 16F shows HEK293T cells expressing Flag tagged YFP or the indicated Wdr5 variants were lysed and Flag- tagged proteins were imunoprecipitated.
  • FIG. 17 shows Wdr5 S49E expression rescues genetic loss of Pask.
  • FIGs. 18A-D shows Pask and phosphomimetic Wdr5 promote H3K4mel to H3K4me3 conversion and MyoD recruitment to the Myog promoter.
  • FIG. 18A is a depiction of the Myog locus showing abundance of position of enhancer and promoter region as well as H3K4me3 levels and MyoD occupancy 60hrs after initiation of differentiation from ENCODE datasets deposited by Barbara Wold lab.
  • FIG. 18B shows ChlP-qPCR of differentiation time course of Control vs Pask silenced C2C12 cells using MyoD, H3K4mel, H3K27ac and H3K4me3 antibodies. Actb negative region was used as normalizer. *p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.001.
  • FIG. 18A is a depiction of the Myog locus showing abundance of position of enhancer and promoter region as well as H3K4me3 levels and MyoD occupancy 60hrs after initiation of differentiation from ENCODE datasets deposited by
  • FIG. 18C shows H3K4mel ChIP of control and Pask silenced C2C12 cells using primers spanning the indicated gene and position relative to the TSS. actb negative control region was used as the normalizer. *P ⁇ 0.05, **P ⁇ 0.005.
  • FIG. 18D shows H3K4me3 ChIP of control and Pask silenced C2C12 cells using primers spanning the indicated gene and position relative to the TSS. actb negative control region was used as the normalizer. *P ⁇ 0.05, **P ⁇ 0.005,
  • FIG. 19 is a table providing cell lines, antibodies and primer sequences used herein.
  • FIG. 20 shows that Pax7+ cell number increases upon PASK inhibition.
  • FIG. 21 depicts the effects of WT, S49A or S49E mutant Wdr5 expression on Pax7 expression in C2C12 myoblasts.
  • FIGS. 22A-C shows the restoration of Pax7 expression and myogenesis program differentiation defective myoblasts after BioE-1197 pretreatment.
  • FIG. 22 A is a schematic representation of the method used to isolate differentiation defective myoblasts and the treatment used in this experiment. Pax7 levels were detected (FIG. 22B) at passage # as indicated in (FIG. 22B).
  • FIG. 22C shows the differentiation capacity of differentiation defective myoblasts.
  • Ranges can be expressed herein as from “about” or “approximately” one particular value, and/or to "about” or “approximately” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” or “approximately,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as "about” that particular value in addition to the value itself. For example, if the value " 10" is disclosed, then “about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11 , 12, 13, and 14 are also disclosed.
  • sample is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein.
  • a sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.
  • the term "subject” refers to the target of administration, e.g., a human.
  • the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • a subject is a mammal.
  • a subject is a human.
  • the term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the term "patient” refers to a subject afflicted with a disease or disorder.
  • the term “patient” includes human and veterinary subjects.
  • the "patient” has been diagnosed with a need for treatment for cancer, such as, for example, prior to the administering step.
  • “Inhibit,” “inhibiting” and “inhibition” as used herein, mean to diminish or decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% inhibition or reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, in an aspect, the inhibition or reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • the inhibition or reduction is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90- 100% as compared to native or control levels. In an aspect, the inhibition or reduction is 0-25, 25-50, 50-75, or 75-100% as compared to native or control levels.
  • Module means a change in activity or function or number. The change may be an increase or a decrease, an enhancement or an inhibition of the activity, function or number.
  • “Promote,” “promotion,” and “promoting” refer to an increase in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the initiation of the activity, response, condition, or disease. This may also include, for example, a 10% increase in the activity, response, condition, or disease as compared to the native or control level.
  • the increase or promotion can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or more, or any amount of promotion in between compared to native or control levels.
  • the increase or promotion is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to native or control levels.
  • the increase or promotion is 0-25, 25-50, 50-75, or 75-100%, or more, such as 200, 300, 500, or 1000% more as compared to native or control levels.
  • the increase or promotion can be greater than 100 percent as compared to native or control levels, such as 100, 150, 200, 250, 300, 350, 400, 450, 500% or more as compared to the native or control levels.
  • determining can refer to measuring or ascertaining a quantity or an amount or a change in activity. For example, determining the amount of a disclosed polypeptide in a sample as used herein can refer to the steps that the skilled person would take to measure or ascertain some quantifiable value of the polypeptide in the sample. The art is familiar with the ways to measure an amount of the disclosed polypeptides and disclosed nucleotides in a sample.
  • disease or “disorder” or “condition” are used interchangeably referring to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person.
  • a disease or disorder or condition can also related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint.
  • Pask PAS domain containing protein Kinase
  • SREBP-1 Sterol Regulatory Element Binding Protein- 1
  • Pask pharmacologic inhibition or genetic ablation of Pask resulted in decreased liver fat content and improved insulin sensitivity in rodent models of diabetes and obesity (Hao et al, 2007; Wu et al, 2014).
  • Pask responds to extracellular glucose and stimulates the transcription of the gene encoding insulin via regulation of the PDX-1 transcription factor (An et al., 2006; Semache et al, 2013).
  • PDX-1 transcription factor As et al., 2006; Semache et al, 2013.
  • Pask is expressed at a low level in most adult tissues (Katschinski et al., 2003). Interestingly, elevated Pask mRNA abundance in stem or progenitor cell types in several transcriptome datasets was observed. As described herein, genetic and pharmacologic methods of modulating Pask activity were used to uncover a function of Pask in regulating the differentiation of stem and progenitor cells into, for example, neuronal, adipocytes or myocytes lineages. As described herein, the mechanism underlying the role of Pask in regulating stem and progenitor cell differentiation can depend upon, for instance, direct phosphorylation of Wdr5.
  • Wdr5 is a component of several chromatin modifying complexes, including mixed lineage leukemia (Mil) histone H3 Lysine 4 (H3K4) methyltransferase complexes (Ruthenburg et al, 2007; Wysocka et al, 2005). Wdr5 is a histone H3 binding protein (Wysocka et al, 2005) that is postulated to present the H3 N-terminal tail to the Mil or Setl enzymes for methylation at lysine 4 (Ruthenburg et al, 2006; Schuetz et al, 2006).
  • Mil mixed lineage leukemia
  • H3K4 histone H3 Lysine 4
  • Wdr5 is a histone H3 binding protein (Wysocka et al, 2005) that is postulated to present the H3 N-terminal tail to the Mil or Setl enzymes for methylation at lysine 4 (Ruthenburg et al, 2006; Schuetz e
  • Lysine 4 of Histone H3 is sequentially methylated to the mono- (H3K4mel), di- (H3K4me2) and tri-methyl (H3K4me3) forms by methyltransferases (Shilatifard, 2012).
  • H3K4mel is typically found at enhancers, which are binding sites for regulatory DNA- binding transcription factors (Rada-Iglesias et al, 2011 ; Shlyueva et al, 2014).
  • H3K4mel functions as a transcriptional repressive mark at the promoters of lineage specifying genes (Cheng et al, 2014).
  • H3K4me3 marks are usually associated with transcriptionally active promoters, or with poised promoters when found together with repressive H3K27me3 marks (Bernstein et al., 2006). These histone modifications collaborate with pioneering transcription factors to elicit programs of gene expression that drive differentiation of stem and progenitor cells (Zaret and Carroll, 201 1). As described herein, myogenic progenitor cells were used as a model of inducible differentiation to show that phosphorylation of a single Wdr5 serine by Pask is needed and sufficient for the conversion of repressive H3K4mel marks to activating H3K4me3 marks at the lineage-specifying myogenin ⁇ Myog) promoter.
  • Pask may function through similar mechanisms in other systems. Specifically, the data described herein shows that Pask phosphorylates Wdr5 to promote H3K4mel to H3K4me3 conversion on the promoters of lineage-specifying genes to facilitate chromatin remodeling, gene expression and differentiation.
  • the method can comprise contacting a stem cell with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • the PASK inhibitor is BioE- 1197.
  • the contact between the stem cell and a PASK inhibitor can be conducted or otherwise carried out in vitro.
  • the stem cell can be a pluripotent stem cell, a mouse embryonic fibroblast cell, a progenitor cell, a C3H10T1/2 mesenchymal stem cell, a mouse C2C12 muscle progenitor cell, or a primary myoblast cell.
  • the stem cell can comprise a PAS domain.
  • the method can comprise contacting mouse embryonic fibroblast cells with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • the PASK inhibitor can be BioE-1197.
  • the contact between the mouse embryonic fibroblast cells and a PASK inhibitor can be conducted or otherwise carried out in vitro.
  • the mouse embryonic fibroblast cells can comprise a PAS domain.
  • the mouse embryonic fibroblast cells can be reprogrammed such that they are less likely to or do not differentiate.
  • the method can comprise contacting a pluripotent embryonic stem cell with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • the PASK inhibitor can be BioE-1197.
  • the contact between the pluripotent embryonic stem cell and a PASK inhibitor can be conducted or otherwise carried out in vitro.
  • the method can comprise contacting a C3H10T1/2 mesenchymal stem cell with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • the PASK inhibitor can be BioE-1197.
  • the contact between the C3H10T1/2 mesenchymal stem cell and a PASK inhibitor can be conducted or otherwise carried out in vitro.
  • the method can comprise contacting a mouse C2C12 muscle progenitor cell with a PAS domain containing protein kinase (PASK) inhibitor.
  • PASK protein kinase
  • the PASK inhibitor can be BioE-1197.
  • the contact between the mouse C2C12 muscle progenitor cell and a PASK inhibitor can be conducted or otherwise carried out in vitro.
  • the method can comprise: a) administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having preadipocytes and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress preadipocytes cell differentiation into adipocytes; and b) suppressing PASK activity in the sample; thereby increasing the number of preadipocytes present in the sample.
  • the PASK inhibitor can be BioE-1197.
  • the sample is a human sample.
  • the increase in the number of preadipocytes can occur in vitro prior to
  • the patient in need of cell therapy can be at risk for developing or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.
  • method can comprise a) administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having neural stem cells and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress neural stem cells differentiation into neurons; and b) suppressing PASK activity in the sample;
  • PASK protein kinase
  • the PASK inhibitor can be BioE-1197.
  • the sample is a human sample.
  • the increase in the number of neural stem cells can occur in vitro prior to transplantation of the neural stem cells into a patient in need of cell therapy.
  • the patient in need of cell therapy can be at risk for developing or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.
  • the method can comprise a) administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having myoblasts and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress myoblasts cell differentiation into myoctyes; and b) suppressing PASK activity in the sample; thereby increasing the number of myoblasts present in the sample.
  • the PASK inhibitor can be BioE-1197.
  • the sample can be a human sample.
  • the increase in the number of myoblasts can occur in vitro prior to transplantation of the myoblasts into a patient in need of cell therapy.
  • the patient in need of cell therapy can be at risk for developing or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.
  • the method can comprise: a) providing a population of preadipocytes having with a PAS domain containing protein kinase (PASK); and b) contacting the population of cells in a) with a PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells; thereby producing a culture of undifferentiated cells.
  • the undifferentiated cells can be mammalian cells.
  • the PASK inhibitor can be BioE- 1197.
  • the method can comprise: a) providing a population of neural stem cells having a PAS domain containing protein kinase (PASK); and b) contacting the population of cells in a) with a PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells; thereby producing a culture of undifferentiated cells.
  • the undifferentiated cells can be mammalian cells.
  • the PASK inhibitor can be BioE- 1197.
  • the method can comprise: a) providing a population of myoblasts having a PAS domain containing protein kinase (PASK); and b) contacting the population of cells in a) with PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells; thereby producing a culture of undifferentiated cells.
  • the undifferentiated cells can be mammalian cells.
  • the PASK inhibitor can be BioE- 1197.
  • the method can comprise contacting a cell expressing a PASK with a short-interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK), thereby inhibiting the expression of PAS K.
  • the cell can be an undifferentiated stem cell.
  • the cell can be a stem cell.
  • the stem cell can a pluripotent stem cell, a mouse embryonic fibroblast cell, a progenitor cell, a C3H10T1/2 mesenchymal stem cell, a mouse C2C 12 muscle progenitor cell, or a primary myoblast cell.
  • the stem cell can comprise a PAS domain.
  • the cell can be a fibroblast, a preadipocyte, neural stem cell or a myoblast.
  • the method can comprise administering to a subject an effective amount of a pharmaceutical composition comprising a short-interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK), thereby inhibiting the expression of PASK in the subject.
  • a pharmaceutical composition comprising a short-interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK), thereby inhibiting the expression of PASK in the subject.
  • the subject can be a human.
  • the subject can be a patient in need of therapy or treatment.
  • the subject can be at risk for developing or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.
  • RNA that comprises or forms a double-stranded structure containing a first strand comprising a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second strand comprising a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene, wherein the first and the second ribonucleotide sequences can be separate complementary sequences that hybridize to each other to form said double-stranded structure, can be delivered to cells in a variety of known mechanisms.
  • siRNA can be delivered using an expression construct that encodes the siRNA.
  • cells can be transfected with the expression construct to get the siRNA inside the cell.
  • contacting the cell or providing the cell with an RNA comprises introducing the RNA comprising a double-stranded structure into the cell using a nanoparticle carrier.
  • a nanoparticle carrier can be used.
  • the naked RNA is delivered.
  • the vehicle RNA and associated delivery agent
  • the vehicle can be small.
  • the vehicle can be, but is not limited to, less than 100 nm in diameter, less than 50 nm, less that 20 nm, or less than 10 nm.
  • Genome editing can be carried out to inhibit or prevent expression of a gene.
  • Genome editing techniques are well-known in the art including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR associated system (CAS) technology.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • CAS CRISPR associated system
  • the method can comprise introducing into a stem cell containing and expressing the gene encoding the PASK, an engineered, non-naturally occurring
  • the stem cell is a mammalian cell.
  • the stem cell can a pluripotent stem cell, a mouse embryonic fibroblast cell, a progenitor cell, a C3H10T1/2
  • the mesenchymal stem cell a mouse C2C12 muscle progenitor cell, or a primary myoblast cell.
  • the cell can be a fibroblast, a preadipocyte, neural stem cell or a myoblast.
  • the Cas protein is a Cas9 protein.
  • the Cas protein is codon optimized for expression in a eukaryotic cell.
  • CRISPR system and “CRISPR-Cas system” refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated (“Cas") genes, including sequences encoding a Cas gene, a guide sequence (also referred to as a "spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.
  • CRISPR-associated CRISPR-associated
  • one or more elements of a CRISPR system can be derived from a type I, type II, or type III CRISPR system.
  • one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a proto spacer in the context of an endogenous CRISPR system).
  • the guide RNA can comprise a guide sequence fused to a tracr sequence.
  • the cell can be a eukaryotic cell.
  • the cell can be a mammalian cell.
  • the cell can be a mammalian cell.
  • the cell can be a human cell.
  • the Cas protein can comprise one or more nuclear localization signals (NLS).
  • the Cas protein can be a type II CRISPR system enzyme.
  • the Cas protein can be a Cas9 protein.
  • the Cas9 protein can S. pneumoniae, S. pyogenes, or S. thermophilus Cas9.
  • the Cas protein can be a mutated Cas9 derived from any of these organisms.
  • the Cas protein can also be a Cas9 homolog or ortholog.
  • the Cas protein is codon optimized for expression in a eukaryotic cell.
  • the Cas protein directs cleavage of one or two strands at the location of the target sequence.
  • target sequence is used to refer to a sequence to which a guide sequence can be designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient
  • a target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence can be located in the nucleus or cytoplasm of a cell.
  • the target sequence can be within an organelle of a eukaryotic cell (e.g., mitochondrion).
  • a sequence or template that can be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template” or “editing polynucleotide” or “editing sequence.”
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). It is believed that the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized the CRISPR complex.
  • PAM protospacer adjacent motif
  • the precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). A skilled person will be able to identify further PAM sequences for use with a given CRISPR enzyme.
  • the PAM comprises NGG (where N is any nucleotide, (G)uanine, (G)uanine).
  • the target sequence corresponds to PASK.
  • the expression of the gene product can decreased.
  • CRISPR system can be introduced into a cell by a delivery system including but not limited to viral particles, liposomes, electroporation, microinjection or conjugation.
  • the gene can be a gene that can encode a PAS domain containing protein kinase (PASK).
  • PASK protein kinase
  • the method of modulating expression can be any of the methods described herein (e.g., CRISPR, siRNA).
  • silencing one or more gene comprise techniques well-known in the art. For example, silencing can be performed by silencing transcription or by silencing translation, both of which result in a suppression of the expression of the gene. Several known techniques can be used for silencing, such as, but not limited to, RNAi, CRISPR, or siRNA. Silencing can comprise administering a silencing agent.
  • the methods disclosed herein can use a variety of cells.
  • Examples of cells include but are not limited to stem cells, such as embryonic stem cells.
  • the subject or patient is a human.
  • the method can comprise administering a therapeutically effective amount of the in vitro produced and cells.
  • the in vitro produced cells can be produced by any of the methods disclosed herein.
  • the cells are
  • the cells can be pluripotent stem cells, a progenitor cells, mesenchymal stem cells, embryonic stem cells, primary myoblast cells, fibroblasts, preadipocytes, neural stem cells or a myoblasts.
  • the subject can be in need of cell therapy or a cellular transplantation or transfusion.
  • the method can comprise administering a therapeutically effective amount of any of the cells produced and/or disclosed herein to the subject or patient.
  • the method can comprise identifying a patient in need of treatment.
  • the method can comprise administering to the patient a therapeutically effective amount of undifferentiated stem cells.
  • These cells can comprise a PASK that is inhibited or with lower or inhibited expression that were produced or generated via any of the methods disclosed herein.
  • Therapeutic administration encompasses prophylactic applications. Based on genetic testing and other prognostic methods, a physician in consultation with their patient can choose a prophylactic administration where the patient has a clinically determined predisposition or increased susceptibility (in some cases, a greatly increased susceptibility) to a type of condition disorder or disease.
  • the patient can be at risk for developing sarcopenia, disease- associated sarcopenia or a stem cell loss disorder or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.
  • the subject can be identified using standard clinical tests known to those skilled in the art.
  • Diseases associated or related to sarcopenia include but are not limited to rheumatologic conditions (e.g., rheumatoid arthritis), obesity, Type 2 diabetes and insulin resistance.
  • Stem cells can be used to treat diseases and conditions of the blood and immune systems.
  • Stem cell loss disorder includes but is not limited to leukemias (acute and chronic), lymphomas, myelodyspalstic syndromes, myeloproliferative disorders (e.g., anemias),
  • lymphoproliferative disorders phagocyte disorders, metabolic disorders (including inherited metabolic disorders), histiocytic disorders, inherited erythrocyte abnormalities, immune system disorders (including inherited immune system disorders), plasma cell disorders, malignancies, disorders associated with the nervous system (e.g., multiple sclerosis), and disorders associated with the muscular system.
  • compositions are administered to a subject (e.g., a human patient) already with or diagnosed with a condition, disorder or disease in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression of (and preferably arrest) the symptoms of the condition, its complications, and
  • a therapeutically effective amount of the cells described herein can be an amount that achieves a cure, but that outcome is only one among several that can be achieved. One or more of the symptoms can be less severe. Recovery can be accelerated in an individual who has been treated.
  • the therapeutically effective amount of the cells described herein and used in the methods as disclosed herein applied to mammals can be determined by one of ordinary skill in the art with consideration of individual differences in age, weight, and other general conditions (as mentioned above).
  • the cells including undifferentiated cells (e.g., stem cells) as described herein can be prepared for parenteral administration.
  • Cells prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., topical) administration. Aerosol inhalation can also be used to deliver the cells.
  • compositions comprising protein kinase
  • compositions further comprise a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” refers to solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants that can be used as media for a pharmaceutically acceptable substance.
  • the pharmaceutically acceptable carriers can be lipid-based or a polymer-based colloid. Examples of colloids include liposomes, hydrogels, microparticles, nanoparticles and micelles.
  • the compositions can be formulated for administration by any of a variety of routes of administration and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration.
  • excipient means any compound or substance, including those that can also be referred to as “carriers” or “diluents.” Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one of ordinary skill in the art can consult numerous authorities for guidance if needed.
  • the compositions can also include additional agents (e.g., preservatives).
  • compositions as disclosed herein can be prepared for, for example, parenteral administration.
  • Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, intervertebral subcutaneous, or intraperitoneal.
  • Paternal administration can be in the form of a single bolus dose, or may be, for example, by a continuous pump.
  • Topical administration includes ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery. Aerosol inhalation can also be used to deliver any of the compositions described herein.
  • Pulmonary administration includes inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal.
  • compositions can be prepared for parenteral administration that includes dissolving or suspending the CRISPR-Cas systems, si-RNA molecules, nucleic acids, polypeptide sequences or vectors in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g., PBS), and the like.
  • an aqueous carrier such as water, buffered water, saline, buffered saline (e.g., PBS), and the like.
  • PBS buffered saline
  • One or more of the excipients included can help approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.
  • compositions include a solid component (as they may for oral administration)
  • one or more of the excipients can act as a binder or filler (e.g., for the formulation of a tablet, a capsule, and the like).
  • a binder or filler e.g., for the formulation of a tablet, a capsule, and the like.
  • one or more of the excipients can be a solvent or emulsifier for the formulation of a cream, an ointment, and the like.
  • the pharmaceutical compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered.
  • Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration.
  • the pH of the pharmaceutical compositions typically will be between 3 and 1 1 (e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8).
  • the resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules.
  • composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
  • the compositions can also be formulated as powders, elixirs, suspensions, emulsions, solutions, syrups, aerosols, lotions, creams, ointments, gels, suppositories, sterile injectable solutions and sterile packaged powders.
  • the active ingredient can be nucleic acids or vectors described herein in combination with one or more
  • pharmaceutically acceptable carriers means molecules and compositions that do not produce or lead to an untoward reaction (i.e., adverse, negative or allergic reaction) when administered to a subject as intended (i.e., as appropriate).
  • Example 1 Pask is required for terminal differentiation in multiple cell lineages in vitro and muscle regeneration in vivo.
  • Pask mRNA abundance was examined in several publicly available gene expression datasets. Elevated Pask mRNA was observed across diverse stem and progenitor cell types compared to differentiated cells and tissues (FIG. 8 A). For example, Pask was more abundant in mouse embryonic stem (ES) cells and progenitor cell types such as C2C12 myoblasts, C3H10T1/2 mesenchymal stem cells, Neuro2a neuroblastoma cells and immune progenitor cells compared to mouse embryonic fibroblasts, other somatic cell types and adult tissues (FIG. 8A) (BioGPS:Pask, GeneAtlas MOE430).
  • ES mouse embryonic stem
  • progenitor cell types such as C2C12 myoblasts, C3H10T1/2 mesenchymal stem cells, Neuro2a neuroblastoma cells and immune progenitor cells compared to mouse embryonic fibroblasts, other somatic cell types and adult tissues (FIG. 8A) (BioGPS:Pask
  • iPSCs induced pluripotent stem cells
  • Progenitor cells such as C3H10T1/2 mesenchymal stem cells and C2C12 myoblasts also robustly express Pask (FIG. 10A). Adipocyte differentiation was induced as described herein. Cells were processed for Oil Red O staining 8 days after
  • C3H10Tl/2 cells differentiate into adipocytes in response to appropriate signaling cues (Pinney and Emerson, 1989; Reznikoff et al, 1973; Zhou et al, 2013).
  • C3H10T1/2 cells were treated with adipogenic stimuli in the presence or absence of BioE-1197.
  • Vehicle-treated control cells efficiently differentiated into mature adipocytes, as evidenced by accumulation of lipid droplets in more than 80% of cells (FIG. 10A, quantified in FIG. 10B).
  • Pask inhibition greatly impaired adipocyte differentiation, as about 10% of cells contained observable lipid droplets.
  • FIG. 11A quantified in FIG. IB
  • CRISPR/Cas9-mediated Pask mutation also resulted in suppression of insulin-induced differentiation as indicated by the absence of multi -nucleated myotubes (FIG. 11 A, quantified in 1 IB) and prevented the induction of MHC protein expression (FIG. 11C).
  • BioE-1197 treatment also blunted the induction of myosin heavy chain in human primary myoblasts in response to both horse serum and insulin treatment (FIG. 1 ID). Protein extracts were prepared 3 days of differentiation and probed for MHC or ⁇ -tubulin.
  • Pask was reversed by the expression of siRNA-resistant human wild type (WT) Pask, but not a K1028R kinase-dead (KD) Pask mutant (FIG. 1C, quantified in FIG. ID) (Kikani et al, 2010), indicating the importance of Pask catalytic activity for myoblast fusion.
  • this effect appears to be cell autonomous as a fraction of cells in the population that express WT Pask (WT + ) show rescue of the fusion defect, whereas cells not expressing hPask within the same culture (WT " ) remained mono-nucleated and fusion defective. Additionally, Mylpf (myosin light chain) and Actal mRNAs, which are both markers of myoblast differentiation, failed to be induced in Pask knockdown cells.
  • the myoblast differentiation process is activated in vivo to repair and replenish the myotome after muscle injury.
  • satellite cells become activated, proliferate and eventually fuse in response to differentiating signals to restore mature myotubes.
  • Pask mRNA (FIG. 1H) and protein (FIG. II) expression was robustly upregulated. This is consistent with high Pask expression in stem cells compared with differentiated cells (FIG. 8A), since muscle stem cells begin to proliferate upon muscle injury before eventually fusing to replenish the injured myotome.
  • the kinetics of muscle regeneration in WT and Pask '1' mice was compared. As shown in FIG.
  • Pask expression is high in stem and progenitor cell types (FIG. 8A-C).
  • the Pask promoter is occupied by the pluripotency promoting transcription factors, Oct4 and Nanog (FIG. 8D).
  • This transcription factor occupancy coincided with Pask transcriptional activation as indicated by the occupancy of RNA PolII as well as the activating H3K4me3 and H3K27ac chromatin modifications.
  • Pask expression is dramatically induced as early as 3 days after injury (FIG. 1H), coincident with expansion of the satellite cell population and stimulation of My oG expression (Braun and Gautel, 2011 ).
  • HEK 293T, C2C12 myoblasts and C3H10T1/2 mesenchymal stem cells were obtained from American Type Culture Collection (ATCC).
  • Human skeletal muscle primary myoblasts Cat# SKB-F were obtained from Zenbio labs Inc. These cell lines were verified for authenticity by ATCC and Zenbio labs and all cell lines were routinely tested to be free of mycoplasma using PlasmoTest (Invivogen Inc.).
  • HEK293T, C2C12 and C3H10T1/2 cells were maintained in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin and streptomycin) (growth media, GM) at ⁇ 60% confluency.
  • FBS fetal bovine serum
  • PS penicillin and streptomycin
  • Human skeletal muscle myoblasts were maintained in proprietary media obtained from Zenbio labs. Mouse embryonic cell culture conditions and differentiation were as described previously (Shaky a et al, 2015). C3H10T1/2 differentiation was as described previously (Villanueva et al., 2011) except that BioE-1197 or DMSO was added 48 h prior to induction of the differentiation regime. C2C12 differentiation was initiated at 95% confluency by switching from GM to DMEM with 2% horse serum and 1%PS (DM) or DMEM + ⁇ Insulin + 1% PS (Insulin differentiation media). Day 0 time-point samples were collected at 95% confluency before switching to DM. During differentiation, fresh DM was applied every 24 h until the end of the experiment.
  • mSTEMCCA construct containing mouse Oct4, Sox2, Klf4 and c-Myc was obtained from R. Mostoslavsky. Lentivirus was produced by co-transfecting 293T cells with l.7 ⁇ g each of packaging plasmids (pMDLg/pRRE,pRSV-Rev) and 1.7 ⁇ g envelope plasmid (pVSVG) and 5 ⁇ g mSTEMCCA.
  • 1X105 Oct4-GFP MEFs generated from Pou5fltm2Jae/J (Jackson Lab) were plated on the feeders in 6-well plates and were infected with lentivirus for 48 hours in the presence of 4 ⁇ g/ml polybrene (Sigma). The ESC medium with or without 50 ⁇ BioE-1197 was changed every day. From day 8, GFP colonies were counted and images were captured with an Olympus 1X51 inverted microscope.
  • pCDNA3 Flag Rbbp5 (Addgene Cat# 15550)
  • pCDNA3-Flag-PTIP (Addgene Cat # 15557) were deposited by Dr. Kai Ge (Cho et al, 2007).
  • S49A and S49E mutations in pCDNA3 Flag-Wdr5 were made using sewing PCR-based mutagenesis in these vectors.
  • WT, S49A and S49E Wdr5 were subcloned into pQCXIP vector.
  • pQCXIP/GFP was described previously (Chen et al, 2014).
  • pCL-Flag- PCAF (KAT2B, Addgene Cat#8941) was deposited by Dr.
  • pCDNA-Set9 (Addgene Cat#24084) was deposited by Dr. Danny Reinberg.
  • pCDNA-Flag-Menin (Addgene Cat# 32079) was deposited by Dr. Matthew Meyerson.
  • pCL-Babe-MyoD (Addgene Cat# 20917) was deposited by Dr. Stephen Tapscott (Yang et al., 2009).
  • Rat MyoG was cloned into the pQCXIP vector after PCR from a rat cDNA library. Retroviral production for infection was as described (Kikani et al, 2012).
  • Satellite cell isolation Satellite cells were isolated from 10-12 weeks old WT and Pask-/- littermates according to published protocol (Danoviz and Yablonka-Reuveni,
  • TA muscles from hind limbs of WT or Pask-/- mice were isolated, minced in DMEM and enzymatically digested with 0.1% Pronase for lhr. After repeated tituration, the cell suspension was filtered through 40uM filter. Cells were plated on matrigel precoated plates and allowed to grow for four days. The differentiation of these satellite cells derived myoblasts was stimulated by addition of ⁇ insulin in serum free DMEM.
  • siRNA-mediated gene knockdown was performed by transfecting 50nM of pooled siRNA against control (Cat #ID-001810-10- 05), mouse Pask (L-065533-00-0005) or mouse Myodl (L-041113-00-0005) purchased from Dharmacon using Lipofectamine RNAimax (Life Technologies). Knockdown was carried out with the cells at 40% confluence in suspension. Cells were allowed to attach and grow for 48 h when they reached 95% confluence, which marked the Day 0 point. Cells were either harvested at this point or differentiation was initiated by switching from GM to DM.
  • C2C12 or human primary skeletal muscle myoblasts were seeded at 40% density in the presence of 25 ⁇ BioE-1 197 or equal volume of DMSO. Cells were allowed to grow for 48 h at which point they reached 95% confluence and differentiation was initiated by switching from GM to DM in presence of 25 ⁇ BioE-1197 or DMSO.
  • RNA was prepared from C2C12 or human myoblasts subjected to differentiation conditions as described.
  • qRT-PCR was performed from cDNA prepared from RNA using mRNA target-specific primers. Three independent experiments, done in triplicate with identical experimental parameters, were used for statistical analysis of mRNA transcript abundance. Student t-test was used for statistical significance with significance value set to P ⁇ 0.05. Sequences for qRT-PCR primers used in this study are listed in FIG. 19.
  • Protein extracts co-immunoprecipitation and western blot analysis .
  • cells were lysed in RIP A buffer, cellular debris was eliminated by centrifugation, and lysates were separated by SDS- PAGE.
  • cells were lysed in native lysis buffer described previously (Kikani et al, 2010). Immunoprecipitation was performed using the designated antibodies bound to Protein G beads (Pierce Cat# 22852). Protein complexes were washed with lysis buffer 5 times, denatured and separated by SDS-PAGE.
  • FIG. 19 provides a list of antibodies used and their resource ID.
  • Chromatin Immunoprecipitation Chromatin Immunoprecipitation (ChIP) from C2C 12 cells was performed according to (Hollenhorst et al, 2007) with the following modifications. 1X107 C2C12 cells/15cm plate were treated for differentiation according to procedures described above. At the appropriate time-point, cells were washed twice with PBS and cross-linked with 1% formaldehyde in PBS for 10 min at room
  • Example 2 Pask and Myogenin establish a positive transcriptional feedback loop that enforces myocyte differentiation.
  • satellite cells execute a transcriptional program that culminates in the formation of syncytial myocytes that constitute a healthy muscle fiber (FIG. 2A) (Bentzinger et al, 2012).
  • Quiescent Pax7+ satellite cells are induced to proliferate and initiate expression of MyoD and/or Myf5.
  • MyoD+ myoblasts MyoD drives the expression of MyoG in response to differentiation cues.
  • MyoG then executes the terminal differentiation program by down-regulating Pax7 expression (Olguin and Olwin, 2004) and activating the genes necessary for myoblast fusion and muscle function, including myosin heavy chain (My Iff) and muscle specific actin (Actal).
  • My Iff myosin heavy chain
  • Actal muscle specific actin
  • Pask+ cells were MyoG+ whereas -15% of Pask- cells were MyoG+ (FIG. 2F, quantified in 2H).
  • inhibition of Pask function with BioE-1197 eliminated this correlation, with both populations losing MyoG staining.
  • MyoG expression was present almost exclusively in those cells in the population that escaped Pask silencing (FIG. 2F, quantified in 2H).
  • Example 3 Pask collaborates with MyoD to drive MyoG expression and myogenesis.
  • C3H10T1/2 mesenchymal stem cells normally express the PPARy2 adipogenic transcription factor and efficiently differentiate into adipocytes in response to adipogenic differentiation cues (FIG. 3 A) (Zhao et al, 2013).
  • the myogenic transcriptional program is epigenetically silenced in C3H10T1/2 cells, but it can be activated in response to MyoD expression (Penn et al., 2004; Tapscott et al., 1988). MyoD stimulates MyoG expression, which then collaborates with MyoD to establish myogenic commitment and repress Ppary2 expression (FIG. 3A).
  • Fusion index was used as a measure of differentiation and was calculated as the percent of nuclei in MHC+ cells in relation with total nuclei. For quantification of microscopic images, at least 100 cells were counted from three separate experiments in a sample-blinded manner. Statistical significance was calculated using Student's t-test with P ⁇ 0.05 set as the significance level.
  • Example 4 Pask phosphorylates Wdr5.
  • Immunoprecipitates were analyzed by western blot for Pask and Wdr5, indicating an enrichment of co-immunoprecipitation at Day 1 of differentiation.
  • V5 or Flag-tagged proteins were immunoprecipitated and examined by western blot using anti- Flag or V5 antibody.
  • Wdr5 is a member of several protein complexes that catalyze histone methylation or acetylation (Migliori et al, 2012; Shilatifard, 2012; Trievel and Shilatifard, 2009). To properly place the Pask-Wdr5 interaction within these complexes and to determine if Pask associates with any intact Wdr5 -containing complexes, exclusive members of each of the major Wdr5- containing complexes were expressed.
  • Wdr5 was phosphorylated efficiently by WT Pask, but not KD Pask, in in vitro kinase reactions (FIG. 4D) and this was abrogated by the BioE-1197 Pask inhibitor (FIG. 16D). BioE-1197 also robustly blunted the in situ phosphorylation of Pask and Pask- bound Wdr5 using in-cell 32P labeling (FIG. 4E). Immunoprecipitates were analyzed by SDS-PAGE and autoradiography or western blot. As Pask association with Wdr5 was enhanced at the onset of differentiation, the next set of experiments were carried out to determine if Pask activity towards Wdr5 is stimulated at this time.
  • Pask exhibits modest activity in the absence of stimulation in C2C12 myoblasts (FIG. 4F). At 12 h post-addition of insulin containing differentiation media, however, Pask was activated as assessed by increased
  • Wdr5 was selected based on it containing a sequence that matched the Pask consensus substrate motif (Kikani et al., 2010). That site, with Serine 49 as the putative phospho-acceptor residue, is strikingly similar to that of the best-characterized substrate of yeast Pask, Ugpl (FIG. 4G), which is also robustly phosphorylated by human Pask (Kikani et al., 2010; Rutter et al, 2002). In particular, the -5His and -3Lys residues, which were shown to be the most important for determining Pask phosphorylation (Kikani et al., 2010), are present in this putative Wdr5 phosphorylation motif.
  • Pask and Wdr5 phosphorylation promote terminal differentiation Pharmacologic inhibition of Pask activity caused a loss of terminal differentiation in three differentiation paradigms: ES cells to a neuronal fate, C3H10T1/2 mesenchymal stem cells to adipocytes and C2C12 myoblasts to myotubes.
  • Wdr5 was identified and described herein as a Pask substrate that mediates these differentiation effects. Wdr5 has been previously implicated in controlling stem cell maintenance. For example, Oct4 associates with Wdr5 and recruits H3K4me3 complexes to its target promoters to induce transcriptional activation and maintenance of pluripotency (Ang et al., 2011).
  • Wdr5 was shown to interact with Pax7 to enforce myogenic specification (Kawabe et al, 2012; McKinnell et al, 2008; Rudnicki et al, 2008). These results provide a new regulatory role for Wdr5 in promoting differentiation via the expression of key target genes. More importantly, these results define a novel regulatory paradigm wherein the function of Wdr5 is modulated by Pask-dependent phosphorylation. While it is likely, it, however, remains to be established whether Wdr5 phosphorylation is a conserved and required mechanism whereby Pask promotes the differentiation of other stem and progenitor cells.
  • Example 5 Wdr5S49E restores Myog expression and myogenesis in the absence of Pask activity.
  • C2C12 myoblasts were transduced with retrovirus carrying WT, S49A or S49E mutants of Wdr5 and were selected with 3 ⁇ g/ml puromycin for 48hrs. Pask (or control) was knocked down at 70% cell density in these populations by pooled Pask siRNA. 24hrs after siRNA treatment, differentiation was initiated with 2% horse serum-containing media. Next, the ability of Wdr5WT, but not Wdr5WT or Wdr5S49A, rescued the morphological features of C2C12 differentiation, including multinucleated myofibers (FIG. 17).
  • C2C12 myoblasts were transduced with retrovirus carrying WT, S49A or S49E mutants of Wdr5 and were selected with 3 ⁇ g/ml puromycin for 48hrs.
  • Pask or control
  • Wdr5S49A and Wdr5S49E to rescue the expression of MyoG and its targets upon Pask knockdown was examined.
  • Wdr5WT and Wdr5S49A had minimal effect on Pax7, Myog, Mylpf, or Actal mRNA abundance in Pask-siRNA cells (FIG. 5A).
  • qRT-PCR analysis was performed for the indicated mRNA on day 3 of differentiation. 18S rRNA was used as normalizer.
  • the Wdr5S49E mutant either partially or completely rescued the defects in expression of each of these genes caused by Pask knockdown.
  • Wdr5S49E caused a modest increase in Pask expression.
  • Wdr5S49A or Wdr5 S49E to rescue the defect in progression from the Pax7+ to MyoG+ state in Pask-siRNA cells was examined.
  • GFP, Wdr5WT, and Wdr5S49A all had no significant effect on the number of Pax7+ cells (FIG. 5B; quantified in 5C).
  • Wdr5 S49E on the other hand, completely reversed the aberrant increase in Pax7+ cells caused by Pask knockdown. Importantly, this decrease in Pax7 positivity was present in those cells in the population that expressed Wdr5S49E (S49E+; FIG. 5B, indicated by arrows), and not in those cells in the population that were uninfected (S49E-).
  • Wdr5S49E like WT Pask, was sufficient to induce expression of MyoG even in the absence of differentiation stimuli (FIG. 5H).
  • Cells were lysed after selection and abundance of the indicated proteins was determined by Western blotting. Taken together, these data show that Wdr5 phosphorylation is a major mechanism whereby Pask promotes MyoG expression and myotube formation.
  • Example 6 Pask phosphorylation of Wdr5 promotes transcriptional derepression of the Myog promoter via H3K4mel to H3K4me3 conversion and MyoD recruitment.
  • the Myog promoter is remodeled with H3K4me3 modification resulting in its transcriptional activation (Asp et al, 2011; Cheng et al., 2014). Because transcriptional induction of Myog is dependent on Pask kinase activity or phospho-mimetic Wdr5S49E, experiments were carried out to test whether Pask might act on the Myog promoter through regulating H3K4me3 accumulation.
  • the Myog locus contains a region of H3K4me3 approximately -150bp upstream of the transcriptional start site (TSS), which overlaps with the peak of MyoD binding marked by E-Box sequences (FIG. 6A).
  • H3K4me3 abundance at two sites within this region of the promoter was strongly increased on Day 1 of differentiation, while a region more distal to the TSS showed little H3K4me3 accumulation (Myog a, FIG. 6B). Because the b amplicon showed the most significant enrichment of H3K4me3 in control samples, it was selected for future studies. This increase in H3K4me3 was markedly blunted (Myog b) or abolished (Myog c) by Pask knockdown or inhibition (FIG. 6B). In contrast, Pask knockdown had no effect on H3K4me3 abundance on the Myod promoter (FIG. 6B).
  • Wdr5 phosphorylation might stimulate H3K4me3 occupancy of the Myog promoter is through enhanced Wdr5 promoter recruitment.
  • Flag-Wdr5 ChlP-qPCR analysis was performed on these same samples to determine Flag-Wdr5 occupancy on the Myog promoter. It was found that Wdr5WT modestly occupied the Myog promoter and this was decreased for the S49A mutant and increased for the S49E mutant (FIG. 6D). Neither WT nor KD Pask occupied the Myog promoter (FIG. 6D), consistent with our observation that Pask is not a stable member of any Wdr5 -containing complexes.
  • the Myog promoter was recently shown to have high H3K4mel occupancy in non-differentiating C2C12 cells (Cheng et al, 2014). These marks are converted to H3K4me3 marks in response to differentiation cues through an unknown mechanism (Cheng et al, 2014). Pask is activated by these same differentiation cues and expression of either WT Pask or phospho-mimetic Wdr5 is sufficient to bypass the requirement of differentiation cues. It was next tested whether Pask-Wdr5 signaling regulates H3K4mel to H3K4me3 conversion on the Myog promoter in response to differentiation cues. In control cells, H3K4mel marks were progressively depleted from the Myog promoter over the differentiation time-course (FIG.
  • MyoD recruitment to the enhancer during differentiation is impaired similarly to the proximal promoter by Pask knockdown, possibly due to the interdependence of the enhancer and promoter for optimal activation (Sanyal et al, 2012).
  • the Mybph and Actal promoters, but not Myod were reported to undergo H3K4mel to
  • phosphorylation status may dictate MyoD recruitment to the Myog promoter, perhaps via H3K4mel to H3K4me3 conversion.
  • H3K4mel Role of Pask-pWdr 5 in H3K4mel to H3K4me3 conversion.
  • H3K4mel was recently demonstrated to be a transcriptionally repressive mark at the promoters of myogenic genes (Cheng et al, 2014).
  • H3K4mel is further methylated to H3K4me3 on these promoters to initiate their expression.
  • H3K4 monomethylation is catalyzed by the Wdr5 -containing M113/M114 complexes (Hu et al, 2013), while the Wdr5 -containing M111/M112 complexes catalyze H3K4 trimethylation (Shilatifard, 2012).
  • Example 7 Pask inhibition is increases the expression of Pax7 in muscle stem cells.
  • results show that PASK inhibition by BioE-1197 stimulates increase in Pax7+ cell number in C2C12 myoblasts (FIG. 20).
  • C2C12 myoblasts were treated with DMSO or BioE-1197 for 2 days.
  • Pax7 was quantified (FIG. 20, right) using immunofluroscence microscopy using anti-Pax7 antibdoy.
  • FIG. 21 shows that PASK phosphorylation of Wdr5 results in a decrease in Pax7 protein expression (Wdr5S49E lane) whereas unphosphorylable Wdr5 (Wdr5S49A) results in increased Pax7 expression, similar to PASK inhibition.
  • FIG. 22 shows that BioE-1197 pretreatment restores Pax7 expression and differentiation capacity of differentiation defective myoblasts.
  • Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network. Cell 145, 183-197.
  • H3K4me3 breadth is linked to cell identity and transcriptional consistency.
  • a bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315- 326.
  • Skeletal muscle satellite cells background and methods for isolation and analysis in a primary culture system. Methods Mol Biol 798, 21-52.
  • Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice. Nature 431, 466-471.
  • Pax7 activates myogenic genes by recruitment of a histone methyltransferase complex. Nature cell biology 10, 77-84.
  • Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development 138, 3625-3637.
  • a MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation. Genes Dev 18, 2348-2353.
  • 10T1/2 cells an in vitro model for molecular genetic analysis of mesodermal determination and differentiation.
  • PAS kinase an evolutionarily conserved PAS domain-regulated serine/threonine kinase. Proc Natl Acad Sci U S A 98, 8991-8996.
  • Pax7 is required for the specification of myogenic satellite cells. Cell 102, 777-786.
  • MyoDl a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts. Science 242, 405-411.
  • TLE3 is a dual-function transcriptional coregulator of adipogenesis. Cell Metab 13, 413-427.
  • PAS kinase drives lipogenesis through SREBP-1 maturation.
  • WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development.
  • MyoD and E-protein heterodimers switch rhabdomyosarcoma cells from an arrested myoblast phase to a differentiated state.
  • acetyltransferase and MLL/SET complexes NSL complex functions in promoting histone H3K4 di-methylation activity by MLL/SET complexes.

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Abstract

La présente invention concerne des méthodes de prévention ou d'inhibition de la différenciation de cellules (par exemple, des cellules souches et des cellules progénitrices) Les méthodes comprennent la mise en contact de cellules (par exemple, des cellules souches et des cellules progénitrices) avec un inhibiteur de protéine kinase contenant un domaine PAS (PASK). Les cellules produites fournissent une méthode utile de traitement ou de prévention de maladies ou de troubles tels que la sarcopénie associée à une maladie ou des troubles de la perte de cellules souches.
PCT/US2017/052781 2016-09-23 2017-09-21 Méthodes de prévention de la différenciation de cellules souches à l'aide d'un inhibiteur de pask Ceased WO2018057782A1 (fr)

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WO2025006571A1 (fr) * 2023-06-26 2025-01-02 University Of Kentucky Research Foundation Destruction ciblée de pask par dégron à base de peptide

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