[go: up one dir, main page]

WO2021119030A1 - Régénération améliorée de cardiomyocytes - Google Patents

Régénération améliorée de cardiomyocytes Download PDF

Info

Publication number
WO2021119030A1
WO2021119030A1 PCT/US2020/063838 US2020063838W WO2021119030A1 WO 2021119030 A1 WO2021119030 A1 WO 2021119030A1 US 2020063838 W US2020063838 W US 2020063838W WO 2021119030 A1 WO2021119030 A1 WO 2021119030A1
Authority
WO
WIPO (PCT)
Prior art keywords
srf
mutated
composition
heart
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2020/063838
Other languages
English (en)
Inventor
Robert Schwartz
Dinakar IYER
Siyu XIAO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Animatus Biosciences LLC
Original Assignee
Animatus Biosciences LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Animatus Biosciences LLC filed Critical Animatus Biosciences LLC
Publication of WO2021119030A1 publication Critical patent/WO2021119030A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates generally to compositions and methods for promoting adult mammalian cardiomyocytes processes and systems for enhancing cardiomyocyte regeneration. More specifically, the invention relates to locally administering a therapeutic agent containing a modified messenger RNA for expressing a mutated serum response factor polypeptide and a modified messenger RNA for expressing a mutated YAP polypeptide into diseased heart muscle to promote its regeneration.
  • CMs can be derived include embryonic stem cells (ESC), Induced Pluripotent Stem Cells (iPSCs), bone marrow derived mesenchymal stem cells and Cardiac Progenitor Cells (CPCs) from adult cardiac tissue.
  • ESC embryonic stem cells
  • iPSCs Induced Pluripotent Stem Cells
  • CPCs Cardiac Progenitor Cells
  • the present invention relates generally to compositions and methods for promoting adult mammalian cardiomyocytes processes and systems for enhancing cardiomyocyte regeneration. More specifically, the invention relates to locally administering a therapeutic agent containing a modified messenger RNA for expressing a mutated serum response factor polypeptide and a modified messenger RNA for expressing a mutated YAP polypeptide into diseased heart muscle to promote its regeneration.
  • a therapeutic agent containing a modified messenger RNA for expressing a mutated serum response factor polypeptide and a modified messenger RNA for expressing a mutated YAP polypeptide into diseased heart muscle to promote its regeneration.
  • One embodiment of the invention is a composition used to enhance cardiomyocyte regeneration.
  • the composition used in the treatment of heart disease, includes: a) a first modified messenger RNA for expressing a mutated serum response factor polypeptide; b) a second modified messenger RNA for expressing a mutated YAP polypeptide; and c) a delivery agent for delivering the first and second modified messenger RNAs into a cell.
  • the mutated serum response factor polypeptide in the composition is generally selected from SRF-141(A3), SRF-144(A3), SRF-147(A3), SRF-150(A3), SRF- 153(A3), or combinations thereof.
  • the mutated YAP polypeptide in the composition is preferably YAP1(5SA).
  • the delivery agent used for the composition is either a viral transduction vector or a liposomal transfection agent.
  • Another embodiment of the invention is a method of inducing cardiomyocyte regeneration comprising: a) preparing a first modified messenger RNA for expressing a mutated serum response factor polypeptide; b) preparing a second modified messenger RNA for expressing a mutated YAP polypeptide; c) delivering the first and second modified messenger RNAs using a delivery agent into a cardiomyocyte.
  • Yet another embodiment of the invention is a method for promoting cardiac repair and regeneration in a subject, the method comprising injecting an effective amount of a therapeutic composition into a site of a heart of the subject, wherein the composition comprises a first modified messenger RNA for expressing a mutated serum response factor polypeptide, a second modified messenger RNA for expressing a mutated YAP polypeptide, and a delivery agent for delivering the first and second modified messenger RNAs into a cardiomyocyte.
  • Another embodiment of the invention is a method for promoting cardiac repair and regeneration in a subject, the method comprising identifying a site of cardiac muscle damage in a heart of the subject and injecting a therapeutic composition into the heart adjacent the site of cardiac muscle damage.
  • a therapeutic composition into the heart adjacent the site of cardiac muscle damage.
  • multiple injections of the therapeutic composition are made at different locations in the heart wherein each injection location is adjacent to an identified site of cardiac damage.
  • FIGURES 1A and IB schematically illustrate the role of the CArG box recruitment of serum response factor and its interaction with various co-factors in cardiomyocyte differentiation and proliferation.
  • FIGURE 2 A illustrates several triplet alanine scanning mutations across the MADS box of SRF that were made and tested.
  • FIGURE 2B illustrates the stimulation of the stem cell factors Nanog and
  • FIGURE 3 shows that SRF 153 mutant (Stemin) works with YAP1(5SA) mutant to activate Nanog, CMyc, Oct4 and Sox 2.
  • FIGURE 4 shows that SRF 153 mutant (Stemin) induced stem cell factors, blocked contractile proteins and assembly factors and activated cyclins.
  • FIGURE 5 illustrates DNA replication in rat cardiomyocytes after transfection with Stemin mmRNA or YAP -mutant mmRNA alone or both together.
  • FIGURE 6 shows the experimental timeline for the in vivo treatment of infarcted adult mouse hearts with Stemin mmRNA and YAP-mutant mmRNA.
  • FIGURE 7 shows the results of DAPI staining and EdU incorporation into adult mouse hearts injected with Stemin mmRNA and YAP-mutant mmRNA.
  • FIGURE 8 A - 8L illustrate the cardiac function in infarcted adult mouse hearts injected with Stemin mmRNA and YAP-mutant mmRNA and in control infarcted adult mouse hearts that were not injected with Stemin mmRNA and YAP-mutant mmRNA.
  • FIGURE 9 illustrates the infarct zone in infarcted adult mouse hearts injected with Stemin mmRNA and YAP-mutant mmRNA and in control infarcted adult mouse hearts that were not injected with Stemin mmRNA and YAP-mutant mmRNA.
  • the term “effective amount”, as used herein, refers to an amount of a composition or compound that is capable of producing a medically desirable result in a treated subject.
  • the medically desirable result may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
  • subject refers to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae.
  • the subject may include humans, dogs, cats, pigs, cows, sheep, horses, goats, rats, and mice, hamsters, and guinea pigs.
  • the term “subject” does not exclude an individual that is normal in all measurements made.
  • treatment may refer to the administration of a substance or composition to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, suppress, inhibit, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to the disorder, or a predisposition toward the disorder.
  • Treating may refer to a therapeutic treatment or prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove, or “treating” may refer only to therapeutic treatment.
  • treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Additionally, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of, or decreasing resistance to alternative therapeutics, or a combination thereof.
  • the present invention relates to compositions and methods for promoting adult mammalian cardiomyocyte processes and systems for enhancing cardiomyocyte regeneration are described.
  • the invention relates to locally administering a therapeutic agent containing a modified messenger RNA for expressing a mutated serum response factor polypeptide and a modified messenger RNA for expressing a mutated YAP polypeptide into diseased heart muscle to promote cardiomyocyte proliferation and cardiac regeneration.
  • Heart development also known as cardiogenesis refers to the prenatal development of the heart.
  • the heart is the first functional organ in vertebrate embryos and beats spontaneously by week 4 of development.
  • Although there is some evidence for a very low level of postnatal cardiomyocyte proliferation which can be increased after injury in mice, it is insufficient to replenish lost cardiomyocytes that are damaged or to re-establish proper heart function.
  • Even that level of postnatal cardiomyocyte proliferation in mice is lost seven days after birth. Not only does cardiomyocyte cell division slow down after birth, but it responds to physiological or pathological challenges thereafter through cardiac hypertrophy (Fan etal ., 2015).
  • This loss of proliferative response corresponds to the exit of cardiomyocytes from the cell cycle and demonstrates that adult mammalian cardiomyocytes are refractory to mitotic activity, unlike those found in either early postnatal mice or zebrafish.
  • sarcomere structure which is the basic contractile unit of muscle fiber.
  • the sarcomeres must be disassembled for cytokinesis to occur (Sanger, et a/., 2010) Such disassembly may require signals for cardiomyocyte de- differentiation, which is accompanied by multiple cellular changes including reactivation of gene expression programs restricted to the embryonic state.
  • sarcomeres proliferate in two consecutive steps. First, sarcomeres must be disassembled to enable chromosome segregation to complete the cell division cycle because the sarcomere occupies a large volume of the mature cardiomyocyte that physically impedes mitosis and cytokinesis. Thus, sarcomere disassembly is a prerequisite task for cardiomyocyte proliferation. Once disassembly is achieved myocyte replication can proceed. Next, the sarcomeres must reassemble after cell division to form its contractile apparatus so that the cells regain the function to contract. Thus, regulatory factors involved in sarcomeres assembly and disassembly are critical for cardiomyocyte proliferation.
  • SRF Serum Response Factor
  • Human serum response factor also known as SRF
  • SRF is a well known protein whose sequence is given as SEQ ID NO: 1.
  • SRF is a member of an ancient DNA binding protein family that shares a highly conserved DNA-binding/dimerization domain of 90 amino acids termed the MADS (MCM1, Agamous, Deficiens, and SRF) box superfamily of transcription factors (Treisman, 1994; Reecy et al ., 1999; Miano, 2003).
  • SRF binds to the serum response element (SRE) in the promoter region of target genes.
  • SRF is known to regulate the activity of many early genes, such as c-fos, whereby it participates in cell cycle regulation, apoptosis, cell growth, and cell differentiation.
  • MADS boxes have similar DNA binding specificities and dimerize to symmetrically contact the serum response element (SRE) with a consensus sequence CC(A/T) 6 GG, also known as the SRE and or the CArG box. As illustrated in Figures 1 A and IB, SRF interaction with cofactors has a regulatory role in both the differentiation and proliferation of cardiomyocytes.
  • CArG boxes recruit the cysteine-rich protein 2 LIM, which bridges the SRF and GATA factors through interaction with the MADS N-terminal extension (Sepulveda et al ., 2002) and myocardin, which competes with Erythroblast Transformation Specific (ETS) factors, the ERS family of transcription factors, that interact with the loop region of the MADS box (Wang et al, 2002; Wang et al., 2004).
  • ETS Erythroblast Transformation Specific
  • MRTFs myocardin related transcription factors
  • RhoA The effect of RhoA on SRF dependent genes is mediated through a TCF independent mechanism (Hill and Treisman, 1995).
  • the myocardin family proteins MRTF-A/B provide the link between RhoA-dependent cytoskeletal regulation and SRF-dependent gene expression (Lockman et al., 2004; Miralles et al., 2003; Wang et al, 2002).
  • MRTF-A associates with G-actin and is thus sequestrated in the cytoplasm under resting conditions.
  • TEAD associates with MRTF-A overlapping the myocardin binding site on the SRF’s MADS box, thus it facilitates signaling through actin tread milling leading to Rho kinase activation and cell replication (Zaromytidou et al, 2006).
  • the recruitment of YAP, TEAD, and other co-factors such as NCOA3/SRC3 and ESRRB may propel the cells further towards cell survival and activate stem factor activity.
  • SRC3 is an essential coactivator required to mediate ESRRB function in embryonic stem cells. SRC3 interacts with ESRRB via its ligand-binding domain and bridges ESRRB to RNA polymerase II complexes (Percharde et al ., 2012).
  • SRC3 is critical for both the induction and maintenance of pluripotency and cell replication.
  • association between SRC3, ESRRB, TEAD, YAP, and SRF may be important in promoting cardiomyocyte proliferation as shown in Figure IB.
  • Figures 1A and IB illustrate the potential competition between transcription factors that facilitate proliferation versus cardiomyocyte differentiation through interactomes with the SRF MADS box.
  • the MADS box domain has been proposed to be an important regulatory nexus for the convergence of crucial cellular signals enabling SRF to recruit specific cofactors to their respective DNA binding sites and enhance transactivation of target genes.
  • SRF has a core domain of 90 amino acids required for dimerization and sequence specific DNA binding (Sharrocks, 1995; Treisman, 1995).
  • the highly conserved MADS box within this core domain is necessary for critical interactions with co-accessory factors (Pellegrini, 1995).
  • all DNA contacts occur within the N-terminal portion of the MADS box, and SRF-SRF dimerization is mediated by elements within the MADS box together with additional residues from the immediately adjacent C terminal region (Pellegrini, 1995).
  • SRF activity controls sarcomerogenesis in higher vertebrate and supports the concept that SRF resides at a high point in the regulator hierarchy governing sarcomerogenesis (Niu, et al ., 2005; Niu, et al ., 2008).
  • SRF mutant named Stemin (SEQ ID NO: 2) has been shown to block the normal SRF- mediated cardiac muscle differentiation program resulting in the production of undifferentiated cardiomyocytes.
  • the MADS box domain is likely to be an important regulatory nexus for the convergence of crucial cellular signals enabling SRF to recruit specific cofactors to their respective DNA binding sites and enhance transactivation of target genes.
  • SRF sequence SEQ ID NO: 4
  • SEQ ID NO: 4 An important SRF sequence (SEQ ID NO: 4), sometimes referred to as the
  • the MADS box is a region for interactions with critical transcription regulating co-accessory factors. There are likely to be distinct sites for interactions with cofactors that regulate the transcription of proliferation genes and others for interactions with cofactors that regulate the transcription of differentiation genes. For example, one key site that modulates SRF’s transcriptional activity towards proliferation genes is well known. That site includes a region in the bII coil of the MADS box (aa 194-198) that binds to the Ets protein Elkl to form a ternary complex with SRF and the c-fos promoter, leading to increased transcription.
  • YAP and the HIPPO Pathway [0045]
  • the Hippo pathway is a highly conserved signaling pathway in mammals that is thought to control organ size during the developmental processes by regulating cell proliferation and cell apoptosis (Zhao et al ., 2010). It has also been suggested that the Hippo pathway may regulate cell self-renewal and expansion.
  • Yes-associated protein a transcription co- activator and a potent growth promoter
  • Hippo pathway kinases Latsl and Lats2 are directly phosphorylated by the Hippo pathway kinases Latsl and Lats2, and then inhibited through cytoplasmic retention and degradation.
  • Chemical and physical signals such as GPCR ligands and matrix stiffness dampen the Hippo pathway, thus leading to YAP activation, which facilitates cell proliferation, evasion from apoptosis, and stem cell self-renewal.
  • Additional studies have indicated that the Hippo pathway is upregulated in heart failure and that a Hippo pathway deficiency reversed systolic heart failure (Leach et al ., 2017). Such results have increased interest in the manipulation of the Hippo pathway as an approach to the treatment of heart failure.
  • Human YAPl (NM_0130145.3. trans 1 ;403- 1917) is important is a central player in a cascade of phosphorylation events in the Hippo pathway.
  • the phosphorylation of YAPl prevents shuttling of YAPl into the nucleus, promotes 14-3-3 binding, and protein degradation.
  • unphosphorylated YAPl enters the nucleus and binds to multiple transcription factors (e.g. TEAD/TEF and MRTFa).
  • YAPl binding to its partners in the nucleus typically promotes gene expression programs that favor survival, proliferation, and tissue growth (Xiao, et al., 2016; Ikeda & Sadoshina, 2016).
  • MST1 Mommalian STE20- like protein kinase 1
  • MST2 MST2
  • SAV1 SAV1
  • LATS1 Large tumor suppressor homologue 1
  • LATS2 LATS2
  • LATS1 and LATS2 then interact with MOB1A (MOB domain kinase activator 1A) and MOB IB (MOB domain kinase activator IB), which further phosphorylate the downstream effectors including YAP (Yes-associated protein) and TAZ (Transcriptional co-activator with PDZ-binding motif).
  • MOB1A MOB domain kinase activator 1A
  • MOB IB MOB domain kinase activator IB
  • Transcription co-activators YAP and TAZ are the key regulators in the Hippo pathway regulated by cell proliferation and cell adhesion through phosphorylation. Activation of the Hippo signaling pathway inhibits YAP and TAZ transcriptional activity by promoting their degradation through phosphorylation, therefore preventing their translocation into the nucleus. When the Hippo signaling pathway is inactivated, TEADs (TEA domain transcription factor family members) serve as the main binding platforms of YAP and TAZ to bind to nuclear targets.
  • Hippo signaling pathway may be a potential strategy for promoting cardiac cell proliferation and cardiac regeneration.
  • YAP and TAZ have been reported to control TBX5-dependent transcription, thus involved in cardiac and limb development.
  • Mice with Savl conditional knockout in the heart showed expanded ventricular myocardial layers without expansion in cardiomyocyte size.
  • YAP5SA an active form of YAP, termed YAP5SA, by mutating all the LATS1/2 phosphorylation sites from serine to alanine (Zhao et al., 2007).
  • the phosphorylation sites mutation of YAP prevents 14-3-3 binding, thus preventing YAP protein degradation.
  • YAP5SA enters nucleus and binds with TEAD to regulate nuclear targets.
  • Recently, YAP5SA has been proven to partially reprogram the highly differentiated adult mouse cardiomyocytes to a more primitive proliferative state (Monroe etal., 2019).
  • Mutated polypeptides including all or at least 22 residues of the sequence within the residues 132-179 (SEQ ID NO: 5) in the MADS box of the SRF core domain sequence (SEQ ID NO: 4).
  • SRF mutants include mutations that insert, remove or change one or more residues within this same region that stimulate a stem cell factor, such as Nanog and Oct4.
  • the mutations may be substitutions of residues 147 and/or 153, deletions of residues 147 and/or 153, and one to three residue insertions adjacent to or within 2-3 residues of residues 147 and/or 153 incorporating triplet alanine scanning mutations across the MADS box of SRF (see Figure 2) were made and tested to determine if they interfered with the association of these co-accessory factors.
  • mutants are referred to herein according to where the alanine triplet was placed within residues 132-179 (SEQ ID NO: 5) such as SRF-141(A3) (SEQ ID NO: 8), SRF-144(A3) (SEQ ID NO: 9), SRF- 147(A3) (SEQ ID NO: 7), SRF-150(A3) (SEQ ID NO: 10), or SRF-153(A3) (SEQ ID NO: 5) such as SRF-141(A3) (SEQ ID NO: 8), SRF-144(A3) (SEQ ID NO: 9), SRF- 147(A3) (SEQ ID NO: 7), SRF-150(A3) (SEQ ID NO: 10), or SRF-153(A3) (SEQ ID NO: 5) such as SRF-141(A3) (SEQ ID NO: 8), SRF-144(A3) (SEQ ID NO: 9), SRF- 147(A3) (SEQ ID NO: 7), SRF-150(A3) (SEQ ID NO: 10
  • Stemin inhibited the induction of sarcomere assembly factors involved in cardiomyocyte differentiation thereby blocking the normal SRF-mediated cardiac muscle differentiation program resulting in the production of undifferentiated, proliferative cells (see Figure 4).
  • Stemin, or SRF-153(A3) showed a powerful activation of at least 15 stem cell marker genes, such as Rexl, Nanog, Oct4, Sox2, Esgl, SFmbt2, Rhox6 and proliferin, but not C-Myc and Klf4 in comparison to SRF null embryonic stem cells (ES cells).
  • Stemin elicited an imperfect or partial pluripotency program.
  • Stemin also inhibited the induction of many cardiac myocyte specified genes such as sarcomeric actins, heavy and light chain myosins, troponins, channels and structural genes.
  • sarcomeric assembly factors such as Actinin2, Nebulin, Titin, Myomesin, Obscurin Filamin, Smydl and SNF1-K2 were blocked from appearing in comparison to wild type ES cells that formed cardiac myocytes following hanging drop formation.
  • evidence for Stemin fostering cell replication was shown by the up regulation of cyclins A2, Bl, El and Dl. The applicants’ observation that a single transcription factor, Stemin, albeit mutated SRF, induced the expression of stem cell factors was unexpected and unprecedented.
  • MAL or MKL myocardin related transcription factors MADS boxes
  • MAL or MKL myocardin related transcription factors MADS boxes
  • RhoA RhoA-dependent cytoskeletal regulation and SRF-dependent gene expression
  • MRTF-A associates with G-actin and is thus sequestrated in the cytoplasm under resting conditions.
  • SRC3 is an essential coactivator, required to mediate Esrrb function in ES cells. SRC3 interacts with Esrrb via its ligand-binding domain and bridges Esrrb to RNA polymerase II homing complexes (Percharde, et al ., 2012). Functionally, SRC3 is critical for both the induction and maintenance of pluripotency and cell replication; thus, their association with SRF, TEAD and YAP may be important in the generation of cardiomyocyte regeneration.
  • YAP/TAZ as transcription co-activators, do not directly bind to DNA.
  • transcriptional enhanced associate domain (TEAD) transcription factors bind to the TEAD binding domain of YAP/TAZ and serve as the main binding platform of YAP/TAZ.
  • TEAD transcriptional enhanced associate domain
  • YAPl and TEADl TEADl (TEA domain family member 1), an SRF cofactor that binds directly to SRF’s MADS box, is required to stimulate cardiomyocyte proliferation.
  • MRTF-A another SRF cofactor that binds to the WW domain of YAP/TAZ, potentiates TEAD-YAP transcriptional activity.
  • YAP-TEAD and SRF-MRTF pathways show mutual dependence.
  • YAP5SA interacts with SRF MADS box through TEADs and MRTF-A, inhibiting interactions between SRF and cardiac specific cofactors to inhibit SRF dependent cardiomyocyte differentiation, to synergistically promote cardiomyocyte proliferation.
  • MADS box (SEQ ID 3), has been shown to inhibit the induction of sarcomere assembly factors involved in cardiomyocyte differentiation. If Stemin could also enhance its binding to cofactors TEAD (TEA domain family member 1) and MRTF-A (Myocardin related transcription factor A), it might promote the interaction between SRF and YAP to manipulate hippo signaling pathways and promote cardiomyocyte proliferation.
  • TEAD TEA domain family member 1
  • MRTF-A Myocardin related transcription factor A
  • mmRNA modified messenger RNA
  • mRNA does not require to be transferred into the nucleus to cause the expression of a target protein. Since the mRNA delivery method will not be affected by the state of the nuclear membrane, transfection efficiency in both dividing and non-dividing cells is guaranteed. Moreover, mRNA-based gene delivery is able to deliver gene combinations with different ratios specifically tailored to patients with a different course of disease.
  • Unmodified mRNAs are recognizable by the innate immune system of the cells via toll-like receptors, thus promoting the degradation of the unmodified mRNA.
  • Modifying mRNA’s secondary structure by substituting uridine with pseudouridine and replacing cytosine with 5-methyl-cytidine can produce mRNAs that lead to less recognition by nucleases and toll-like receptors.
  • Modified mRNAs were synthesized that would provide for the intracellular expression of mutated polypeptide sequences of the SRF core domain or the residues 132-179 of the SRF core domain. Likewise mmRNA that would provide for the intracellular expression of YAP1(5SA) mutants were synthesized as described by Zhao, et al. in 2007).
  • mmRNA The utilization of mmRNA for therapeutic applications requires a nontrivial intracellular delivery of the mmRNA.
  • the challenge of efficient intracellular delivery has promoted the development of various delivery systems.
  • Lenti-viral vectors have demonstrated inefficient nucleic acid delivery, as well as immunogenicity and safety concerns.
  • lipid particles such as liposomes or lipid nanoparticles provide an efficient, non-immunogenic, and safe delivery alternative.
  • Lipofectamine MaxTM was shown to have a transfection efficiency approaching about 45% of mmRNA transfection in rat myocytes.
  • RNA for SRF SEQ ID NO: 11
  • YAPl SEQ ID NO: 13
  • the mRNA for SRF was modified to produce the mRNA for Stemin (SEQ ID NO: 12).
  • Synthetic mmRNAs consist of an anti-reverse cap analog (ARC A), 5’ and
  • UTRs untranslated sequences
  • polyA tail 3’ untranslated sequences
  • Antireverse cap analog (ARCA) are modified guanosine nucleotides that are incorporated into the 5’ end of the transcript and ensure that synthesis proceeds in only one direction.
  • Capping mRNAs with ARCA simulates the natural capping process and improves transcript stability and enhances translation.
  • mmRNAs are synthesized using modified nucleotides (typically, 5-methycytidine-5’ -triphosphate and pseudouridine-5 ’ ⁇ triphosphate) (Kariko, et al ., 2008; Kariko, et al ., 2005).
  • mmRNAs are typically a fusion between the gene of interest’s coding DNA and the 5’ and 3’UTRS from a different gene such as beta-globin; the 5’ and 3’UTR sequences that are typically selected are chosen because their sequences are known to increase transcript stability when they are used in place of the native UTRs of otherwise unstable transcripts.
  • Stemin and YAP1(5SA) mRNA were synthesized. To provide these mRNAs additional selectivity, the substitution of 5’UTR and 3’UTR of the cardiac alpha actin gene onto the Stemin and
  • YAP1(5SA) mmRNA to enhance the selective translatability and stability in cardiac myocytes.
  • Myocytes from controls versus synthetic Stemin and YAP1(5SA) induced myocytes were FACS isolated following washing and refeeding of the cells to remove synthetic RNA in the switched off myocytes.
  • Stemin in synergy with YAP1(5SA) induces many stem cell marker genes, such as, Nanog, Oct4, Sox2 and C-Myc as shown in Figure 3; inhibits cardiac myocyte specified genes such as sarcomeric contractile proteins; and represses sarcomeric assembly factors, such as Actinin2, Nebulin, Titin, Myomesin, Obscurin Filamin, Smydl and SNF1-K2.
  • stem cell marker genes such as, Nanog, Oct4, Sox2 and C-Myc as shown in Figure 3
  • cardiac myocyte specified genes such as sarcomeric contractile proteins
  • sarcomeric assembly factors such as Actinin2, Nebulin, Titin, Myomesin, Obscurin Filamin, Smydl and SNF1-K2.
  • sarcomeric assembly factors such as Actinin2, Nebulin, Titin, Myomesin, Obscurin Filamin, Smydl and SNF1-K2.
  • RNA induction will require simple replacement of culture media. Replicated myocytes are allowed to re-differentiate over time in myocyte differentiation media that does not contain fetal bovine serum (FBS) to allow investigators to determine if myocytes revert to their myocyte identity or differentiated state after synthetic RNA induction.
  • FBS fetal bovine serum
  • Myocytes were assessed for expression of Troponin T (TnnT) and aEdu staining by immunofluorescence assays. Nuclei were stained with DAPI. Quantification of immunofluorescence microscopic images was done with imageJTM software. An equal number of myocytes were plated in triplicate for each transfection condition. At the end of day three after a 6 hour pulse of aEdu, the cells were fixed and the fluorescently labeled cells counted. The number of troponinT marked myocytes stained with anti TnnT that were also labeled for DNA synthesis with a-Edu (RED) and coincided with nuclear Dapi stain were quantitated (see Figure 5).
  • SRF153(A3)(Stemin) with constitutively active mutant YAP1(5SA), TEAD, SRC3 into murine SRF null ES cells that had been reprogrammed to cardiomyocytes.
  • Figure 3 shows the induction of Nanog, Oct4, CMyc and Sox2 by SRF153(A3) and YAP1(5SA) mutants. These factors were detected on protein blots with specific antibodies.
  • the co- infection of YAP 1(5 S A) and SRF153(A3) in SRF null ES cells showed a synergistic co induction of Nanog, Oct4, C-Myc and Sox2.
  • Bioinformatic analysis revealed the upregulation of multiple cell cycle gene clusters with the co-expression of SRF mutants and YAP5SA; while gene clusters associated with cardiomyocyte differentiation, sarcomeric assembly and cardiac muscle contraction were profoundly down regulated.
  • ATAC-seq of the cells under SRF mutant and YAP5SA treatment was performed to create a bioinformatics landscape of interactomes of SRF mutants.
  • ATAC seq Assay for Transposase- Accessible Chromatin using sequencing) is a technique used in molecular biology to assess genome-wide chromatin accessibility, i.e. which genes are being actively transcribed.
  • Rat neonatal cardiomyocytes were harvested 32 hours, 40 hours, and 48 hours after treatment with SRF mutant mRNA, YAP5SA mRNA, and a combination of SRF mutant and YAP5SA mRNA delivered by Lipofectamine MessengerMAXTM (ThermoFisher).
  • RNA was extracted and underwent quality control assessment using the RNA tape on Tapestation 4200TM (Agilent) and were quantified with Qubit FluorometerTM (ThermoFisher). KallistoTM was used for pseudo alignment and transcript quantification.
  • RNA-seq data showed that several spindle assembly factors such as Bubl (mitotic checkpoint serine/threonine-protein kinase BEIB1) were upregulated after the synthetic RNA induction, suggesting the neonatal cardiomyocytes were pushed into cell division by the synthetic mRNA induction.
  • Bubl mitotic checkpoint serine/threonine-protein kinase BEIB1
  • PLK1 Poly-like kinase 1
  • ANLN (Anillin) was also highly upregulated in the combination treatment group.
  • the presence of ANLN is required in multiple stages of cytokinesis, and ANLN acts as a key mediator of cytokinesis.
  • Upregulation of crucial cell cycle genes such as Plkl and ANLN suggested that SRF mutants and YAP5SA fostered cell replication by promoting several steps of the cell-division cycle of the cardiomyocyte.
  • CDC20 Cell Division Cycle 20
  • MPF maturation-promoting factor
  • Cardiac specific factors were identified to be down regulated after mRNA treatment. SRF mutants alone could decrease the expression level of specific cardiac specific genes, but the groups with the SRF mutant and YAP5SA combination treatment at all three time points presented lower expression of the cardiac differentiation factors than both single mRNA treatment groups, which may result from the synergistic effect of SRF mutants and YAP5SA.
  • Genes involved in cardiac muscle cell differentiation were down regulated in the combination treatment group.
  • ACTN2 Actinin Alpha 2 encodes a muscle- specific alpha actinin isoform that is expressed in cardiac muscles and skeletal muscles.
  • MYH6 Myosin Heavy Chain 6 is one of the two alpha heavy chain subunits of cardiac myosin.
  • TNNT2 Cardiac muscle Troponin T
  • Troponin 13 Troponin 13
  • Troponin 13 Troponin 13
  • Some embodiment of the invention will use mutant SRF or a mutated SRF polypeptide in conjuction with a mutant YAP1 or a mutated YAP1 polypeptide to treat heart disease.
  • preferred embodiments of the invention described herein combine the use of mmRNAs designed to produce intracellular YAPl and SRF mutants in the treatment of heart disease.
  • Therapeutic agents or compositions envisioned herein include a modified messenger RNA for expressing Stemin or a modified SRF polypeptide, as well as a modified messenger RNA for expressing a modified YAP polypeptide, and a liposomal delivery agent.
  • MI Myocardial infarction
  • LAD left anterior descending
  • Mice were put under 1.5% isoflurane inhalation anesthesia and body temperature kept at 36 °C while the surgeries were conducted.
  • a 3% isoflurane was initially used to anesthetize the animal for intubation.
  • a 20G intravenous catheter connected to a ventilator was inserted into the mouse trachea through the oral cavity, conducting artificial ventilation at 120 strokes/min, 20 mL/kg/stroke using room air.
  • the mouse heart was exposed through the thoracic cavity opened through the left fourth intercostal space.
  • An 8-0 polypropylene ligature was used to tie the LAD by fine, smooth tipped forceps. Alteration of heart color was observed after the ligation of the LAD.
  • a 6-0 polypropylene ligature was used to suture the animal thoracic cavity by layered stitches. The lungs were inflated to displace air. Animals were removed from artificial ventilation and remained in a supervised setting until fully conscious. Animals in both experimental groups and control groups were housed in separate warm cages until recovery.
  • Myocardial Echocardiography Myocardial echocardiographs (echos) were conducted under anesthesia with 1% isoflurane. Each mouse was placed on a warm pad to keep the body temperature around 36 °C. Warmed echo gel was placed on the chest of the mouse and the heart was imaged with a linear transducer. The heart rate was controlled at a similar level within each strain of measurement. LV ejection fraction (EF), LV fractional shortening (FS), stroke volume, LV septal thicknesses, heart rate, left ventricular posterior wall, and left ventricle internal dimension were measured.
  • EF LV ejection fraction
  • FS LV fractional shortening
  • stroke volume stroke volume
  • LV septal thicknesses heart rate
  • Myocardial echocardiography was measured on day (-2) (i.e., -48 hours; baseline), first week (+1) (i.e., +7 days; midpoint), second week (+2) (i.e., +14 days; midpoint), third week (+3) (i.e., +21 days; midpoint), and fourth week (+4) (i.e., +28 days; final) time-point intervals relative to the injection of the mRNA as shown in Figure 6
  • EdU 10 pg/g of mouse body weight
  • EdU 10 pg/g of mouse body weight
  • the intact heart was fixed in 4% paraformaldehyde, stored in 70% ethanol and then embedment into paraffin for histological assessment.
  • the heart was cross-sectioned and EdU was detected by Click-iT EdU Cell Proliferation KitTM (ThermoFisher). Slides were visualized by a LeicaTM SP8 confocal microscope.
  • DAPI and EdU Quantification were detected by a LeicaTM SP8 confocal microscope. Confocal microscopy pictures were analyzed by Image JTM to get the quantification analysis on DAPI staining and EdU incorporation. Pictures were divided into 90 equal divisions and the integrated density was measured for each division. The same threshold was used for all the divisions in different treatment groups.
  • Stemin and YAP5SA mRNA injection could increase cell number and promote EdU incorporation in the left ventricle of adult mouse heart in vivo.
  • a long term experiment was performed to detect if the injection of Stemin and YAP5SA mRNA would improve the mouse heart function after myocardial infarction and repair of the damaged heart.
  • Echocardiography was done two days before surgery to create a base line of the heart function in both experimental and control groups.
  • Myocardial infarction was induced by LAD ligation before mRNA injection through an open-chest surgery on the starting day of the experiment ( Figure 6).
  • LV ejection fraction (EF), LV fractional shortening (FS), stroke volume, LV septal thicknesses, heart rate, left ventricular posterior wall, and left ventricle internal dimension were measured every seven days after injection. The mice were sacrificed on the 28th day post injection to harvest the heart for imaging.
  • Ejection fraction and fractional shortening dropped in both control and Stemin and YAP5SA mRNA injection groups due to the myocardial infarction induction in the first week ( Figure 8 A and 8B).
  • the ejection fraction and fractional shortening at week 2 were increased more than 60% and 40% respectively in the combination mRNA injection group, and were kept at a higher level compared to the control, which indicated a significantly improved heart function promoted by Stemin and YAP5SA mRNA injection.
  • Stroke volume and cardiac output in the combination treatment group was higher than control group from the first week post-surgery, and an increase can be observed in the second week post-surgery in both groups ( Figure 8C and 8D).
  • Cardiac output was calculated by stroke volume times heart rate.
  • LV interventricular septal thicknesses (IVS) of the combination mRNA treatment group was narrower than the control group at the first week post-surgery, but an increase in IVS at both systole and diastole was observed at the second week after injection in the combination group, while the IVS of control group dropped to a relatively low level ( Figure 8E and 8F).
  • the heart rate of both groups was within an acceptable range, and the LV mass correction in the combination injection group was close to the control group, which indicated that there was no abnormal LV mass change (Figure 8G and 8H).
  • LVPW left ventricular posterior wall
  • mice hearts were harvested at the end of the four weeks of the long term experiment. Tissues were sectioned and stained with hematoxylin and eosin to observe morphological change in the heart after surgery and injection. The infarct zone can be seen and marked in the control group heart, while the infarct zone in the Stemin and YAP5SA was undetectable in all the sections of the combination treatment group ( Figure 9). This indicated that the injection of Stemin and YAP5SA after mouse myocardial infarction induction could repair the infarcted adult mouse heart in vivo. [00115] Thus, injections of Stemin/YAPl mmRNA repaired mouse infarcted hearts by increasing myocyte replication and reducing wall thinning. As the myocytes gained maturity after treatment, the heart gained in its pumping function.
  • Embodiments of the composition of the therapeutic agent to be used in treating heart disease include a modified messenger RNA for expressing a SRF mutated polypeptide, a modified messenger RNA for expressing a modified YAP polypeptide, and a liposomal delivery agent.
  • Preferred embodiments of a mmRNA designed to produce an intracellular mutant of the amino acid residues 147-153 of the core domain of SRF include the mutants 141A 3 (SQ ID 5), 144A 3 (SQ ID 6), 147A 3 (SQ ID 4), 150A 3 (SQ ID 7), and 153A 3 (SQ ID 3).
  • a preferred embodiment of the mmRNA for expressing a modified YAP polypeptide is the YAP5SA mRNA reported by Zhao, et al. in 2007.
  • a number of methods can be used to deliver the mmRNAs to the cells, such methods include liposomes, polymers, microspheres, gene therapy vectors, and naked DNA vectors.
  • transducing viral e.g., retroviral, adenoviral, lentiviral, and adeno-associated viral
  • transducing viral have often been used been used because of their high efficiency of infection and stable integration and expression.
  • Lipofection also known as “lipid transfection” or “liposome-based transfection,” uses a lipid complex to deliver DNA to cells and is the preferred embodiment of the mmRNA delivery system.
  • Lipids are a broad class of fat-soluble biomolecules.
  • the cell membranes of animal cells are composed of a bilayer of phospholipids with hydrophilic surfaces facing the cytoplasm and extracellular environment.
  • Lipofection technology uses tiny vesicular structures called liposomes that have the same composition as the cell membrane.
  • the mRNA to be introduced into the heart is encapsulated into a liposome. Depending on the liposome and cell type, the liposome can be endocytosed or directly fuse with the cell membrane to release the mRNA into the target cell.
  • LipofectamineTM or Lipofectamine Messenger MAXTM are common transfection reagents, produced and sold by Invitrogen.
  • Lipid nanoparticles (LPNs) have also been designed to provide a safe, safe alternative for protecting mRNAs from degradation and immune activation and to facilitate their release from endosomal compartments to the cytosol.
  • LPNs have been specifically targeted for cell specific expression by coating the LNPs entrapping the mmRNAs with monoclonal antibodies.
  • One preferred embodiment of the therapeutic agent includes a Stemin mRNA, a YAP5SA mRNA, and a Lipofectamine transfection agent.
  • cardiomyocytes underlies most causes of heart failure. Normal repair processes are inadequate to deal with extensive myocardial damage.
  • the ability to regenerate myocytes in a patient by enhancing the replication of cardiomyocytes with the therapeutic agents described above will be a new standard of care for damaged heart muscle in the future.
  • the therapy described herein will increase the number of myocytes derived from the patient’s own heart to increase heart wall thickness following an infarct or increase function in the setting of heart failure.
  • this treatment is designed to treat patients having suffered from a myocardial infarction or other type of heart disease, it may also be used to treat pediatric cardiac diseases, to repair congenital cardiac birth defects, and to expand the heart wall in hypoplastic hearts.
  • the heart tissue of a patient with heart disease will be visualized using echocardiography and/or ultrasound to identify areas of infarct or tissue damage.
  • the mRNAs of the therapeutic agents are introduced into the border of the damaged cardiac tissue by a cardiac catheter.
  • the therapeutic agent is injected at multiple sites in the border area between visualized healthy and damaged tissue.
  • a typical treatment will include 1-10 injection sites as the catheter is moved to different areas of cardiac muscle damage. Echocardiograms will be performed to assess the success of the treatment.
  • the treatment regimen will typically begin when the myocardial infarction has been diagnosed or within two weeks of the diagnosis.
  • the use of the therapeutic agent to treat infracted heart tissue may be useful over a much longer extension of time.
  • the described treatment regimen may be helpful in the treatment of a number of types of heart disease.
  • compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
  • Li F Wang X, Capasso JM, Gerdes AM. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996; 28: 1737-1746.
  • Lats2 is a negative regulator of myocyte size in the heart. Circ Res 103, 1309-1318 (2008).
  • Vartiainen MK Guettler S, Larijani B, Treisman R. Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL. Science Jun 22 2007; 316:1749-1752.
  • Zaromytidou AI, Miralles F, Treisman R MAL and ternary complex factor use different mechanisms to contact a common surface on the serum response factor DNA- binding domain. Mol Cell Biol. 2006; 2611 :4134-4148

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des compositions et des méthodes pour favoriser des procédés et des systèmes de cardiomyocytes de mammifères adultes pour améliorer la régénération des cardiomyocytes. L'invention concerne l'administration locale d'un agent thérapeutique contenant un ARN messager modifié pour exprimer un polypeptide de facteur de réponse sérique muté et un ARN messager modifié pour exprimer un polypeptide YAP muté dans le muscle cardiaque malade pour favoriser la prolifération des cardiomyocytes et la régénération cardiaque.
PCT/US2020/063838 2019-12-09 2020-12-08 Régénération améliorée de cardiomyocytes Ceased WO2021119030A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962945513P 2019-12-09 2019-12-09
US62/945,513 2019-12-09

Publications (1)

Publication Number Publication Date
WO2021119030A1 true WO2021119030A1 (fr) 2021-06-17

Family

ID=76330461

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2020/063838 Ceased WO2021119030A1 (fr) 2019-12-09 2020-12-08 Régénération améliorée de cardiomyocytes

Country Status (1)

Country Link
WO (1) WO2021119030A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018170172A1 (fr) * 2017-03-14 2018-09-20 Baylor College Of Medicine Yap active dominante, effecteur hippo, induisant l'accès à la chromatine et le renouvellement des cardiomyocytes
US20190175690A1 (en) * 2016-08-16 2019-06-13 Children's Medical Center Corporation Compositions and methods for cardiac repair
WO2019136031A1 (fr) * 2018-01-05 2019-07-11 University Of Houston System Facteur de réponse sérique régénère les cellules sénescentes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190175690A1 (en) * 2016-08-16 2019-06-13 Children's Medical Center Corporation Compositions and methods for cardiac repair
WO2018170172A1 (fr) * 2017-03-14 2018-09-20 Baylor College Of Medicine Yap active dominante, effecteur hippo, induisant l'accès à la chromatine et le renouvellement des cardiomyocytes
WO2019136031A1 (fr) * 2018-01-05 2019-07-11 University Of Houston System Facteur de réponse sérique régénère les cellules sénescentes

Similar Documents

Publication Publication Date Title
Song et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors
Leone et al. Advances in heart regeneration based on cardiomyocyte proliferation and regenerative potential of binucleated cardiomyocytes and polyploidization
JP6162104B2 (ja) 細胞及び遺伝子に基づく心臓機能改善方法
Lumb et al. The AAA ATPase spastin links microtubule severing to membrane modelling
Bang et al. Understanding the molecular basis of cardiomyopathy
D'Amore et al. A journey through the cytoskeleton with protein kinase CK2
US11179479B2 (en) Enhanced cardiomyocyte regeneration
EP3280429B1 (fr) Fstl1 pour la réparation de tissu cardiaque
WO2019006512A1 (fr) Régénération de cardiomyocytes
WO2018052374A1 (fr) Modulation de l'expression de tjp1 pour réguler la régénération de cellules cardiaques
Greenberg et al. Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes
Fang et al. High mobility group box 2 regulates skeletal muscle development through ribosomal protein S6 kinase 1
WO2021119030A1 (fr) Régénération améliorée de cardiomyocytes
Raeker et al. Targeted deletion of the zebrafish obscurin A RhoGEF domain affects heart, skeletal muscle and brain development
Fu et al. MUSTN1 interaction with SMPX regulates muscle development and regeneration
Holaska Diseases of the nucleoskeleton
US20250283080A1 (en) Gene therapy of hippo signaling improves heart function in a clinically relevant model
US20240301355A1 (en) Methods of inducing cardiac cell proliferation and inducing heart regeneration
WO2009108772A1 (fr) Compositions morphogéniques de myocyte cardiaque et procédés d’utilisation de celles-ci
Wei et al. Desmoplakin mutations in cardiac fibroblasts cause TGFβ1-mediated pathological fibrogenesis in desmoplakin cardiomyopathy via beclin-1 regulation
Xiao Synergistic Effect of Cardio-Stemin and YAP5SA Promotes Cardiomyocyte Regeneration in vivo
KR20250057176A (ko) Arid4b 유전자 발현을 통한 신경세포로의 직접교차분화 유도 방법
MX2011008764A (es) Uso del modulador de actividad de vgii3 para la modulacion de adipogenesis.
Vuerich Studio del cross-talk tra cardiomiociti e cellule endoteliali per promuovere la rivascolarizzazione e la rigenerazione cardiaca
Vigil Garcia Therapeutic RNA regulation during cardiac remodeling and disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20899584

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20899584

Country of ref document: EP

Kind code of ref document: A1