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WO2024189038A1 - Methods of preventing or treating cardiomyopathy by redirecting mislocalized pathogenic rbm20 protein variants - Google Patents

Methods of preventing or treating cardiomyopathy by redirecting mislocalized pathogenic rbm20 protein variants Download PDF

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Publication number
WO2024189038A1
WO2024189038A1 PCT/EP2024/056577 EP2024056577W WO2024189038A1 WO 2024189038 A1 WO2024189038 A1 WO 2024189038A1 EP 2024056577 W EP2024056577 W EP 2024056577W WO 2024189038 A1 WO2024189038 A1 WO 2024189038A1
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Prior art keywords
protein
rbm20
amino acid
acid sequence
mutated
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Inventor
Lars Steinmetz
Julia KORNIENKO
Kai FENZL
Marta RODRÍGUEZ-MARTÍNEZ
Michael Gotthardt
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Europaisches Laboratorium fuer Molekularbiologie EMBL
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Leland Stanford Junior University
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Europaisches Laboratorium fuer Molekularbiologie EMBL
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Leland Stanford Junior University
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Publication of WO2024189038A1 publication Critical patent/WO2024189038A1/en
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Definitions

  • the present invention relates to agents that increase the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the same protein in the cytoplasm of said cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence.
  • the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNPO3) or an ortholog thereof.
  • the disease or condition is myopathy, in particular dilated cardiomyopathy (DCM).
  • DCM dilated cardiomyopathy
  • the present invention further relates to a method for identifying suitable agents used to increase the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or to decrease the amount of the same protein in the cytoplasm of said cell.
  • Correct protein localization is fundamentally based on the recognition of a targeting signal within the nascent protein by a targeting factor for the destination organelle. Gene variants impairing these targeting signals often result in severe diseases. For example, cytoplasmic mislocalization of p53 leads to various types of cancer, and cytoplasmic mislocalization of TDP-43 is associated with Amyotrophic Lateral Sclerosis. Details about the transport mechanisms involved are required for developing targeted therapies and are still elusive for many cases.
  • DCM Dilated cardiomyopathy
  • RBM20 variants can result in a particularly severe form of the disease often causing arrhythmia and progressive heart failure, and account for about 3% of familial DCM cases [7-10].
  • Current guidelines for RBM20 patients suggest evaluation for primary prophylactic placement of implantable cardioverter defibrillators based on individual predicted risk [9-11], RBM20 is predominantly expressed in the heart, and is involved in regulation of tissue-specific alternative splicing [12], Among its targets are genes involved in sarcomere structure (e.g. TTN), mitochondrial function (e.g. IMMT), calcium handling (e.g. RYR2 and ion channels (e.g.
  • CACNA1C CACNA1C
  • RBM20 Heterozygous mutations in RBM20 result in haploinsufficiency with respect to transcriptional splicing [13, 14, 17, 18], where alternative splicing of RBM20’s targets is proportional to the amount of wild-type (WT) versus mutated RBM20 expressed [19],
  • WT wild-type
  • iPSC-CMs induced pluripotent stem cell-derived cardiomyocytes
  • R636S RBM20 protein preferably bound to the 3 ’ UTR of transcripts in the cytoplasm, and co-localized with the P-body marker DDX6 [22], Altogether, aberrant localization of RBM20 was shown for mutations of four out of six residues in the PRSRSP stretch: S635, R636, S637, and P638 [19-24], leaving the remaining two (P633 and R634) uncharacterized. Overall, it is unclear whether these mutations affect RBM20's intrinsic role as a mediator of spliceosome activity, or whether the splicing haploinsufficiency results from protein mislocalization alone. This remains uncertain because the mechanism driving RBM20 cellular localization is unknown.
  • W02015042308A2 relates to RNA-based HIV-inhibitors, such as a viral entry inhibiting RNA encoding sequence encoding a nuclear receptor siRNA, which can be a transportin 3 (TNP03) siRNA.
  • Tnpo3 binding partners contain arginine-serine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran- and alternative splicing factor/splicing factor 2 (ASF/SF2)- bound forms.
  • the flexible 0-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo.
  • a constellation of charged residues on and around the arginine- rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2.
  • Tnpo3 Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. The results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.
  • CPSF6 polyadenylation specificity factor 6
  • WO 2010/149332 relates to RBM20 polynucleotides, preferably for use in medicine.
  • these polynucleotides can be used to diagnose cardiac diseases, like cardiomyopathies or sudden cardiac death.
  • the invention pertains to methods for diagnosing a subject suffering from a cardiac disease and to treating such a subject.
  • RBM20 proteins comprising a P638L (Homo sapiens) or a P641L mutation (Rattus norvegicus).
  • US20110281260A1 relates to methods and materials for using nucleic acid and amino acid sequence variants of ribonucleic acid binding motif protein 20 (RBM20).
  • RBM20 ribonucleic acid binding motif protein 20
  • methods and materials for using nucleic acid sequence variants and/or their corresponding amino acid variants of RBM20 that are associated with dilated cardiomyopathy to identify mammals (e.g., humans) at risk of having dilated cardiomyopathy that is likely to progress to heart failure are provided.
  • WO 2020/092171 relates to methods of treatment, genetic screening, and disease models for heart conditions associated with RBM20 deficiency.
  • methods of treating heart conditions associated with RBM20 deficiency, including RBM20-dependent dilated cardiomyopathy and heart failure with compounds that upregulate expression of RBM20, such as all-trans retinoic acid are provided.
  • methods of genetic screening to detect the presence of a P633L mutation in RBM20 in order to identify individuals having a genetic predisposition to developing RBM20-dependent DCM.
  • IPC-CMs Induced pluripotent stem cell-derived cardiomyocytes (IPSC-CMs) produced by differentiation of IPSCs comprising at least one RBM20 allele encoding a P633L mutation and methods of using them in screening for therapeutics for treating RBM20-dependent DCM are also disclosed.
  • Claimed is a method of treating a subject for a heart condition associated with RBM20 deficiency, the method comprising administering a therapeutically effective amount of all-trans retinoic acid (ATRA) to the subject.
  • ATRA all-trans retinoic acid
  • RBM20 variants display gain-of- function effects related to mislocalization of the mutated protein outside the nucleus.
  • RBM20 mislocalized to the cytoplasm and formed potentially detrimental RNP-granules [19]
  • S637A mutation S635A in humans
  • RBM20-dependent dilated cardiomyopathy and heart failure methods of treating heart conditions associated with RBM20 deficiency, including RBM20-dependent dilated cardiomyopathy and heart failure with compounds that upregulate expression of RBM20 appear to have undesired side-effects.
  • New and improved strategies are therefore sought in order to prevent or treat heart conditions associated with RBM20 deficiency. It is therefore an object of the present invention to provide such strategies. Other objects and advantages will readily become apparent for the person of skill from studying the following more detailed description and examples.
  • the problem of the present invention is solved by providing an agent that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence.
  • the agent for use is selected from the group consisting of an agent specifically binding to the protein comprising a mutated RS domain amino acid sequence and comprising at least one nuclear localization signal (NLS), an agent for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, a compound that improves the binding of the protein comprising a mutated RS domain amino acid sequence to its nuclear transporter protein, and a genetic construct for expressing or overexpressing the nuclear transporter protein or of the protein comprising a mutated RS domain amino acid sequence.
  • NLS nuclear localization signal
  • the agent for use is selected from the group consisting of a proteinaceous binding domain that is specifically binding to the protein comprising a mutated RS domain amino acid sequence, such as an antibody or binding fragment thereof, fused or linked to the at least one NLS, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof, or an NLS-RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9 fusion or an RBM20 L-rich region binding fragment thereof, a nanobody -NLS fusion, an expression construct for expressing a polynucleotide encoding the binder-NLS fusion and/or the nuclear transporter protein, a genetic integration construct for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as, for example, a prime editing construct containing at least one NLS, and a small molecule binding to
  • agent for use according to the present invention wherein the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNPO3) or an ortholog thereof.
  • the agent for use according to the present invention wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • the problem of the present invention is solved by providing a method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell, comprising the steps of i) providing at least one mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, ii) providing at least one nuclear transporter protein comprising a domain binding to an RS domain, and/or at least one nuclear transporter protein RS domain binding domain amino acid sequence, and iii) detecting the binding of the mutated RS domain amino acid sequence of i) to the domain of ii) binding to an RS domain in the absence and presence of at least one candidate compound, wherein an increase of the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the presence of at least one candidate compound indicates a compound that increases the amount of a protein comprising a mutated RS domain
  • mutated RS domain amino acid sequence is derived from RBM20 or an ortholog thereof, and/or the domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof.
  • TNPO3 transportin 3
  • the amino acid sequences of i) and/or ii) are provided in a cell, and are preferably provided as recombinantly expressed amino acid sequences.
  • examples are the provision using mRNA delivery of the coding sequence(s), so that cells express ii) or i) or a binder themselves, such as, for example, as mRNA vaccines or “naked” mRNA provided to the heart.
  • the RNA could be encapsulated into nanoparticles to increase the delivery/stability.
  • DNA delivery of the sequence(s) can be performed. Viral delivery of DNA or RNA is also possible.
  • a change in the biological function and/or intracellular localization of i) the at least one protein comprising a mutated RS domain amino acid sequence, and/or an increase of the expression and/or a change in the biological function of ii) the at least one nuclear transporter protein comprising a domain binding to an RS domain is detected instead or in addition to the binding of the mutated RS domain amino acid sequence in the absence and presence of the at least one candidate compound, wherein a change in the biological function and/or intracellular localization of a protein of i) and/or an increase of the expression and/or change in the biological function of a protein of ii) indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of a protein comprising a mutated RS domain amino acid sequence in the cytoplasm of said cell.
  • the biological function is tested based on detecting cytoplasmatic mislocalization of the protein comprising the mutated RS domain amino acid sequence, the formation of cytoplasmatic granules of the protein comprising the mutated RS domain amino acid sequence, and/or the splicing activity of the protein comprising the mutated RS domain amino acid sequence.
  • the compound as identified may be selected from the group consisting of natural compound, plant extract, a peptide, such as an artificial in silico designed peptide binder, a protein, a small molecule (less than about 500 Da), an RNA, an antibody or antigen binding fragment thereof, and an agent for use according to the present invention.
  • the problem of the present invention is solved by a method for producing a pharmaceutical composition, comprising performing a method according to the present invention, and admixing the compound as identified with at least one pharmaceutically carrier.
  • the problem of the present invention is solved by a compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention for use in medicine, in particular for use in the prevention or treatment of a disease or condition in a cell of a subj ect that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising a mutated RS domain amino acid sequence.
  • the cell is a cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, and the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • the problem of the present invention is solved by a method for increasing the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreasing the amount of the protein in the cytoplasm of said cell, comprising providing to a cell an effective amount of the compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention, whereby the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell is increased and/or the amount of the protein in the cytoplasm of said cell is decreased.
  • the problem of the present invention is solved by a method for preventing or treating a disease or condition related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of a protein comprising a mutated RS domain amino acid sequence in a subject in need of said prevention or treatment, comprising administering to the subject an effective amount of the compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention.
  • the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • the object of the present invention is solved by an agent that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence.
  • a protein comprising a mutated RS domain amino acid sequence shall relate to a domain as found in a protein member of the arginineserine-rich protein family (SR proteins) that are multifunctional RNA-binding proteins that have emerged as key determinants for mRNP formation, identity and fate. They bind to pre- mRNAs early during transcription in the nucleus and accompany bound transcripts until they are translated or degraded in the cytoplasm.
  • SR proteins are mostly known for their essential roles in constitutive splicing and as regulators of alternative splicing (see, for example, Irena Sliskovic, Hannah Eich, Michaela Muller-McNicoll; Exploring the multifunctionality of SR proteins.
  • a preferred protein providing the RS domain amino acid sequence is human RBM20 (UniProt Q5T481) or an ortholog thereof.
  • the amino acid sequence of human RBM20 RNA binding motif protein 20 has Gene ID: 282996 and can be found at https://www.ncbi.nlm.nih.gov/gene/282996.
  • a preferred RS domain amino acid sequence according to the present invention comprises an amino acid sequence that is to at least 90%, preferably to at least 95%, and most preferred to at least 99% or even to 100% identical to the amino acids 613-673 of the human non-mutated RBM20 protein.
  • Orthologs of the RS domain amino acid sequence according to the present invention may be selected from bovine, dog, equine, cat, chicken, monkey, mouse, and rat.
  • the rbm20 gene encodes a protein that binds RNA and regulates splicing. Mutations in this gene have been associated with familial dilated cardiomyopathy.
  • the majority of DCM-causing RBM20 variants are heterozygous missense mutations, many of which cluster in a conserved stretch of six amino acids PRSRSP (amino acid position 633 - 638) in the protein’s arginine/serine (RS)-rich domain [7, 13, 16, 17], This stretch further constitutes a preferred embodiment of the invention.
  • a protein comprising a mutated RS domain amino acid sequence shall further include mutated amino acid sequences in a protein member of the arginine-serine-rich protein family (SR proteins), where the mutation or mutations are found outside of the above-described RS-domain, as long as these mutations cause or are involved in an aberrant protein localization and/or splicing activity of the respective SR protein.
  • SR proteins arginine-serine-rich protein family
  • mutations in the RRM region such as, for example, V535I, or the glutamate-rich region (AA 839-945 of human RBM20), such as, for example, D888N or E913K (see Lennermann D, Backs J, van den Hoogenhof MMG. New Insights in RBM20 Cardiomyopathy. Curr Heart Fail Rep. 2020 Oct;17(5):234-246. doi: 10.1007/sl l897-020-00475-x. PMID: 32789749; PMCID: PMC7495990, and the references as cited therein, incorporated herein by reference).
  • the inventors thus uncovered the direct mechanism of RBM20 nuclear transport and demonstrated how RS-domain mutations disrupt this process.
  • the inventor’s findings enable a new therapeutic strategy, targeted at improving the nuclear import of mislocalized RBM20. This strategy is more likely to be successful in treating RBM20-mediated DCM caused by mislocalizing mutations, since it will compensate both for alternative splicing (AS) haploinsufficiency and alleviate the dominant-negative effects of the cytoplasmic granules.
  • AS alternative splicing
  • RBM20 is a splicing factor with two canonical domains, an RNA recognition motif (RRM) and an arginine-serine rich (RS) domain.
  • RRM loss-of-function disrupts the splicing of RBM20 target transcripts and leads to systolic dysfunction without overt DCM, while mutations in the RS domain precipitate DCM.
  • mice lacking the RS domain manifest DCM with mis-splicing of RBM20 target transcripts.
  • RBM20 is mis-localized in Rbm20 ARS mice but not in mice lacking the RRM, which are also deficient in RBM20 splicing.
  • the RS domain not other domains including the RRM, is critical for RBM20 nuclear import and define the core nuclear localization signal (NLS) within this domain.
  • Mutation analysis of phosphorylation sites within the RS domain indicated that phosphorylation is dispensable for RBM20 nuclear import. They establish disruption of the NLS in RBM20 as a causative mechanism in DCM through nucleocytoplasmic transport.
  • Gene therapy is an alternative option to repair RBM20 mutations.
  • the majority of existing gene editing methods apart from prime editing, are mutation-dependent. This will require to develop and optimize a personalized gRNA for every new mutation, which is an inefficient time- and finance-wise approach.
  • introducing genome editing in humans has big ethical and societal complications, which may result in longer times for translation into clinical practice.
  • the inventors showed that restoring the nuclear localization of RBM20 RS-domain variants both compensates for the splicing deficiency, and results in granule clearance. They proposed a couple of principal strategies to achieve this in patients, as is described herein.
  • the agent for use according to the present invention wherein the agent is selected from agents specifically binding to the protein comprising a mutated RS domain amino acid sequence and comprising at least one nuclear localization signal (NLS).
  • the agent specifically binding to the protein comprises a proteinaceous binding domain that is specifically binding to the protein comprising a mutated RS domain amino acid sequence.
  • the binder can be an antibody or protein binding fragment thereof, or an artificially designed peptide binder, fused or otherwise suitably linked to the at least one NLS, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof, or an NLS-RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9 fusion or an RBM20 L-rich region binding fragment thereof, a nanobody - NLS fusion construct as described herein.
  • Nanobodies can be generated via in vivo llama/alpaca immunization, the term shall also include sybodies, i.e.
  • binders include single-domain antibodies, e.g., VHH antibodies with a single variable domain on a heavy chain (see, for example, Henry KA, MacKenzie CR. Editorial: Single-Domain Antibodies-Biology, Engineering and Emerging Applications. Front Immunol. 2018 Jan 23;9:41. doi: 10.3389/fimmu.2018.00041. PMID: 29410670; PMCID: PMC5787064).
  • the agent for use according to the present invention wherein the agent is selected from an agent for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as a genetic integration construct for genetically fusing the at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as, for example, a prime editing construct containing at least one NLS.
  • NLS nuclear localization signal
  • the agent for use according to the present invention wherein the agent is selected from a compound that improves the binding of the protein comprising a mutated RS domain amino acid sequence to its nuclear transporter protein, such as a small molecule binding to the nuclear transporter protein and/or the protein comprising a mutated RS domain amino acid sequence and thereby improving the nuclear transport of the protein comprising a mutated RS domain amino acid sequence.
  • the agent for use according to the present invention wherein the agent is selected from a genetic construct for expressing or overexpressing the nuclear transporter protein of the protein comprising a mutated RS domain amino acid sequence, such as integrating or other expression construct for expressing a polynucleotide encoding the binder-NLS fusion and/or the nuclear transporter protein.
  • the agent for use according to the present invention wherein the at least one NLS sequence is selected from the group consisting of a non-classical or classical NLS, such as, for example a monopartite or bipartite classical NLS, in particular an NLS of SV40, C-myc, nucleoplasmin, EGL-13, or TUS-protein, the acidic M9 domain of hnRNP Al, the sequence KIPIK in yeast transcription repressor Mata2, and the complex signals of U snRNPs (see also Lu, J., Wu, T., Zhang, B. et al. Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Commim Signal 19, 60 (2021). https://doi.org/10. 1186/s 12964-021 -00741-y).
  • a non-classical or classical NLS such as, for example a monopartite or bipartite classical NLS, in particular an NLS of SV40, C-myc, nucleoplasm
  • agent for use according to the present invention wherein the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNPO3) or an ortholog thereof.
  • the agent for use according to the present invention wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • the inventors show that introducing a SV40 NLS tag restores nuclear localization and splicing activity of RBM20 in HeLa and HEK293 cell lines, as well as in iPSC-CMs.
  • the transfection of HEK293T and iPSC-CMs cells with plasmids encoding for RBM20 RS-domain variants with an NLS tag resulted in significant restoration of splicing activity for all variants in comparison to the non-tagged variants (Fig. 2b, c).
  • RNA-Seq analysis showed that the expression of 1,751 genes was altered in cells expressing R634Q-RBM20, compared to WT (Fig. 2d).
  • the genes are consistent with severe cardiac impairment and similar to the genes altered in expression in cells expressing P633L in the cytoplasm.
  • RBM20 target gene expression was consistently either unchanged or down-regulated. Strikingly, the inventors found only few differentially expressed genes between WT and NLS-tagged-R634Q expressing cells (Fig. 2d).
  • splicing of RBM20 target genes was restored in NLS-R634Q expressing iPSC-CMs to similar levels seen in WT (Fig. 2e).
  • the inventors started to test this as proof-of-principle in the inventor’s eGFP-RBM20-WT and -R634Q HeLa reporter cells, with an eGFP specific nanobody.
  • the initial results suggest that the inventors can regulate the localization of RBM20 with such a nanobody-NLS fusion as alternative nuclear transporter.
  • the inventors plan to design and produce now a set of RBM20-specific nanobodies that should recognize all RBM20 variants and/or a set of artificial in silico designed peptide binders.
  • the targeting efficiency of such newly generated RBM20 specific nanobodies and/or binders will be then validated and tested in the inventor’s established HeLa, iPSC-CMs and RBM20-DCM-mouse models.
  • the inventors supply an RBM20 specific nanobody-NLS fusion and/or peptide binder-fusion either by delivering its cDNA with AAV-based vectors, or its mRNA with lipid bioparticles specifically to cardiomyocytes. These are tested and optimized in iPSC-CMs. Work is focused on optimizing the delivery methods in vivo using the inventor’s mouse models. As an alternative, the inventors are also investigating the possibility to introduce the NLS sequence via prime editing into the endogenous RBM20 locus. The advantage is that this approach can potentially repair the localization of all RBM20 variants, and does not require a personalized gRNA per patient. The exact position and design for this approach is investigated and is further optimized.
  • the inventors demonstrate that supplying a full TNP03 cDNA both in vitro and in vivo enhances the probability of mislocalized RBM20 variants to interact with TNP03.
  • TNP03 overexpression rescued the localization and splicing activity of all tested RBM20 variants in iPSC-CMs.
  • the nuclear localization of P633L was completely rescued to the level of the WT protein; similar results were obtained for R634Q-RBM20, although a small fraction of the protein remained in the cytoplasm (Fig. 5a, b).
  • Restoring nuclear localization of the mutant RBM20 variants also up-regulated the splicing activity (Fig. 5c, d).
  • the inventors delivered Tnpo3 cDNA via AAV9 to mouse hearts bearing homozygous P635L-RBM20 (P635L +/+ ) mutations (P633L in humans).
  • the inventors analyzed the RBM20 localization and splicing function four weeks after AAV9 injection (Fig. 5e). Strikingly, increasing Tnpo3 levels (Fig. 5f) resulted in partial rescue of RBM20 localization (Fig. 5g) and Tin alternative splicing (Fig. 5h, i) independently of differences in Rbm20 expression levels.
  • This preclinical model of RBM20-DCM reveals a novel therapeutic strategy for mislocalizing RBM20 variants for future studies. See also Figure 16.
  • preferred strategies of the present invention to achieve an effective prevention and/or treatment by improving the mislocalization of RBM20 are at least one of i) increasing TNPO3 levels in a desired cell to rescue the RBM20 mislocalization, for example using a TNPO3 expression construct; ii) using a compound, for example a small molecule, or a co-factor, to mediate, improve or increase the binding of the (mutated) RBM20 protein and/or the RS domain thereof to the TNP03 protein in order to improve the RBM20 transport and RBM20 mislocalization; and/or iii) modifying the mutated RBM20 protein and/or the RS domain thereof in order to improve the RBM20 transport and RBM20 mislocalization by using a genetic construct to fuse at least one NLS to the RBM20 and/or by attaching (tagging) at least one NLS to the RBM20 using a binder that also comprises at least one NLS, for example as a nanobody -NLS format
  • the object of the present invention is solved by providing a method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of a cell, comprising the steps of a) providing at least one L-rich domain amino acid sequence of a protein comprising a mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, b) providing at least one candidate compound prospectively binding to the L-rich domain amino acid sequence, and c) detecting the binding of the at least one candidate compound to the L-rich domain amino acid sequence, wherein a binding of the at least one candidate compound to the L-rich domain amino acid sequence indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said
  • the object of the present invention is solved by providing a method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell, comprising the steps of i) providing at least one mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, ii) providing at least one nuclear transporter protein comprising a domain binding to an RS domain, and/or at least one nuclear transporter protein RS domain binding domain amino acid sequence, and iii) detecting the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the absence and presence of at least one candidate compound, wherein an increase of the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the presence of at least one candidate compound indicates a compound that increases the amount of a protein comprising
  • the present invention uses the binding of a compound, such as a peptide, to the L-rich region and/or formation and structure of the TNPO3-RBM20 complex in order to screen for compounds, such as small molecules, as prospective therapeutics through further stabilizing the interaction of TNPO3 with RBM20 variants. It was surprisingly found in the context of the present invention that a loss of interaction of RBM20 with TNPO3 results in the formation of undesired RBM20 RNP granules, regardless of the mutation present. Hence, an improvement of the binding of RBM20 and TNPO3 will both reduce granules and improve mislocalization of RBM20.
  • a compound such as a peptide
  • the method according to the present invention wherein the at least one protein comprising a mutated RS domain amino acid sequence further comprises at least one added NLS sequence, as discussed above and herein. This will revert and rescue the mislocalization further.
  • the method according to the present invention wherein at least one of the amino acid sequences as used, i.e., comprising the RS domain or the nuclear transporter binding sequence is labelled, and/or wherein the at least one candidate compound is labelled. This allows for a detection in the assay, i.e., detecting the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the absence and presence of at least one candidate compound.
  • Labelling can be done with any suitable label that optimally does not substantially interfere with the binding of the components.
  • the label may be attached directly or indirectly, for example directly labelled with a GFP fusion or indirectly labelled using an antibody or fragment thereof that specifically binds to an amino acid sequence of the components, the antibody being suitably labelled.
  • a suitable protein such as importin or small GTPase Ran
  • RNA molecule such as a RNA molecule
  • mutated RS domain amino acid sequence is derived from RBM20 or an ortholog thereof, and/or the domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof.
  • a preferred RS domain amino acid sequence according to the present invention whether individually or included in the protein, such as RBM20, comprises an amino acid sequence that is to at least 90%, preferably to at least 95%, and most preferred to at least 99% or even to 100% identical to the amino acids 613-673 of the human non-mutated RBM20 protein.
  • Orthologs of the RS domain amino acid sequence according to the present invention may be selected from bovine, dog, equine, cat, chicken, monkey, mouse, and rat.
  • a preferred domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof, or comprises an amino acid sequence that is to at least 90%, preferably to at least 95% identical to the amino acids around the arginine-rich helix of Tnpo3 HEAT repeat 15 of the human transportin 3 (Tnpo3, Transportin-SR2, UniProt Q9Y5L0) protein (see Maertens GN, et al. Structural basis for nuclear import of splicing factors by human Transportin 3. Proc Natl Acad Sci U S A. 2014 Feb 18; 111(7):2728-33. doi: 10.1073/pnas.l 320755111. Epub 2014 Jan 21.
  • Orthologs of the transportin 3 amino acid sequence according to the present invention may be selected from bovine, dog, equine, cat, chicken, monkey, mouse, and rat.
  • the method according to the present invention may be performed in vitro or in vivo. While the above binding-assays may be performed in a cell-free model, preferred is a method that is performed in a cellular assay. In a preferred method according to the present invention, the amino acid sequences of a) i) and/or ii) are therefore provided in a cell and are preferably provided as recombinantly expressed amino acid sequences.
  • any suitable cellular assay may be used in the context of the present invention.
  • the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells (see also example, below).
  • a heart muscle cell or cardiomyocyte such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells (see also example, below).
  • iPSC-CMs induced pluripotent stem cell-derived cardiomyocytes
  • HeLa cells HEK293 cells
  • An advantage of the cellular assay is that detecting a change in the biological function of the components as involved can be readily achieved.
  • Preferred is the method according to the present invention, wherein further a change in the biological function of i) the at least one protein comprising a mutated RS domain amino acid sequence, and optionally an increase of the expression and/or a change in the biological function of ii) the at least one nuclear transporter protein comprising a domain binding to an RS domain is detected instead or in addition to the binding of the mutated RS domain amino acid sequence in the absence and presence of the at least one candidate compound, wherein a change in the biological function of a protein of i) and/or an increase of the expression and/or change in the biological function of a protein of ii) indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell.
  • all these parameters are expected to be suitable to indicate an improvement of the pathological situation, and thus can indicate a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell.
  • any suitable candidate compound can be used for the method of the present invention.
  • the method according to the present invention wherein the compound is selected from the group consisting of natural compound, plant extract, a peptide, such as an artificial in silico designed peptide binder, a protein, a small molecule (molecular weight of less than about 500 Da), an RNA, an antibody or antigen binding fragment thereof.
  • the screening can be used to identify and/or test an agent for use according to the present invention as described above.
  • the object of the present invention is solved by providing a method for producing a pharmaceutical composition, comprising performing a method according to the present invention, and admixing the compound or agent as identified with at least one pharmaceutically carrier.
  • composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.
  • a pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
  • compositions according to the invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • compositions according to the invention may be in liquid, dry or semi-solid form, such as, for example, in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granulate, sugar-coated tablet, lozenge, pill, ampoule, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, moist compress, gargling solution, plant juice, nasal agent, inhalation mixture, aerosol, mouthwash, mouth spray, nose spray, or room spray.
  • the object of the present invention is solved by providing a compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention for use in medicine, in particular for use in the prevention or treatment of a disease or condition in a cell of a subject that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising a mutated RS domain amino acid sequence.
  • the compound or pharmaceutical composition for use according to the present invention wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • the object of the present invention is solved by a method for increasing the amount of a protein in the nucleus of a cell and/or decreasing its amount in the cytoplasm of said cell, comprising providing to a cell an effective amount of the compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention.
  • An effective amount is sufficient to increase the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell as treated.
  • the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
  • a heart muscle cell or cardiomyocyte such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
  • a heart muscle cell or cardiomyocyte such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
  • iPSC-CMs induced pluripotent stem cell-derived cardiomyocytes
  • the object of the present invention is solved by providing a method for preventing or treating a disease or condition related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of a protein comprising a mutated RS domain amino acid sequence in a subject in need of said prevention or treatment, comprising administering to the subject an effective amount of the compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention.
  • the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • the term “subject”, as used in certain embodiments, preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human, and a patient.
  • the term “patient” preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, horse, cattle, cow, cat, dog, monkey, or preferably a human, for example a human patient, for whom diagnosis, prognosis, or therapy is desired.
  • the subject of the invention may be at danger of suffering from a disease, such as CM, in particular DCM.
  • CM in particular DCM.
  • treating means stabilizing or reducing an adverse symptom associated with a condition; reducing the severity of a disease symptom; slowing the rate of the progression of a disease; inhibiting or stabilizing the progression of a disease condition; or changing a metric that is associated with the disease state in a desirable way.
  • preventing means the avoidance of the occurrence of an adverse symptom associated with a condition or disease before they occur.
  • the compounds/agents of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound/agent to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 to 1000 pg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 pg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 pg to 10 mg units per kilogram of body weight per minute, respectively.
  • compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; the pharmaceutical composition may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.
  • the inventors provided the proof-of-principle, that introducing a S V40 NLS tag restores nuclear localization and splicing activity of RBM20 in HeLa and HEK293 cell lines, as well as in iPSC- CMs.
  • the transfection of HEK293T cells with plasmids encoding for RBM20 RS-domain variants with an NLS tag resulted in significant restoration of splicing activity for all variants in comparison to the non-tagged variants (Fig. 2b).
  • the term “about” shall indicate a deviation of +/- 10% of a given value, if not stated otherwise. Preferred is the value as given, +/- 5%, more preferred is the value as given and determined according to a method according to the state of the art.
  • DCM-causing variants in the RS-domain of RBM20 have been shown, by the inventor’s group and others, to result in aberrant RBM20 localization and RNP granule formation in the cytoplasm [24-29], These mutations are associated with a more severe disease phenotype than an RBM20 KO in vivo [25, 26] Prior to this study, it was unknown why these single point mutations lead to RBM20 protein mislocalization and whether restoration of nuclear localization could restore the splicing activity in vivo.
  • the inventors demonstrate that all tested mislocalizing RBM20 RS-domain mutants regain their splicing activity upon addition of the SV40 NLS tag, as previously shown for S635A [40], This was done also in iPSCs for at least one RS mutant, and not only in HeLa cells with a reporter gene.
  • TNPO3 as the main nuclear importer of RBM20 with the genome-wide ICS CRISPR-Cas9 screen.
  • TNPO3 belongs to the 0-karyopherin family of importins, which directly interact with their cargo via amino acid sequence recognition.
  • TNPO3 specifically recognizes arginine-serine (RS) repeats that are present in many splicing factors like SRSF1, ASF/ SF2, and others [34, 37, 38, 41-43],
  • RS arginine-serine
  • the applied ICS screen is well suited to be used in a targeted fashion with iPSC-derived cardiomyocytes in the future. This may reveal additional factors involved in nuclear import or in regulating post-translational modifications of RBM20. It has been observed that the target-specific binding of TNP03 to its cargo can be phosphorylation-dependent [34, 37, 38] or independent [42], In the case of RBM20, serine residues in the RS-domain are normally phosphorylated [26, 31, 40, 44], however, phosphomimetic mutations do not rescue the localization phenotype [26], Importantly, both pooled and individual CRISPR KOs of kinases AKT2, CLK1, and SPRK1 - previously shown to phosphorylate RBM20 [33] - did not impact RBM20 localization (Fig. 3d). This result suggests that either the kinases complement each other, or that the RBM20-TNPO3 interaction is potentially phosphorylation-independent.
  • the inventors further used in vitro and in vivo models to demonstrate that the overexpression of TNPO3 can improve the nuclear import of RBM20 mutants and restore the splicing deficiency. Importantly, the inventors observed that cytosolic granules were reduced after enhancing nuclear transport. Further investigations will be needed to understand the nature and the effect of RBM20 granule formation.
  • the inventor’s data provide the first evidence that cytoplasmic granule formation of RBM20 variants is the result of mislocalization, and not its cause.
  • the inventors show that WT RBM20, when forced to remain in the cytoplasm, also forms granules of the same nature as the mutant variants. These results demonstrate that RS- domain mutations of PBM20 do not confer pro-aggregative qualities.
  • TNPO3 Mutations in TNPO3 have been linked to impaired myogenesis [43] and myopathies [45, 46], which includes one mutation in TNPO3 that was linked to familial DCM [47], Detailed structural studies will be needed to further decipher the RBM20-TNPO3 interaction and the direct impact of different mutations on both partners.
  • the inventor’s data reveals a new therapeutic avenue for DCM patients with disease causing variants in the RS-rich region of RBM20. Enhancing RBM20 nuclear import could be achieved by endogenous tagging of PBM20 with another NLS to be recognized by other importins. Alternatively, developing a small molecule to stabilize the interaction between the mutant RS-domain variants and TNPO3 could be beneficial too.
  • the inventor’s data demonstrates that these actions can alleviate the known splicing deficiency and abolish cytoplasmic granule formation via enhanced nuclear import.
  • the inventors showed that RS-domain mutations do not affect RBM20’s intrinsic splice regulatory activity, as previously believed, but rather its import into the nucleus.
  • RBM20 variants By genetically tagging RBM20 variants with the SV40 nuclear localization sequence (NLS), the inventors restored their nuclear localization.
  • the inventors show that they are functionally equivalent to the wild type and that the main factor driving their pathological phenotype is their cellular localization.
  • the inventors identify TNPO3 as the main nuclear importer of RBM20 and demonstrate that their interaction is disrupted by mutations in RBM20’s RS-domain, in proportion to the severity of the mislocalization phenotype.
  • the inventors show that enhancing the interaction between TNPO3 and RBM20 by increasing the amount of available transporter, rescues the aberrant localization and splicing phenotypes in vitro and in vivo.
  • the inventor’s results open up an undiscovered avenue for developing therapeutic strategies for DCM. Firstly, by enhancing the interaction between RBM20 and TNPO3 to up-regulate the physiological import mechanism. Secondly, by introducing the SV40 NLS-tag to the RBM20 gene via gene therapy, or by employing an RBM20-specific nanobody -NLS fusion protein as an alternative nuclear importer. The inventors show that both of these approaches restore RBM20’s nuclear localization, splicing activity, and resolve cytoplasmic granules.
  • the present invention relates to the following items:
  • Item 1 An agent that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence.
  • Item 2 The agent for use according to Item 1, wherein the agent is selected from the group consisting of an agent specifically binding to the protein comprising a mutated RS domain amino acid sequence and comprising at least one nuclear localization signal (NLS), an agent for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, a compound that improves the binding of the protein comprising a mutated RS domain amino acid sequence to its nuclear transporter protein, and a genetic construct for expressing or overexpressing the nuclear transporter protein of the protein comprising a mutated RS domain amino acid sequence.
  • NLS nuclear localization signal
  • Item 3 The agent for use according to Item 2, wherein the agent is selected from the group consisting of a proteinaceous binding domain that is specifically binding to the protein comprising a mutated RS domain amino acid sequence, such as an antibody or binding fragment thereof, or an artificially designed peptide binder, fused or linked to the at least one NLS, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof, or an NLS-RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9 fusion or an RBM20 L-rich region binding fragment thereof, a nanobody-NLS fusion, an expression construct for expressing a polynucleotide encoding the binder-NLS fusion and/or the nuclear transporter protein, a genetic integration construct for genetically fusing the at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as, for example,
  • Item 4 The agent for use according to any one of Items 1 to 3, wherein the at least one NLS sequence is selected from the group consisting of a non-classical or classical NLS, such as, for example a monopartite or bipartite classical NLS, in particular an NLS of SV40, C-myc, nucleoplasmin, EGL-13, or TUS-protein, the acidic M9 domain of hnRNP Al, the sequence KIPIK in yeast transcription repressor Mata2, and the complex signals of U snRNPs.
  • a non-classical or classical NLS such as, for example a monopartite or bipartite classical NLS, in particular an NLS of SV40, C-myc, nucleoplasmin, EGL-13, or TUS-protein, the acidic M9 domain of hnRNP Al, the sequence KIPIK in yeast transcription repressor Mata2, and the complex signals of U snRNPs.
  • Item 5 The agent for use according to any one of Items 1 to 4, wherein the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNP03) or an ortholog thereof.
  • Item 6 The agent for use according to any one of Items 1 to 5, wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
  • Item 7 The agent for use according to any one of Items 1 to 6, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • a method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of a cell comprising the steps of a) providing at least one L-rich domain amino acid sequence of a protein comprising a mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, b) providing at least one candidate compound prospectively binding to the L-rich domain amino acid sequence, and c) detecting the binding of the at least one candidate compound to the L-rich domain amino acid sequence, wherein a binding of the at least one candidate compound to the L-rich domain amino acid sequence indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
  • a method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of a cell comprising the steps of i) providing at least one mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, ii) providing at least one nuclear transporter protein comprising a domain binding to an RS domain, and/or at least one nuclear transporter protein RS domain binding domain amino acid sequence, and iii) detecting the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the absence and presence of at least one candidate compound, wherein an increase of the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the presence of at least one candidate compound indicates a compound that increases the amount of a protein
  • Item 10 The method according to Item 8 or 9, wherein the at least one protein comprising a mutated RS domain amino acid sequence further comprises at least one added NLS sequence and/or wherein at least one of the amino acid sequences of a), i) or ii) is labelled, and/or wherein the at least one candidate compound is labelled, such as, for example, labelled with GFP or a labelled antibody or fragment thereof specifically binding to the amino acid sequence.
  • Item 11 The method according to any one of Items 8 to 10, wherein at least one binding cofactor is present, in particular a suitable RNA molecule.
  • Item 12 The method according to any one of Items 8 to 11, wherein the mutated RS domain amino acid sequence is derived from RBM20 or an ortholog thereof, and/or the domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof.
  • TNPO3 transportin 3
  • Item 13 The method according to any one of Items 8 to 12, wherein the amino acid sequences of a), i) and/or ii) are provided in a cell, and are preferably provided as recombinantly expressed amino acid sequences.
  • Item 14 The method according to Item 13, wherein a change in the biological function of i) the at least one protein comprising a mutated RS domain amino acid sequence, and optionally an increase of the expression and/or a change in the biological function of ii) the at least one nuclear transporter protein comprising a domain binding to an RS domain is detected instead or in addition to the binding in the absence and presence of the at least one candidate compound, wherein a change in the biological function of a protein of i) and/or an increase of the expression and/or change in the biological function of a protein of ii) indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
  • Item 15 The method according to Item 13 or 14, wherein the biological function is tested based on detecting cytoplasmatic mislocalization of the protein comprising the mutated RS domain amino acid sequence, the formation of cytoplasmatic granules of the protein comprising the mutated RS domain amino acid sequence, and/or the splicing activity of the protein comprising the mutated RS domain amino acid sequence.
  • Item 16 The method according to Item 13 or 14, wherein the biological function is tested based on detecting cytoplasmatic mislocalization of the protein comprising the mutated RS domain amino acid sequence, the formation of cytoplasmatic granules of the protein comprising the mutated RS domain amino acid sequence, and/or the splicing activity of the protein comprising the mutated RS domain amino acid sequence.
  • a heart muscle cell or cardiomyocyte such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
  • a mammalian heart muscle cell or cardiomyocyte such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
  • iPSC-CMs induced pluripotent stem cell-derived cardiomyocytes
  • HeLa cells HEK293 cells
  • Item 17 The method according to any one of Items 8 or 13 to 16, wherein the compound is selected from the group consisting of natural compound, plant extract, a peptide, such as an artificial in silico designed peptide binder, a protein, a small molecule (less than about 500 Da), an RNA, an antibody or antigen binding fragment thereof, and an agent for use according to any one of claims 1 to 7, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof.
  • a peptide such as an artificial in silico designed peptide binder
  • a protein a small molecule (less than about 500 Da)
  • an RNA an antibody or antigen binding fragment thereof
  • Item 18 A method for producing a pharmaceutical composition, comprising performing a method according to any one of Items 8 to 17, and admixing the compound as identified with at least one pharmaceutically carrier.
  • Item 19 A compound as identified according to the method according to any one of Items 8 to 17, or a pharmaceutical composition as produced according to Item 18 for use in medicine, in particular for use in the prevention or treatment of a disease or condition in a cell of a subject that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising a mutated RS domain amino acid sequence.
  • Item 20 The compound or pharmaceutical composition for use according to Item 20, wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
  • a heart muscle cell or cardiomyocyte such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
  • Item 21 The compound or pharmaceutical composition for use according to Item 19 or 20, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • Item 22 A method for increasing the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreasing the amount of the protein in the cytoplasm of said cell, comprising providing to a cell an effective amount of the compound as identified according to the method according to any one of Items 8 to 17, or a pharmaceutical composition as produced according to Item 18, whereby the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell is increased.
  • Item 23 The method according to Item 22, wherein the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
  • a heart muscle cell or cardiomyocyte such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
  • Item 24 A method for preventing or treating a disease or condition related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of a protein comprising a mutated RS domain amino acid sequence in a subject in need of said prevention or treatment, comprising administering to the subject an effective amount of the compound as identified according to the method according to any one of Items 8 to 17, or a pharmaceutical composition as produced according to Item 18.
  • Item 25 The method according to Item 24, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
  • CM cardiomyopathy
  • HCM hypertrophic
  • DCM dilated cardiomyopathy
  • Figure 1 shows that the splice regulatory activity of RBM20 variants is proportional to their nuclear localization
  • iPSC-CMs normalized to GAPDH expression and displayed as a mean fold change of the RBM20-WT line (standard errors are indicated).
  • Three independent iPSC-CM differentiations were used as biological replicates, (e) Left ventricular ejection fraction (LVEF) for patients with P633L-RBM20 or with other pathogenic or likely pathogenic (P/LP) mutations in the RSRSP-stretch at the time of diagnosis, (f) Initial LVEF as a function of the age at presentation, (g) Internal Left Ventricular Diastolic Dimension (LVIDd) corrected for body size for analyzed patients, (h) ICS-sorting strategy for P633L-RBM20.
  • LVEF Left ventricular ejection fraction
  • P/LP pathogenic or likely pathogenic
  • Figure 2 shows that restoring nuclear localization of RS-domain RBM20 variants rescues their splicing function
  • Each bar shows a mean of three technical replicates, (c) iPSC-CMs with S635FS mutation transduced with eGFP-WT, eGFP-R634Q, or eGFP-NLS-R634Q, representative images, (d) Numbers of significantly (absolute value of log2FC > 1 and adjusted p-value ⁇ 0.1, Benjamini- Hochberg) differentially expressed genes in pairwise comparisons of S635FS iPSC-CMs expressing eGFP-WT vs eGFP-R634Q and eGFP-WT vs eGFP-NLS-R634Q.
  • PSI-values for differentially spliced exons of known RBM20 targets between eGFP-WT and eGFP-R634Q cells (delta PSI > 0.1, p-value ⁇ 0.05, t-test).
  • Figure 3 shows that genome-wide ICS screen identifies TNPO3 as the main nuclear transporter of RBM20.
  • Figure 4 shows that mislocalization of RS-domain RBM20 mutants is caused by loss of interaction with TNPO3.
  • Each dot represents a Pearson correlation coefficient R for at least five cells, data are combined from three biological replicates, (c) qPCR analysis of TIN exon 242 splicing in WT or P633L iPSC-CMs transfected with control siRNA or siRNA targeting TNPO3 normalized to GAPDH expression and displayed as mean fold changes versus the RBM20-WT line transfected with siRNA control with the standard errors, from two biological replicates with two technical replicates for each of them, (d) ICS-measured correlations between eGFP-RBM20 and DRAQ5 for cells expressing eGFP-WT-, -P633L-, -R634Q-, or - RSS-RBM20, transfected with control siRNA or siRNA targeting TNP03.
  • Each dot represents a Pearson correlation coefficient R for at least five cells, data are combined from three biological replicates, (f) Superimposed AlphaFold2 models of the RRM-RS domain of RBM20 (amino acid 511-673) as wild type (grey), or with single point mutations: P633L (light blue) or R634Q (salmon), (g) Representative AlphaFold2 model of RBM20’s RRM-RS wild type domain (blue, amino acid 511-673) in complex with TNPO3 (grey, full length, amino acid 1-923).
  • Ns not significant, * - p ⁇ 0.05, ** - p ⁇ 0.01, *** - p ⁇ 0.001, **** - p ⁇ 0.0001, Student’s t-test for (b), and one-way ANOVA with Tukey’s HSD post-test for (c), (e).
  • Figure 5 shows that enhancing RBM20-TNPO3 interaction restores nuclear localization and splicing in vitro and in vivo
  • Figure 6 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • RBM20 lower right part
  • RBM20 alpha fold predicted ribbon model of RBM20 with the same domain color code
  • Figure 6 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top).
  • the bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 1.
  • the interacting amino acids are not all conserved in mice, and therefore this binder may not bind to mouse RBM20.
  • Figure 7 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • Figure 8 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • Figure 9 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • RBM20 lower right part
  • RBM20 alpha fold predicted ribbon model of RBM20 with the same domain color code
  • Figure 9 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top).
  • the bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 4.
  • the interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20.
  • Figure 10 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • RBM20 lower right part
  • RBM20 alpha fold predicted ribbon model of RBM20 with the same domain color code
  • the bold dark green bars indicate the alpha helical parts
  • red boxes indicate the amino acids that were predicted by the model to interact with binder 5.
  • the interacting amino acids are not all conserved in mice, and therefore this binder may not bind to mouse RBM20.
  • Figure 11 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • RBM20 lower right part
  • RBM20 alpha fold predicted ribbon model of RBM20 with the same domain color code
  • the bold dark green bars indicate the alpha helical parts
  • red boxes indicate the amino acids that were predicted by the model to interact with binder 6.
  • the interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20.
  • Figure 12 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • RBM20 lower right part
  • RBM20 alpha fold predicted ribbon model of RBM20 with the same domain color code
  • Figure 12 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top).
  • the bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 7.
  • the interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20.
  • Figure 13 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • RBM20 lower right part
  • RBM20 alpha fold predicted ribbon model of RBM20 with the same domain color code
  • Figure 13 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part).
  • Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top).
  • the bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 8.
  • the interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20.
  • Figure 14 shows the quantification of RBM20:DAPI co-localization, measured by Pearson R correlation coefficient. Fiji plugin Coloc2 was used to quantify this parameter. Each dot represents at least one cell. At least 10 cells per genotype per treatment were quantified. The higher the RBM20:DAPI correlation is, the more the RBM20 signal is in the nucleus. It can be seen that in cells (both HeLa and HEK293FT) expressing the binder 2 ( Figure 7) (as shown by mScarlet expression), localization of RBM20-WT is not changed compared to non-expressing binder cells. However, the RBM20:DAPI correlation for RBM20-R634Q is significantly higher, and as high as observed for RBM20-WT, upon binder expression. **** - P ⁇ 0,0001, one-way ANOVA with Tukey’s post test. Ns - p > 0.05.
  • Figure 15 shows splicing activity of RBM20 WT or R634Q in cells with or without binder.
  • IMMT splicing one of the well-documented RBM20 targets, Koelemen et al., 2021
  • IMMT spliced and unspliced isoform expression was measured via qPCR, using primers targeting the alternative exon junctions (published in Kornienko et al., 2023).
  • expression of the binder restores the alternative splicing of IMMT to the levels observed in WT cells, in line with the restoration of nuclear localization showed in Figure 14.
  • Figure 16 shows qPCR analysis of in vivo Tnpo3 (panel a) and Tin spliced isoform (panel b) expression in the left ventricles of RBM20-WT, -P635L +/+, or -R636Q +/+ and +/- mice treated with either AAV9- pCAG-iCre-T2A-eGFP (Ctr) or AAV9-Tnpo3 (Tnpo3 ⁇ Data is normalized to Gapdh and displayed as fold change versus one of the RBM20-WT Ctr mice.
  • Each dot represents a mean of two technical replicates per mouse, (a) demonstrates an elevated level of Tnpo3 expression in the hearts of Tnpo3 -treated mice, (b) demonstrates that these elevated levels of Tnpo3 expression are sufficient to restore the alternative splicing of Ttn in P635L +/+ and R636Q +/- mice to the levels that are not statistically different from those observed in WT mice.
  • Figure 17 shows the nuclear localization of (mutated) RMB20 and the effect of a binder in iPSC-CMs. a) images of stainings of wild type and mutated RBM20. b) box plot of colocalization of DAPI and RBM20 stainings. The binder 2 ( Figure 7) causes RBM20 (mutated) to be localized in the nucleus.
  • SEQ ID NO: 1 shows the sequence of the NLS-sequence as used, PKKKRKVGGGS (SEQ ID NO: 1).
  • SEQ ID NOs 2 to 9 show the amino acid sequences of the L-rich region binding peptides.
  • the inventors showed that pathogenic RS-domain variants do not affect the splice regulatory activity, and that the splicing defect is only due to mislocalization of RBM20.
  • the inventors uncovered the molecular basis of RBM20’s nuclear transport, and demonstrate how RS-domain mutations disrupt this process.
  • the inventor’s findings have implications for the development of therapeutic strategies targeted at improving the nuclear import of mislocalized RBM20.
  • the inventors use an NLS nanobody (here an SV40 NLS tag) to stimulate the nuclear import of mutant RPM20 and to also restore proper splicing.
  • an eGFP-NLS nanobody is used to test nuclear import and restored splicing of an eGFP-R634Q and eGFP-P633L-RBM20.
  • RBM20 specific nanobodies are generated and the reliability of such nanobody -NLS fusions in iPSC-CMs with R634Q or P633L mutations is tested.
  • the RBM20-NLS delivery method in vivo is optimized by testing AAV9; AAVMAYO (cDNA delivery) or lipid nanoparticles (mRNA delivery). Also an optimization of the treatment dosage and duration in vivo is performed.
  • gene editing to introduce the endogenous NLS-tag is performed as follows
  • the gRNA sequence and genome positioning of the tag is optimized with prime editing in iPSC- CMs.
  • AAVMAYO or AAV9 are used for testing in vivo .
  • an optimization of the treatment dosage and duration in vivo is performed.
  • the inventors target the TNPO3-RBM20 interaction as identified as unique in the context of the present invention.
  • TNP03 gene delivery optimization of the delivery method is performed, testing AAV9, and AAVMAYO. Then an optimization of the treatment dosage and duration in vivo takes place, followed by an analysis of potential side effects and risks related to unwanted upregulation of transport of other TNP03 targets.
  • a kindred with four members carrying the P633L variant in RBM20 was identified through cascade screening. The first identification of this variant of uncertain significance was found in a proband with dilated cardiomyopathy and heart failure in the seventh decade of life. Given ascertainment bias associated with comparing variant carriers identified by family screening to probands who generally present with more severe disease, the inventors compared these four RBM20 P633L relatives to individuals with pathogenic and likely pathogenic variants in the RS-domain identified through family screening only (i.e., non-probands).
  • genotype-positive family members who were diagnosed after family screening from 9 contributing inherited cardiomyopathy centers (Stanford Center for Inherited Cardiovascular Disease, the University of Heidelberg, the University Hospital Zurich, Johns Hopkins University, Brigham and Women’s Hospital at Harvard Medical School, the University of Pennsylvania, the University of Michigan, the University of British Columbia, and Children’s Hospital Atlanta). Data on age at presentation and initial echocardiogram was collected retrospectively by chart review. The study was approved by the independent internal review board (IRB) at each site, and patient consent was obtained as required by each individual institution.
  • IRS independent internal review board
  • iPSCs Induced pluripotent stem cells
  • iPSC line used in this study was obtained from the Stanford Cardiovascular Institute Biobank, and single point mutations were engineered as described in [22] .
  • iPSCs were cultured in monolayer in cell culture dishes coated for 1 h at room temperature with Vitronectin (VTN- N) Recombinant Human Protein, Truncated (Gibco, A14700).
  • VTN- N Vitronectin
  • Truncated Truncated
  • Cells were cultivated in the E8- Flex medium (Gibco A2858501) and split twice per week using Versene solution (Gibco, 15040066). For splitting, cells were washed once with PBS and incubated with Versene for 5- 10 minutes at room temperature.
  • iPSCs were cultured as monolayer, and Wnt signaling was modulated as previously described 50 . Briefly, iPSCs were plated at low confluency on vitronectin-coated plates, to reach 70-80% 4 days post plating. On Day 0, the media was changed to RPMI 1640 (Gibco 21875034) + B27-insulin supplement (Gibco, A1895601) (RPMI+B27-ins) with addition of 4pM CHIR 99021 (LC Laboratories C-6556) in DMSO. On day 1, the medium from the day before was diluted by addition of the equal volume of RPMI+B27-ins.
  • HeLa Tet-Cas9 [51] and HEK293FT were maintained in DMEM, high glucose (Gibco, 11965084) supplemented with 10% FBS Supreme (Pan Biotech, P30- 3031), 1% Sodium Pyruvate (Gibco, 11360070), and 1% Penicillin-Streptomycin (Gibco, 15140122).
  • DMEM high glucose
  • FBS Supreme Pan Biotech, P30- 3031
  • 1% Sodium Pyruvate Gibco, 11360070
  • Penicillin-Streptomycin Gibco, 15140122
  • cells were washed 1 time with PBS, and incubated with Trypsin- EDTA (0.25%) (Gibco 25200056) for 5 minutes at 37°C. After that, cells were resuspended in fresh media, diluted to the desired concentration, and plated. Cry opreservation of cells was done in the culturing media supplemented with 10% DMSO
  • HEK293FT cells were grown to 80-90% confluency and transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific) with two lentiviral packaging plasmids (pMD2.G and psPAX2), and a plasmid carrying the gene of interest mixed at 1 :1 : 1 ratio to obtain in total 2.5 ug of DNA per well of a 6-well plate.
  • pMD2.G and psPAX2 two lentiviral packaging plasmids
  • pMD2.G and psPAX2 lentiviral packaging plasmids
  • a plasmid carrying the gene of interest mixed at 1 :1 : 1 ratio to obtain in total 2.5 ug of DNA per well of a 6-well plate.
  • cells were split in approximately 1 :6 ratio (one well of a 6-well plate into a 10 cm tissue culture dish), and cultured at 37°C.
  • the supernatant was collected, and remaining cell debris was filtered through the 0.45
  • TetO-eGFP-GGSG-NLS-Flag-RBM20 plasmid was cloned via site directed mutagenesis-based insertion of the SV40 NLS sequence into the TetO-eGFP-GGSG-FLAG-RBM20 plasmid using GeneArt Site Directed Mutagenesis Kit (Thermo Fisher Scientific). The latter was cloned via Gibson assembly of the eGFP-GSSG cDNA, Flag-RBM20 cDNA (GenScript), as well as the fragment of TetO-lenti backbone (gift from Dr.
  • HeLa Kyoto cells were co-transduced with TetO-eGFP- FLAG-NLS-RBM20-WT, -P633L, or -R634Q, as well as rtTA (Addgene 20342), cultured in the presence of 2 ug/ml of Doxycycline (Sigma) for at least 7 days, single cell sorted for eGFP fluorescence with FACS, and used for the experiment at least two weeks after the sort.
  • TetO-eGFP- FLAG-NLS-RBM20-WT, -P633L, or -R634Q as well as rtTA (Addgene 20342)
  • Doxycycline Sigma
  • pEFa-eGFP-GGSG-Flag-RBM20 plasmids were cloned via Gibson assembly (NEB) of the amplified TetO-backbone (see above), eGFP-GSSG-Flag-RBM20 cDNA from the TetO- plasmids (described above), and pEFa promoter sequence from the Addgene #125592 plasmid.
  • NEB Gibson assembly
  • HeLa Tet-Cas9 [51] were transduced with lentivirus delivering pEFa-eGFP-Flag-RBM20-WT, P633L, R634Q, or R634Q-S635E-S637E in 6-well plates by adding 20 pl of lOOx concentrated virus per well, and single-cell derived colonies were obtained by FACS. Two weeks after single cell sorting, the established lines were further analyzed for their purity with FACS and ICS, and the most stable and pure clonal lines were used for downstream applications.
  • HeLa Tet-Cas9 [51] cells were cultured until they reached a confluency of 20% in medium without Penicillin Streptomycin, and transfected using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) and 20 nM of either non-targeting control siRNA (D-001810-02), or siRNA targeting TNPO3 (L-019949-01) (Dharmacon, Horizon Discovery Group). Medium was replaced with the normal HeLa culture medium described above 24 hours later, and cells were harvested for downstream analyses 72 hours post transfection as described above.
  • iPSC-CMs were cultured at 70-90% confluency in RPMI+B27 media and transfected using LipofectamineStem reagent (Thermo Fisher Scientific) and 100 nM of either control siRNA or siRNA targeting TNPO3. Media was changed to fresh RPMI+B27 media 24 hours later. Four days post transfection, cells were transfected again in the same way, changing medium 24 hours afterwards. After 72 hours post the second transfection, cells were harvested for downstream analyses as described above.
  • eGFP-TNPO3 cDNA was purchased from Addgene (167590), and lentivirus was produced as described above.
  • iPSC-CMs were cultured at 70-90% confluency in RPMI+B27 media, and transduced with 1 : 1000 volume of the virus. Media was changed to a fresh RPMI+B2724 hours after transfection, and again, 3 days later. Seven days post transduction, cells were either fixed for microscopy analysis (see below), or FACS sorted in bulk into 1.5 ml reaction tubes for further RNA extraction (see below), to directly compare eGFP-TNPO3 positive and negative cells.
  • TetO-eGFP-FLAG-RBM20-WT, -R634Q, or NLS-R634Q were cloned and packaged into lentiviral particles, as described above.
  • iPSC-CMs with a frameshift mutation in RBM2(Ps RS- domain 22 were cultivated at 70-90% confluency in RPMI+B27 medium, and co-transduced with 1 : 1000 volume of the virus delivering TetO-RBM20, as well as rtTA (Addgene 20342). 24 hours later, the media was changed to a fresh RPMI+B27 with addition of 2 pg/ml of Doxycycline (Sigma). Medium was changed again 3 days later to the RPMI+B27 with Doxycycline, and after a total of 7 days post infection, cells were harvested for downstream analyses as described above.
  • DAPI Thermo Fisher Scientific
  • DRAQ5 Biostatus
  • iPSC-CMs were harvested with TrypLE Select Enzyme (10X) (Gibco A1217701) (see above), resuspended in the Passaging media (see above), washed once with PBS, and fixed with 4% PFA in PBS at room temperature (RT) for 10 minutes. Then, cells were washed once with PBS, and permeabilized with 0.1% Triton-XlOO (Merck) in 1% BSA (Sigma) in PBS for 5 minutes at RT.
  • TrypLE Select Enzyme (10X) Gibco A1217701
  • PFA room temperature
  • Cells were cultured either in glass bottom plates, or on coverslips, for microscopy analysis, following cell culture conditions described above. Cells were washed once with PBS, fixed with 4% PFA in PBS for 10 minutes at RT, washed once with PBS, and permeabilized with 0.5% Triton-XlOO (Merck) in PBS for 5 minutes at RT. Then, the potential nonspecific antibody binding sites were blocked by incubation with 2% BSA (Sigma) in PBS for 1 hour at RT.
  • BSA BSA
  • Microscopy plates were then washed once with PBS and stored at 4°C in the dark until imaged.
  • Coverslip were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and stored at 4°C in the dark until imaged.
  • cells were filtered through a 35 pM cell strainer to avoid clumping, and kept on ice until analyzed.
  • single-cell FACS sorting cells were sorted based on the desired fluorophore expression into 96-well plates containing culture media, one cell per well.
  • bulk FACS sorting cells were sorted into 1.5 ml microcentrifuge tubes containing DMEM with 10% FBS, based on the desired fluorophore expression.
  • BD FACS AriaTM Fusion was used, using a 100 pm sort nozzle. Viability staining with DAPI was used to sort out dying cells.
  • BD LSRFortessaTM was used for routine checking of transfection/transduction efficacies, as well as for optimizing experimental conditions.
  • Image-enabled cell sorting (ICS) used the BD CellViewTM Imaging Technology as previously described 5 . ICS experiments were performed with a 100 pm sort nozzle, with the piezoelectric transducer driven at 34 kHz, automated stream setup by BD FACSChorusTM Software, and a system pressure of 20 psi. All sorts were performed in purity mode.
  • GaAsP-PMT Gallium Arsenide Phosphid-PMT
  • Fiji (v.2.1.0/1.53c) plugin Coloc2 was used to reflect Pearson correlation coefficient R.
  • Z-stack images were max projected, and fluorescent channels were split. Area covering at least five cells was selected in RBM20 channel, and was used as ROI/mask for quantification of its correlation with DAPI channel.
  • RNA extraction and DNAsel treatment was performed using the Direct-zol RNA Miniprep Plus Kit (Zymo Research), according to the manufacturer's instructions.
  • iPSC-CMs (at least 5000 cells per sample) were pelleted by centrifugation at 500g for 3 minutes and resuspended in 16 pl of a 1 : 16 proteinase K in PKD buffer (Qiagen), incubated at RT for 5 minutes, briefly spun down, and incubated at 56°C for one hour. The solution was then resuspended in 100 pl of TRIzol LS Reagent (Thermo Fisher Scientific). Then, 20 pl of chloroform was added to the TRIzol-sample, and phase separation was achieved at RT by vigorous shaking and centrifugation at 12,500 r.p.m. for 5 min.
  • the SuperScript IV (Thermo Fisher Scientific) kit was used according to the manufacturer's instruction, with addition of 0.5 mM of each dNTP (NEB), 2.5 pM oligo-dT (Thermo Fisher Scientific), 1.25 pM random hexamer primers (Invitrogen), 5 mM DTT (Thermo Fisher Scientific), 2 u/pl RNAse inhibitor (Invitrogen), IX SSIV buffer (Thermo Fisher Scientific), and 10 u/pl SSIV RT (Thermo Fisher Scientific), per each reaction. At least 10 ng of total RNA was used per reaction, but not more than 1 pg.
  • a one-step qPCR reaction (95°C for 10 minutes, 40 cycles of [95°C 15 seconds, 60°C 1 minute]) was performed using SYBR Green Master Mix (Thermo Fisher Scientific), and primers listed in Supplementary Table 2, using Applied Biosystems StepOnePlus Real-Time PCR System (272006365), and StepOne Software v2.3. Delta-delta cT values were quantified versus GAPDH as a housekeeping gene, and versus a control sample for each experiment.
  • RNA quality was checked using the 2100 RNA pico Bioanalyzer (Agilent) kit, and 1-10 ng of RNA were used as input.
  • RNA sequencing libraries from ultra low-input and highly degraded RNA (RIN 1- 7) extracted from fixed samples (see above), SMARTer RNA-Seq Kit v3 - pico (Takara Bio) was used, according to the manufacturer's instructions. The fragmentation step was omitted.
  • Prepared libraries with unique dual index barcodes for each sample were double checked on Bioanalyzer (Agilent), and pooled together at equimolar concentrations, with six libraries per pool. Each pool was sequenced individually, with final concentrations of 8-10ng/ml for each pool.
  • PSI-values percentage of spliced-in values
  • HEK293 cells were seeded on 96-well plates and transfected at 50% confluence with PEI40 at a 1 :3 ratio (DNA: PEI40) and a total of 200 ng of plasmid DNA (1 ng splice reporter TTN-IG Ex241-243 and a 20x molar excess of the RBM20 expression plasmids (compare [57] for WT, mutations were introduced by site-directed mutagenesis in a two-step cycle PCR approach) or control plasmid pcDNA3.1 (Invitrogen, Cat# V79520).
  • the genome-wide CRISPR/Cas9 gRNA libraries were designed as described in [5], Briefly, the library targets 18,408 protein-coding genes listed in the Consensus Coding Sequence Database [58], It consists of six independent sub-libraries, each containing one gRNA per gene [5], Each of these sub-libraries contains the same 118 targeting and 487 nontargeting controls.
  • the library was cloned into the CROPSeq-guide(F+E)-Puro backbone (see sequences), as described in [5], gRNA representation of the genome-wide library at the plasmid stage was checked previously [5], and used as a plasmid stock for the lentivirus generation for this work. These data were also used as a reference for gRNA representation at the plasmid stage for the downstream data processing (see below).
  • gRNAs were synthesized as two short oligos with flanking sequences resembling the Esp3I sticky ends of CROPSeq-guide(F+E)-Puro vector.
  • the oligos (10 mM each) were phosphorylated and annealed using 1 U/pl T4 PNK (NEB), and IX T4-ligase buffer (NEB) in a thermocycler with the following program: 37°C 30 min, 65°C 20 min, 95°C 5 min, ramp down to 25°C at 5°C/min.
  • the phosphorylated and annealed oligos (1 pl) were then ligated with 25 ng of Esp3I-digested (NEB) and gel-extracted (Qiagen) CROPSeq-guide(F+E)-Puro backbone using 1 pl of T4 ligase (NEB), and IX T4 ligase buffer (NEB) in total volume of 10 pl, for 10 minutes at RT, inactivated for 10 min at 65 °C followed by transformation into the NEB Stable competent E. coli (NEB), according to the manufacturer's instructions. Lentivirus for cell transductions was produced as described above.
  • HeLa Tet-Cas9 pEFa-eGFP-RBM20-WT were plated with a density of 750 000 cells per 15 cm tissue culture dish, and cultured for three days until a confluency of 40% was reached (6,000,000 cells per 15 cm dish).
  • 60xl0 6 of cells were transduced per each genome-wide library (ten 15-cm dishes per library).
  • Cells were infected with lentivirus delivering the genome-wide library, with 25 l of 100X concentrated virus per plate, at a low infectivity rate to allow only single qRNA integrations per cell.
  • the culturing medium containing only Puromycin was used until cells were harvested for ICS.
  • cells infected with the same library from all plates were pooled together after trypsinization, and 1,500,000 of cells were plated per each new of seven 15-cm dishes, keeping the coverage at >500X for each individual genome-wide library.
  • 1,500,000 of cells were plated to fifteen 15-cm dishes for each library, and all of them were used for sorting.
  • cells were cultured in 6-well plates until 40% confluency (150,000 cells per well), and 10 pl of 50X concentrated virus were added per each well.
  • cells were trypsinized, resuspended in the medium containing 2 ng/pl Puromycin (Thermo Fisher Scientific), and plated back to the same wells.
  • the medium was changed to a fresh Puromycin-containing medium.
  • cells Once cells reached 90-100% confluency, they were split and cultured in medium containing 2 ng/pl Puromycin (Thermo Fisher Scientific) and 2 pg/ml Doxycycline (Sigma) for the first seven days, followed by seven days of only Puromycin-containing medium. Cells were split twice per week. At least 16 days post transduction, cells were harvested for downstream analyses as described above.
  • Cells were prepared as described above using lentiviral transduction. Samples were kept at 4°C at all times between harvest and genomic DNA isolation after sorting. Sorting was performed as described before 5 with the following modifications. Cells were sorted in batches of 100,000 cells in the collection fractions and the input samples were refreshed regularly by the addition of concentrated cell suspension to a total volume of 1 ml. For the selection of the populations from the eGFP-DRAQ5 correlation parameter, ranged gates were drawn comprising the 7% of cells with the lowest or highest correlation index. From each batch of cells used for sorting, an input sample containing the same number of cells as present in the sorted upper and lower sample was collected.
  • Sorted samples and input samples were collected by centrifugation for 5 min at 500g at 4 °C, and pellets were either frozen at -20 °C or stored on ice until gDNA preparation.
  • One million cells were collected per library and pooled into a single tube for gDNA preparation.
  • Genomic DNA was isolated from the sorted cells using NEB Monarch genomic DNA purification kit (New England Biolabs), including the RNase treatment and elution in 50 pl elution buffer. DNA concentration was measured using Qubit High sensitivity dsDNA kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
  • PCR1 was done with 125-525 ng of gDNA (per reaction, 6 reactions per library), 1.5 pl of 10 pM pU6 fwd, 1.5 pl of 10 pM pLTR-CROP-rev, and 25 pl KAPA HiFi Hotstart Readymix (Roche) in 50 pl total volume.
  • gRNA sublibrary six 50 pl reactions were set up using the total amount of gDNA recovered from sorted cells and the input samples. Cycling conditions for PCR1 were one cycle at 95 °C for 3 min; 24 cycles at [98 °C for 20 s, 67 °C for 15 s, 72 °C for 15 s]; one cycle at 72 °C for 1 min; and cooling to 4 °C.
  • PCR reactions of the same template were pooled (six PCR products into one of total volume 300 pl) and the product was purified with 0.8X volume of AMPure XP (Beckman) with two 80 % ethanol washes, and elution in 40 pl water. Concentrations were then measured with Qubit High sensitivity dsDNA kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
  • PCR2 was done with 10 ng PCR1 product (per reaction, 6 reactions per library), 5 pl of 3 pM CROPseq_libQC_i5_s:n staggered primer 59 (different primer for each reaction for one sample), 5 pl of 3 pM CROPseq_i7:n barcoded primer (same for all reactions for one sample, but unique to every sample), and 25 pl KAPA HiFi Hotstart Readymix (Roche) in 50 pl total volume. Same primers were used as in 5 , and are shown in Supplementary Table 4.
  • Cycling conditions for PCR2 were one cycle at 95 °C for 3 min; 8 cycles at [98 °C for 20 s, 67 °C for 15 s, 72 °C for 15 s]; one cycle at 72 °C for 1 min; and cooling to 4 °C.
  • Product was purified as above and eluted in 40 pl H2O. Concentrations were then measured with Qubit High sensitivity dsDNA kit (Thermo Fisher Scientific), and ready libraries were checked using DNA 1000 Bioanalyzer (Agilent) to yield a single product around 300bp.
  • NP-40 lysis buffer 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% (v/v) NP-40. All protein extraction buffers contain PhosSTOP (Sigma-Aldrich, 04906837001) and Protease Inhibitor Cocktail (Sigma-Aldrich, 05056489001).
  • Cell fractionation was performed as follows. First, cells were resuspended in 2 pellet volumes of hypotonic buffer (10 mM HEPES pH 7.5, 10 mM KC1, 1.5 mM MgCU) incubated on ice for 15 min and homogenized with 20 strokes using a loose pestle.
  • hypotonic buffer 10 mM HEPES pH 7.5, 10 mM KC1, 1.5 mM MgCU
  • Nuclei and insoluble cellular compartments were pelleted at 3,900 rpm for 15 min and supernatant collected as soluble cytoplasmic fraction, which was corrected to 10% (v/v) glycerol, 3 mM EDTA, 0.05% (v/v) NP-40 and 150 mM NaCl final concentration.
  • the remaining pellet was resuspended in chromatin digestion buffer (20 mM HEPES pH 7.9, 1.5 mM MgCh, 10% (v/v) glycerol, 150 mM NaCl, 0.1% (v/v) NP-40 and 125 U benzonase (MerckMillipore, 70746-4) and incubated for 1 h at 4°C.
  • NaCl concentration was then increased to 500 mM and samples were incubated on ice for 30 minutes. Prior to centrifugation at 20,000 g for 20 min at 4°C, the salt concentration was diluted back to 150 mM NaCl by addition of high salt dilution buffer (20 mM HEPES pH 7.9, 3 mM EDTA, 1.5 mM MgCl 2 , 10% (v/v) glycerol, 500 mM NaCl and 0.1% (v/v) NP-40) and the supernatant was kept as nuclear and insoluble fraction.
  • high salt dilution buffer (20 mM HEPES pH 7.9, 3 mM EDTA, 1.5 mM MgCl 2 , 10% (v/v) glycerol, 500 mM NaCl and 0.1% (v/v) NP-40
  • GFP immunoprecipitations 1 mg of the whole cell extracts, cytoplasmic fraction or nuclear and insoluble fractions were incubated with 30 pl of GFP-Trap® Magnetic Particles M-270 (ChromoTek) for at 4°C for 3 h.
  • IP wash buffer 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 1.5 mM MgCl 2 , 3mM EDTA, 10% (v/v) glycerol, 0.1% (v/v) NP-40, phosphatase inhibitors (PhosSTOP, Sigma-Aldrich, 04906837001) and protease inhibitor cocktail (Sigma-Aldrich, 05056489001) and eluted in 30 pl of laemmli buffer with 100 pM DTT.
  • IP wash buffer 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 1.5 mM MgCl 2 , 3mM EDTA, 10% (v/v) glycerol, 0.1% (v/v) NP-40, phosphatase inhibitors (PhosSTOP, Sigma-Aldrich, 04906837001) and protease inhibitor cocktail (Sigma-Aldrich, 05056489001) and
  • Samples were subjected to an in-solution tryptic digest using a modified version of the SinglePot Solid-Phase-enhanced Sample Preparation (SP3) protocol (PMID: 25358341, PMID: 29565595). Eluates were added to Sera-Mag Beads (Thermo Scientific, #4515-2105-050250, 6515-2105-050250) in 10 pl 15% formic acid and 30 pl of ethanol. Binding of proteins was achieved by shaking for 15 min at room temperature. SDS was removed by four subsequent washes with 200 pl of 70% ethanol.
  • SP3 SinglePot Solid-Phase-enhanced Sample Preparation
  • Proteins were digested overnight at room temperature with 0.4 pg of sequencing grade modified trypsin (Promega, #V5111) in 40 pl Hepes/NaOH, pH 8.4 in the presence of 1.25 mM TCEP and 5 mM chloroacetamide (Sigma- Aldrich, #C0267). Beads were separated, washed with 10 pl of an aqueous solution of 2% DMSO and the combined eluates were dried down.
  • TMT6plex Form inputs, Thermo Scientific, #90066
  • TMTpro Form eluates, Thermo Scientific, #A44522
  • TMT reagent was quenched by the addition of 4 pl of an aqueous 5% hydroxylamine solution (Sigma, 438227).
  • Peptides were reconstituted in 0.1 % formic acid, mixed to achieve a 1 : 1 ratio across all TMT-channels and purified by a reverse phase clean-up step (OASIS HLB 96-well pElution Plate, Waters #186001828BA).
  • the peptides were loaded onto the trap column at 30 pl per min using solvent A (0.1% formic acid) and eluted using a gradient from 2 to 40% Solvent B (0.1% formic acid in acetonitrile) over 2 h at 0.3 pl per min (all solvents were of LC-MS grade).
  • the Orbitrap Fusion Lumos was operated in positive ion mode with a spray voltage of 2.4 kV and capillary temperature of 275 °C.
  • Full scan MS spectra with a mass range of 375-1500 m/z were acquired in profile mode using a resolution of 120,000 (maximum fill time of 50 ms or a maximum of 4e5 ions (AGC) and a RF lens setting of 30%.
  • Fragmentation was triggered for 3 s cycle time for peptide like features with charge states of 2-7 on the MS scan (data-dependent acquisition). Precursors were isolated using the quadrupole with a window of 0.7 m/z and fragmented with a normalized collision energy of 38. Fragment mass spectra were acquired in profile mode and a resolution of 30,000 in profile mode. Maximum fill time was set to 64 ms or an AGC target of le5 ions). The dynamic exclusion was set to 45 s.
  • Peptides were subjected to an off-line fractionation under high pH conditions (PMID: 25358341). The resulting 12 fractions were then analyzed on a QExactive plus. Peptides were separated on an UltiMate 3000 RSLC nano LC system (Dionex) fitted with a trapping cartridge (p-Precolumn C18 PepMap 100, 5pm, 300 pm i.d. x 5 mm, 100 A) and an analytical column (nanoEaseTM M/Z HSS T3 column 75 pm x 250 mm C18, 1.8 pm, 100 A, Waters).
  • Trapping was carried out with a constant flow of trapping solution (0.05% trifluoroacetic acid in water) at 30 pL/min onto the trapping column for 6 minutes. Subsequently, peptides were eluted via the analytical column running solvent A (3% DMSO, 0.1% formic acid in water) with a constant flow of 0.3 pL/min, with increasing percentage of solvent B (3%DMSO, 0.1% formic acid in acetonitrile). The outlet of the analytical column was coupled directly to an Orbitrap QExactiveTM plus Mass Spectrometer (Thermo Fisher Scientific) using the Nanospray FlexTM ion source in positive ion mode.
  • solvent A 3% DMSO, 0.1% formic acid in water
  • solvent B 3%DMSO, 0.1% formic acid in acetonitrile
  • the peptides were introduced into the QExactive plus via a Pico-Tip Emitter 360 pm OD x 20 pm ID; 10 pm tip (CoAnn Technologies) and an applied spray voltage of 2.2 kV.
  • the capillary temperature was set at 275°C.
  • Full mass scan was acquired with mass range 375-1200 m/z in profile mode with resolution of 70000.
  • the filling time was set at maximum of 100 ms with a limitation of 3x106 ions.
  • Data dependent acquisition (DDA) was performed with the resolution of the Orbitrap set to 17500, with a fill time of 50 ms and a limitation of 2xl0 5 ions. A normalized collision energy of 32 was applied. Dynamic exclusion time of 20 s was used.
  • the peptide match algorithm was set to ‘preferred’ and charge exclusion ‘unassigned’, charge states 1, 5 - 8 were excluded.
  • MS2 data was acquired in profile mode
  • the replicate information was added as a factor in the design matrix given as an argument to the ‘ImFit’ function of limma. Also, imputed values were given a weight of 0.05 in the ‘ImFit’ function.
  • a protein was annotated as a hit with a false discovery rate (fdr) smaller 0.05 and a fold-change of at least 100 % and as a candidate with an fdr below 0.02 and a fold-change of at least 50 %.
  • fdr false discovery rate
  • Raw TMT reporter ion intensities (‘signal sum’ columns) were first cleaned for batch effects using limma (PMID: 25605792) and further normalized using vsn (variance stabilization normalization - PMID: 12169536). Proteins were tested for differential expression using the limma package. The replicate information was added as a factor in the design matrix given as an argument to the ‘ImFit’ function of limma. Also, imputed values were given a weight of 0.05 in the ‘ImFit’ function. A protein was annotated as a hit with a false discovery rate (fdr) smaller 0.05 and a fold-change of at least 100 % and as a candidate with an fdr below 0.02 and a foldchange of at least 50 %.
  • fdr false discovery rate
  • the inventors employed AlphaFold2 (AF2) [35, 36] within the JupyterHub on the EMBL Hamburg HYDE cluster.
  • AF2 AlphaFold2
  • the default settings of AF2 in Multimer mode (v2.2.2) were used with three recycling rounds per model, with enabled amber relaxation and a total of 5 predictions per model. This resulted in a total of twenty-five predictions per AlphaFold2 run.
  • the predictions were performed with the full-length amino acid sequence of the canonical TNPO3 sequence (amino acid 1-923 of Q9Y5L0, NM_012470.4) and with full-length RBM20 (amino acid 1- 1227 of Q5T481, NM_001134363.3) or only with amino acid sequence 511-673 to predict the RRM-RS domain.
  • RBM20-WT or -P635L-Hom strains in a C57BL/6J genetic background were used for experimental procedures.
  • the animals were maintained in individually ventilated plastic cages (Tecniplast) in an air-conditioned (temperature 22 °C ⁇ 2 °C, humidity 50% ⁇ 10%) and light- controlled room (illuminated from 07:00 to 19:00 h).
  • Mice were fed 1318 P autoclavable diet (Altromin, Germany) ad libitum. All animal care and procedures performed in this study conformed to the EMBL Guidelines for the Use of Animals in Experiments and were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • AAV9 production pCMV-Tnpo3 was cloned into the AAV9 packaging backbone (derived from Addgene 137177, gift from the Genetic and Viral core facility, EMBL Rome, Italy) by digesting the backbone with Bglll and Agel (NEB), and amplifying the murine Tnpo3 cDNA (GenScript), followed by Gibson assembly of the two fragments (NEB).
  • AAV9 packaging backbone derived from Addgene 137177, gift from the Genetic and Viral core facility, EMBL Rome, Italy
  • the serotype 9 rAAV containing pCMV-Tnpo3 cDNA was produced in HEK-293T/17 cells using the triple-transfection method with linear PEI (25 kDa) in a Corning Hyperflask. After 72 hours, the cells were lysed and DNA was degraded by adding Triton X-100 (final concentration of 1%) and 19 pl Bensonase (25-35 U/pl) for Bit at 37°C with 200 rpm shaking 62 . The cell debris/virus mix was removed and the Hyperflask was washed with 200 ml, sterile IxPBS. The washing solution and the cell suspension were centrifuged at 4000 xg for 20 min.
  • the supernatant was filtered with a 0.45 pm PES filter and then concentrated to a total volume of 30 ml using tangential flow filtration [62], The concentrated virus was then purified by standard methods with an iodixanol gradient.
  • the 200pl final volume of virus in PBS with 0.001% pluronic F-68 was aliquoted and the titer (3.3xl0 13 ) was determined by qPCR using primers within the CMV promoter.
  • Microscopy slides were coated with 10 pl/ml of laminin (Gibco, 23017015) in PBS overnight.
  • EDTA buffer 130 mM NaCl, 5 mM KC1, 0.5 mM NaH2PC>4, 10 mM HEPES, 10 mM Glucose, 10 mM 2,3 Butanedione monoxime, 10 mM Taurine, 5 mM EDTA
  • Perfusion buffer 130 mM NaCl, 5 mM KC1, 0.5 mM NaH 2 PO 4 , 10 mM HEPES, 10 mM Glucose, 10 mM 2,3 Butanedione monoxime, 10 mM Taurine, 1 mM MgCl 2
  • Collagenase buffer Perfusion buffer, 1,5 mg/ml Collagenase II (Gibco, 17101015), 1,5 mg/ml Collagenase IV (Gibco, 17104019), 0,15 mg/
  • the Collagenase buffer was pre-warmed to 37°C prior usage. Mice were anesthetized in a CO 2 chamber, opened up, and the descending aorta and vena cava were both cut. Then, 7 ml of the EDTA buffer were injected steadily for about one minute into the basis of the right ventricle, after which the ascending aorta was clamped. The heart was removed and transferred to a 60- mm dish containing 10 ml of the EDTA buffer. A syringe was used to push 10 ml of EDTA buffer through the left ventricle steadily for approximately 2 minutes.
  • the heart was then transferred to a 60-mm dish containing 10 ml of the Perfusion buffer, and the left ventricle was steadily injected with 10 ml of the Perfusion buffer to flush the remaining EDTA.
  • the heart was transferred to a 60-mm dish containing 10 ml of the Collagenase buffer, and the left ventricle was steadily perfused five to six times with 10 ml of Collagenase buffer.
  • the heart was cut into the desired regions. After a small piece of the left ventricle was saved and snap frozen to be further used for RNA extraction (see above), the rest of the left ventricle was transferred to the dish containing 3 ml Collagenase buffer for CM isolation.
  • the tissue was teared apart into 1 mm x 1 mm pieces and pipetted up and down for about 5 minutes to dissociate the cells, after which the collagenase reaction was stopped by adding 5 ml of the Stop solution.
  • the cells were filtered through a 100 pm filter and pelleted by gravity for about 20 minutes, after which the pellet was gently resuspended in Perfusion buffer and the filtering procedure was repeated one more time.
  • the pellet was then resuspended in pre-warmed DMEM media containing 10% FBS Supreme (Pan Biotech, P30-303), plated onto the pre-coated microscopy slides, and let in the cell culture incubator for three hours. Slides were then washed twice with PBS, followed by fixation and staining protocols described above.
  • RBM20 variants Splice regulatory activity of RBM20 variants is proportional to their nuclear localization
  • the inventors analyzed the localization of P633L- and R634Q-RBM20 variants that the inventors engineered before [22] in iPSC-CMs by endogenous immunofluorescence (IF) followed by confocal microscopy (Fig. 1 a, b) or ICS (Fig. 1c).
  • IF endogenous immunofluorescence
  • Fig. 1 a, b confocal microscopy
  • ICS Fig. 1c
  • the inventors collected data from patients with pathogenic or likely pathogenic (P/LP) variants in the RSRSP-stretch of RBM20 who were identified by cascade family screening.
  • the inventors compared patients with the P633L variant to the rest of the cohort (Fig. le-g).
  • ventricular remodeling a characteristic of RBM20-DCM, was less severe [14]
  • LVEF Left ventricular ejection fraction
  • LVIDd Internal Left Ventricular Diastolic Dimension
  • BSA body surface area
  • the mixed phenotype (nuclear and cytoplasmic) of the P633L-RBM20 variant allowed the inventors to differentiate between the consequences of nuclear and cytoplasmic RBM20 localization in the same genetic background. To that end, the inventors compared gene expression, alternative splicing, and protein interactor changes between differentially-localized P633L-RBM20, WT-RBM20, and R634Q-RBM20.
  • ICS Using ICS, the inventors sorted iPSC-CMs with differentially-localized RBM20 based on correlation with nuclear staining (Fig. Ih). This was followed by RNA-sequencing of the sorted populations.
  • the inventors identified 1,415 differentially expressed genes in P633L- cytoplasmic (P633L-cyt) compared to WT (Fig. li). Surprisingly, there were only 50 differentially expressed genes between P633L-nuclear (P633L-nuc) and WT (Fig. li). Down- regulated genes in both P633L-cyt and R634Q impacted the cardiac-related processes.
  • PSI spliced-in
  • the inventors investigated whether the protein binding partners of nuclear or cytoplasmic P633L-RBM20 match the nuclear WT or the cytoplasmic R634Q variant respectively.
  • the inventors performed mass-spectrometry analysis of the interactors that co-immunoprecipitated with WT-, P633L-, and R634Q-RBM20 in the nuclear or cytoplasmic fraction in HeLa cells (see methods).
  • the inventors found that the majority of interactors are common for WT and variant RBM20 in the nucleus (Fig. 11).
  • the inventors also observed restored splicing of endogenous IMMT, an RBM20 target gene, in cells expressing NLS-tagged P633L and R634Q.
  • addition of an NLS to the V914A variant which resides outside the RS-domain and does not mislocalize to the cytoplasm [29], had no effect on splicing.
  • RNA-Seq analysis showed that the expression of 1,751 genes was altered in cells expressing R634Q-RBM20 compared to WT (Fig. 2d). The genes were consistent with severe cardiac impairment (Supplementary Fig.
  • Genome-wide ICS screen identifies TNP03 as the major transporter of RBM20
  • the inventors performed a genome-wide CRISPR-Cas9 screen applying ICS technology [5] (Fig. 3a).
  • the inventors transfected HeLa cells expressing Tet-Cas9 and eGFP-RBM20-WT with a guide RNA library targeting 18,408 protein-coding genes with six gRNAs per gene.
  • the inventors collected the 7% of cells exhibiting the most cytoplasmic (lower fraction) or the most nuclear (higher fraction) eGFP-RBM20 signal, as well as the unsorted input sample (input).
  • the inventors identified only one gene enriched in the lower fraction (TNPO3, positive regulator of RBM20 import), while 56 genes were enriched in the upper fraction (negative regulators) (FDR ⁇ 0.01) (Fig. 3b, c). Since WT-RBM20 localization is solely nuclear, the inventors assumed that negative regulators - gene knockouts that induce a stronger nuclear translocation of RBM20 - are not of direct relevance for the scope of this study. In addition, based on previous experiments [5], cellular shape amongst other parameters can influence the sorting decision of ICS. Therefore, the inventors discarded all negative regulators for further validation.
  • the inventors selected other hits with less significant FDR scores (CLDN14, GALE, ADAMTS16, SLC29A2, CEBPB, UBQLNL, TRIM33, PMM2, TRIM24, IPPK, XPO6) and based on prior knowledge about their relevance for RBM20 biogenesis [18, 32, 33] (TTN, AKT2, SPRK1, CLK1, LMNA).
  • TNPO3 Transportin-3, transportin-SR
  • TNPO3 was also one of the only two proteins the inventors identified by mass- spectrometry as interacting specifically with WT-RBM20 and losing this interaction with mutants (Fig. 11).
  • the inventors did not detect CHD1 as a positive regulator in the ICS screen.
  • TNPO3 is known to specifically recognize and transport other RS-domain containing proteins in humans. Together, these results show that TNPO3 is the essential nuclear transporter ofRBM20.
  • KD siRNA knock-down
  • RSS siRNA knock-down
  • TNP03 KD significantly decreased nuclear localization of WT, P633L, and even R634Q-RBM20, as measured by ICS (Fig. 4d) and confocal microscopy (Fig. 4e). RSS localization did not change. These data show that TNPO3 is responsible for localizing mutant RBM20 protein to the nucleus, and its effectiveness is mutation-dependent.
  • the inventors used AlphaFold2 [35, 36] to predict the complex.
  • the inventors observed no structural rearrangements within the intrinsically disordered RS-domain in the AlphaFold2 models of the RRM-RS (amino acid 511-673) domain from WT-, P633L- or R634Q-RBM20 proteins (Fig. 4f).
  • the predicted complex of TNPO3 and RBM20’s RRM-RS domain indicated that the PRSRSP region is classified as interaction interface residues in all obtained models (Fig. 4g). This was also the case for the prediction of TNPO3 in complex with full-length RBM20.
  • TNP03 KD resulted in the accumulation of cytoplasmic granules of RBM20-WT in HeLa cells and iPSC-CMs, indicating that granule formation is not specific to the RBM20 mutant proteins.
  • This finding is in agreement with the inventor’s obtained AlphaFold models that showed no structural rearrangements within the RRM-RS domain of WT- or mutant-RBM20 (Fig. 4f).
  • the present study and previous work [31] have identified MOVIO as one of the main interactors of mutant RBM20 in the cytoplasm (Fig. 4j).
  • the inventors analyzed RBM20-WT interaction with MOVIO in the cytoplasm upon TNP03 KD.
  • the inventors observed a significant gain of interaction between WT RBM20 and MOVIO upon loss of TNPO3.
  • the inventors observed partial colocalization of MOVIO and RBM20-WT upon TNP03 KD by confocal microscopy, similar to the mutant variants. This concludes that loss of interaction with TNPO3 results in formation of RBM20 RNP granules, regardless of the mutation present.
  • Enhancing RBM20-TNPO3 interaction restores nuclear localization and splicing in vitro and in vivo
  • the inventors delivered Tnpo3 cDNA via AAV9 to mouse hearts bearing homozygous P63 SERB M20 (P635L +/+ ) mutations (P633L in humans).
  • the inventors analyzed RBM20 localization and splicing function four weeks after AAV9 injection (Fig. 5e). Strikingly, increasing Tnpo3 levels (Fig. 5f) resulted in partial rescue of RBM20 localization (Fig. 5g) and Ttn alternative splicing (Fig. 5h, i) independently of Rbm20 expression.
  • the inventors results show that increasing TNPO3 expression restores splicing and mislocalization of RS-domain mutant RBM20. Moreover, the inventors provide first proof- of-principle that this strategy can serve as a promising therapeutic avenue for developing future therapies for RBM20-mediated DCM.
  • the L-rich region and an alpha-helix in the RS-rich region of RBM20 were chosen. Since the binders will presumably remain bound to RBM20, binding to a domain being involved in crucial functionalities of RBM20 was avoided. Both the L-rich or the helix within the RS-rich region are furthermore highly conserved in eukaryotes, and therefore good targets to generate a universal binder that can be used both in mouse and human DCM model systems.
  • the inventors started with the L-rich region as a target.
  • the structured alpha helical part of the L rich region shows only three amino acids that are not conserved between human and mouse ( Figure 6, blue amino acids below dark green bars).
  • the inventors anticipate antigen binders, such as nanobodies, to function with this model, however, artificial binders were pursued as easier to synthetize and to produce, and not requiring animal immunizations or long phage display screenings.
  • the amino acid sequence of the 13 selected binders was translated into a DNA sequence for humans (codon optimized) and cloned into a plasmid for transient transfection into human cells.
  • Two NLS sequences were attached to the N-terminus of the binder, SV40 NLS and/or myc NLS. The cloning of 8 binders was successful, and these binders were tested further for their activity to cause RBM20 to be localized in the nucleus.
  • Binder 1
  • Binder 2 (preferred):
  • Binder 3 APTLEQLAHDAQLAALRAQLAALEAERAALRAAAAAEGDALDEQLLAALEAQLAA LRAQLAALEAAR (SEQ ID NO: 4)
  • AAA (SEQ ID NO: 5)
  • iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy.
  • Rbm20 regulates titin alternative splicing as a splicing repressor.

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Abstract

The present invention relates to agents that increase the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the same protein in the cytoplasm of said cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence. In particular, the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNPO3) or an ortholog thereof. Preferably, the disease or condition is myopathy, in particular dilated cardiomyopathy (DCM). The present invention further relates to a method for identifying suitable agents used to increase the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or to decrease the amount of the same protein in the cytoplasm of said cell.

Description

Methods of preventing or treating cardiomyopathy by redirecting mislocalized pathogenic RBM20 protein variants
The present invention relates to agents that increase the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the same protein in the cytoplasm of said cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence. In particular, the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNPO3) or an ortholog thereof. Preferably, the disease or condition is myopathy, in particular dilated cardiomyopathy (DCM). The present invention further relates to a method for identifying suitable agents used to increase the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or to decrease the amount of the same protein in the cytoplasm of said cell.
Background of the invention
Correct protein localization is fundamentally based on the recognition of a targeting signal within the nascent protein by a targeting factor for the destination organelle. Gene variants impairing these targeting signals often result in severe diseases. For example, cytoplasmic mislocalization of p53 leads to various types of cancer, and cytoplasmic mislocalization of TDP-43 is associated with Amyotrophic Lateral Sclerosis. Details about the transport mechanisms involved are required for developing targeted therapies and are still elusive for many cases.
Dilated cardiomyopathy (DCM) is a heart condition characterized by enlargement of the cardiac left ventricle and systolic dysfunction. It is a highly prevalent disease affecting 1 in 250-500 individuals and eventually leading to heart failure or sudden cardiac death [1, 2], Besides heart failure therapy, targeted approaches are largely lacking in clinics [3], and the only available curative treatment is heart transplantation. About half of DCM cases are familial with primarily autosomal dominant inheritance [4], Variants of several genes have been classified with high confidence as DCM-causing in humans [5, 6], including those in the RNA binding motif protein 20 RBM20). RBM20 variants can result in a particularly severe form of the disease often causing arrhythmia and progressive heart failure, and account for about 3% of familial DCM cases [7-10], Current guidelines for RBM20 patients suggest evaluation for primary prophylactic placement of implantable cardioverter defibrillators based on individual predicted risk [9-11], RBM20 is predominantly expressed in the heart, and is involved in regulation of tissue-specific alternative splicing [12], Among its targets are genes involved in sarcomere structure (e.g. TTN), mitochondrial function (e.g. IMMT), calcium handling (e.g. RYR2 and ion channels (e.g. CACNA1C) [13-15], The majority of DCM-causing RBM20 variants are heterozygous missense mutations, many of which cluster in a conserved stretch of six amino acids PRSRSP (amino acid position 633 - 638) in the protein’s arginine/serine (RS)-rich domain [7, 13, 16, 17],
Heterozygous mutations in RBM20 result in haploinsufficiency with respect to transcriptional splicing [13, 14, 17, 18], where alternative splicing of RBM20’s targets is proportional to the amount of wild-type (WT) versus mutated RBM20 expressed [19], The inventors previously demonstrated that a compound up-regulating RBM20 expression alleviates the disease phenotype in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) [17], However, recent studies suggested that some RBM20 variants display dominant-negative effects related to mislocalization of the mutated protein outside the nucleus. In a porcine DCM model, as well as in patient-derived iPSC-CMs harboring the R636S mutation, RBM20 mislocalized to the cytoplasm and formed potentially-detrimental RNP -granules [19], In a murine DCM model, the S637A mutation (S635A in humans) caused similar mislocalization, lower survival and higher levels of fibrosis compared to an Rbm20 KO [20], Unlike a full KO in vivo, mutant RBM20 mice showed changes in global expression of genes involved in cardiac function [20, 21 ] . In human iPSC-CMs, R636S RBM20 protein preferably bound to the 3 ’ UTR of transcripts in the cytoplasm, and co-localized with the P-body marker DDX6 [22], Altogether, aberrant localization of RBM20 was shown for mutations of four out of six residues in the PRSRSP stretch: S635, R636, S637, and P638 [19-24], leaving the remaining two (P633 and R634) uncharacterized. Overall, it is unclear whether these mutations affect RBM20's intrinsic role as a mediator of spliceosome activity, or whether the splicing haploinsufficiency results from protein mislocalization alone. This remains uncertain because the mechanism driving RBM20 cellular localization is unknown. W02015042308A2 relates to RNA-based HIV-inhibitors, such as a viral entry inhibiting RNA encoding sequence encoding a nuclear receptor siRNA, which can be a transportin 3 (TNP03) siRNA.
Maertens GN, et al. (in: Structural basis for nuclear import of splicing factors by human Transportin 3. Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2728-33. doi: 10.1073/pnas.l320755111. Epub 2014 Jan 21. PMID: 24449914; PMCID: PMC3932936) disclose that transportin 3 (Tnpo3, Transportin- SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. They show that the majority of cellular Tnpo3 binding partners contain arginine-serine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran- and alternative splicing factor/splicing factor 2 (ASF/SF2)- bound forms. The flexible 0-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine- rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. The results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.
WO 2010/149332 relates to RBM20 polynucleotides, preferably for use in medicine. In particular, these polynucleotides can be used to diagnose cardiac diseases, like cardiomyopathies or sudden cardiac death. Further, the invention pertains to methods for diagnosing a subject suffering from a cardiac disease and to treating such a subject. Disclosed are RBM20 proteins comprising a P638L (Homo sapiens) or a P641L mutation (Rattus norvegicus).
US20110281260A1 relates to methods and materials for using nucleic acid and amino acid sequence variants of ribonucleic acid binding motif protein 20 (RBM20). For example, methods and materials for using nucleic acid sequence variants and/or their corresponding amino acid variants of RBM20 that are associated with dilated cardiomyopathy to identify mammals (e.g., humans) at risk of having dilated cardiomyopathy that is likely to progress to heart failure are provided. Granted US8563705B2 relates to a fusion nucleic acid comprising a ribonucleic acid binding motif protein 20 (RBM20) nucleic acid sequence encoding a fragment of a RBM20 polypeptide that is 22 to 100 amino acid residues in length, a nucleic acid sequence heterologous to said RBM20 nucleic acid sequence, and a fluorescent label, wherein said fragment of said RBM20 polypeptide comprises a mutation with respect to a reference sequence, wherein said reference sequence is set forth in SEQ ID NO:3, and wherein said mutation is Arg636Ser or Arg636His.
WO 2020/092171 relates to methods of treatment, genetic screening, and disease models for heart conditions associated with RBM20 deficiency. In particular, methods of treating heart conditions associated with RBM20 deficiency, including RBM20-dependent dilated cardiomyopathy and heart failure with compounds that upregulate expression of RBM20, such as all-trans retinoic acid are provided. Also disclosed are methods of genetic screening to detect the presence of a P633L mutation in RBM20 in order to identify individuals having a genetic predisposition to developing RBM20-dependent DCM. Induced pluripotent stem cell-derived cardiomyocytes (IPSC-CMs) produced by differentiation of IPSCs comprising at least one RBM20 allele encoding a P633L mutation and methods of using them in screening for therapeutics for treating RBM20-dependent DCM are also disclosed. Claimed is a method of treating a subject for a heart condition associated with RBM20 deficiency, the method comprising administering a therapeutically effective amount of all-trans retinoic acid (ATRA) to the subject.
As mentioned above, recent studies suggested that some RBM20 variants display gain-of- function effects related to mislocalization of the mutated protein outside the nucleus. In a porcine DCM model, as well as in patient-derived iPSC-CMs harboring the R636S mutation, RBM20 mislocalized to the cytoplasm and formed potentially detrimental RNP-granules [19], In a murine DCM model, the S637A mutation (S635A in humans) caused similar mislocalization, lower survival and higher levels of fibrosis compared to an Rbm20 KO [20], Therefore, methods of treating heart conditions associated with RBM20 deficiency, including RBM20-dependent dilated cardiomyopathy and heart failure with compounds that upregulate expression of RBM20 appear to have undesired side-effects.
New and improved strategies are therefore sought in order to prevent or treat heart conditions associated with RBM20 deficiency. It is therefore an object of the present invention to provide such strategies. Other objects and advantages will readily become apparent for the person of skill from studying the following more detailed description and examples.
In a first aspect of the present invention, the problem of the present invention is solved by providing an agent that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence. Preferably, the agent for use is selected from the group consisting of an agent specifically binding to the protein comprising a mutated RS domain amino acid sequence and comprising at least one nuclear localization signal (NLS), an agent for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, a compound that improves the binding of the protein comprising a mutated RS domain amino acid sequence to its nuclear transporter protein, and a genetic construct for expressing or overexpressing the nuclear transporter protein or of the protein comprising a mutated RS domain amino acid sequence. More preferably, the agent for use is selected from the group consisting of a proteinaceous binding domain that is specifically binding to the protein comprising a mutated RS domain amino acid sequence, such as an antibody or binding fragment thereof, fused or linked to the at least one NLS, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof, or an NLS-RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9 fusion or an RBM20 L-rich region binding fragment thereof, a nanobody -NLS fusion, an expression construct for expressing a polynucleotide encoding the binder-NLS fusion and/or the nuclear transporter protein, a genetic integration construct for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as, for example, a prime editing construct containing at least one NLS, and a small molecule binding to the nuclear transporter protein and/or the protein comprising a mutated RS domain amino acid sequence and thereby improving the nuclear transport of the protein comprising a mutated RS domain amino acid sequence.
Further preferred is the agent for use according to the present invention, wherein the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNPO3) or an ortholog thereof. Even further preferred is the agent for use according to the present invention, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
In a second aspect of the present invention, the problem of the present invention is solved by providing a method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell, comprising the steps of i) providing at least one mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, ii) providing at least one nuclear transporter protein comprising a domain binding to an RS domain, and/or at least one nuclear transporter protein RS domain binding domain amino acid sequence, and iii) detecting the binding of the mutated RS domain amino acid sequence of i) to the domain of ii) binding to an RS domain in the absence and presence of at least one candidate compound, wherein an increase of the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the presence of at least one candidate compound indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
Preferred is the method according to the present invention, wherein the mutated RS domain amino acid sequence is derived from RBM20 or an ortholog thereof, and/or the domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof.
Further preferred is the method according to the present invention, wherein the amino acid sequences of i) and/or ii) are provided in a cell, and are preferably provided as recombinantly expressed amino acid sequences. Examples are the provision using mRNA delivery of the coding sequence(s), so that cells express ii) or i) or a binder themselves, such as, for example, as mRNA vaccines or “naked” mRNA provided to the heart. Furthermore, the RNA could be encapsulated into nanoparticles to increase the delivery/stability. Similarly, DNA delivery of the sequence(s) can be performed. Viral delivery of DNA or RNA is also possible.
Even further preferred is the method according to the present invention, wherein a change in the biological function and/or intracellular localization of i) the at least one protein comprising a mutated RS domain amino acid sequence, and/or an increase of the expression and/or a change in the biological function of ii) the at least one nuclear transporter protein comprising a domain binding to an RS domain is detected instead or in addition to the binding of the mutated RS domain amino acid sequence in the absence and presence of the at least one candidate compound, wherein a change in the biological function and/or intracellular localization of a protein of i) and/or an increase of the expression and/or change in the biological function of a protein of ii) indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of a protein comprising a mutated RS domain amino acid sequence in the cytoplasm of said cell.
In a preferred embodiment of the method according to the present invention, the biological function is tested based on detecting cytoplasmatic mislocalization of the protein comprising the mutated RS domain amino acid sequence, the formation of cytoplasmatic granules of the protein comprising the mutated RS domain amino acid sequence, and/or the splicing activity of the protein comprising the mutated RS domain amino acid sequence.
The compound as identified may be selected from the group consisting of natural compound, plant extract, a peptide, such as an artificial in silico designed peptide binder, a protein, a small molecule (less than about 500 Da), an RNA, an antibody or antigen binding fragment thereof, and an agent for use according to the present invention.
In a third aspect of the present invention, the problem of the present invention is solved by a method for producing a pharmaceutical composition, comprising performing a method according to the present invention, and admixing the compound as identified with at least one pharmaceutically carrier.
In a fourth aspect of the present invention, the problem of the present invention is solved by a compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention for use in medicine, in particular for use in the prevention or treatment of a disease or condition in a cell of a subj ect that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising a mutated RS domain amino acid sequence. Preferably, the cell is a cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, and the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
In a fifth aspect of the present invention, the problem of the present invention is solved by a method for increasing the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreasing the amount of the protein in the cytoplasm of said cell, comprising providing to a cell an effective amount of the compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention, whereby the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell is increased and/or the amount of the protein in the cytoplasm of said cell is decreased.
In a sixth aspect of the present invention, the problem of the present invention is solved by a method for preventing or treating a disease or condition related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of a protein comprising a mutated RS domain amino acid sequence in a subject in need of said prevention or treatment, comprising administering to the subject an effective amount of the compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention. Preferred is the method according to the present invention, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
As mentioned above, in a first aspect of the present invention, the object of the present invention is solved by an agent that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence.
In the context of the present invention, the term “a protein comprising a mutated RS domain amino acid sequence” shall relate to a domain as found in a protein member of the arginineserine-rich protein family (SR proteins) that are multifunctional RNA-binding proteins that have emerged as key determinants for mRNP formation, identity and fate. They bind to pre- mRNAs early during transcription in the nucleus and accompany bound transcripts until they are translated or degraded in the cytoplasm. SR proteins are mostly known for their essential roles in constitutive splicing and as regulators of alternative splicing (see, for example, Irena Sliskovic, Hannah Eich, Michaela Muller-McNicoll; Exploring the multifunctionality of SR proteins. Biochem Soc Trans 28 February 2022; 50 (1): 187-198. doi: https://doi.org/10.1042/BST20210325). A preferred protein providing the RS domain amino acid sequence is human RBM20 (UniProt Q5T481) or an ortholog thereof. The amino acid sequence of human RBM20 RNA binding motif protein 20 has Gene ID: 282996 and can be found at https://www.ncbi.nlm.nih.gov/gene/282996. A preferred RS domain amino acid sequence according to the present invention comprises an amino acid sequence that is to at least 90%, preferably to at least 95%, and most preferred to at least 99% or even to 100% identical to the amino acids 613-673 of the human non-mutated RBM20 protein. Orthologs of the RS domain amino acid sequence according to the present invention may be selected from bovine, dog, equine, cat, chicken, monkey, mouse, and rat. The rbm20 gene encodes a protein that binds RNA and regulates splicing. Mutations in this gene have been associated with familial dilated cardiomyopathy. The majority of DCM-causing RBM20 variants are heterozygous missense mutations, many of which cluster in a conserved stretch of six amino acids PRSRSP (amino acid position 633 - 638) in the protein’s arginine/serine (RS)-rich domain [7, 13, 16, 17], This stretch further constitutes a preferred embodiment of the invention.
In a preferred embodiment of the present invention, the term “a protein comprising a mutated RS domain amino acid sequence” shall further include mutated amino acid sequences in a protein member of the arginine-serine-rich protein family (SR proteins), where the mutation or mutations are found outside of the above-described RS-domain, as long as these mutations cause or are involved in an aberrant protein localization and/or splicing activity of the respective SR protein. Further preferred examples are mutations in the RRM region (AA 518-593 of human RBM20), such as, for example, V535I, or the glutamate-rich region (AA 839-945 of human RBM20), such as, for example, D888N or E913K (see Lennermann D, Backs J, van den Hoogenhof MMG. New Insights in RBM20 Cardiomyopathy. Curr Heart Fail Rep. 2020 Oct;17(5):234-246. doi: 10.1007/sl l897-020-00475-x. PMID: 32789749; PMCID: PMC7495990, and the references as cited therein, incorporated herein by reference). Other preferred mutations that are included are involved in the phosphorylation of the SR protein, which seems required for nuclear import (see, for example, Maertens GN, et al. Structural basis for nuclear import of splicing factors by human Transportin 3. Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2728-33. doi: 10.1073/pnas.1320755111. Epub 2014 Jan 21. PMID: 24449914; PMCID: PMC3932936).
It was surprisingly found in the context of the present invention, while using image-enabled cell sorting (ICS), which allows for isolation of cells harboring aberrant protein localization phenotypes from a heterogeneous population in high throughput, that the nuclear relocalization of many pathogenic RS-domain mutations can lead to an improved splice regulatory activity of RBM20, and that the severe splicing defect are mostly caused due to mislocalization.
The inventors thus uncovered the direct mechanism of RBM20 nuclear transport and demonstrated how RS-domain mutations disrupt this process. The inventor’s findings enable a new therapeutic strategy, targeted at improving the nuclear import of mislocalized RBM20. This strategy is more likely to be successful in treating RBM20-mediated DCM caused by mislocalizing mutations, since it will compensate both for alternative splicing (AS) haploinsufficiency and alleviate the dominant-negative effects of the cytoplasmic granules.
A previous study on subcellular localization of RBM20 revealed that the RRM domain and the RS-rich region were required for nuclear retention yet did not identify a critical residue (Filippello A, Lorenzi P, Bergamo E, Romanelli MG. Identification of nuclear retention domains in the RBM20 protein. FEBS Letters. 2013;587:2989-2995. doi: 10.1016/j.febslet.2013.07.018).
Murayama, R., et al. (in: Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization. Sci Rep 8, 8970 (2018). https://doi.org/10.1038/s41598-018-26624-w) disclose that mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), and that most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. They identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. Their results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20S637A mouse as a good model for in vivo study. The substitution mutants RBM20S637A, RBM20S639A and RBM20S637A/S639A were excluded from the nuclei of cells, indicating that these residues are essential for the nuclear localization. They also showed in HeLa cells ectopically expressing an RBM20 S637A/S639A variant that the addition of three tandem copies of SV40 NLSs restores nuclear localization and splicing of a TTN splicing reporter. These results further indicated that the RSRSP stretch was critical for nuclear localization of and not splicing regulation by RBM20. Finally, they speculate that the effects of these mutations as well as other DCM-related mutations in the hotspot of RBM20 is likely due to loss of recognition of the RSRSP stretch by protein kinase(s) that phosphorylate the serine residues or by a partner protein that specifically binds to RBM20 only after phosphorylation of the RSRSP stretch. It would therefore be interesting to identify a protein kinase and an importin protein for RBM20 in vivo, which would elucidate physiological and pathological relevance of the phosphorylation of RBM20.
Zhang Y, Gregorich ZR, Wang Y, et al. (in: Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy. bioRxiv; 2022. DOI: 10.1101/2022.12.08.519616) disclose that RBM20 is a splicing factor with two canonical domains, an RNA recognition motif (RRM) and an arginine-serine rich (RS) domain. RRM loss-of-function disrupts the splicing of RBM20 target transcripts and leads to systolic dysfunction without overt DCM, while mutations in the RS domain precipitate DCM. They show that mice lacking the RS domain (Rbm20 ARS) manifest DCM with mis-splicing of RBM20 target transcripts. They found that RBM20 is mis-localized in Rbm20 ARS mice but not in mice lacking the RRM, which are also deficient in RBM20 splicing. They determined that the RS domain, not other domains including the RRM, is critical for RBM20 nuclear import and define the core nuclear localization signal (NLS) within this domain. Mutation analysis of phosphorylation sites within the RS domain indicated that phosphorylation is dispensable for RBM20 nuclear import. They establish disruption of the NLS in RBM20 as a causative mechanism in DCM through nucleocytoplasmic transport.
A previously published therapeutic approach demonstrated that up-regulation of the RBM20 wild-type allele rescues the splicing deficiency [Briganti F, Sun H, Wei W, et al. iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy. Cell Rep. 2020;32(10): 108117. doi: 10.1016/j.celrep.2020.108117], However, this strategy was allele-unspecific and upregulated the mutated RBM20 allele as well, which seems to worsen the disease phenotype by increasing the probability of RNP granule formation in the cytoplasm. Therefore, this strategy is not applicable for patients with mislocalized RBM20 variants, and can only be beneficial for patients with variants that do not affect RBM20’s nuclear localization.
Gene therapy is an alternative option to repair RBM20 mutations. However, the majority of existing gene editing methods, apart from prime editing, are mutation-dependent. This will require to develop and optimize a personalized gRNA for every new mutation, which is an inefficient time- and finance-wise approach. In addition, introducing genome editing in humans has big ethical and societal complications, which may result in longer times for translation into clinical practice.
The inventors showed that restoring the nuclear localization of RBM20 RS-domain variants both compensates for the splicing deficiency, and results in granule clearance. They proposed a couple of principal strategies to achieve this in patients, as is described herein.
Preferred is the agent for use according to the present invention wherein the agent is selected from agents specifically binding to the protein comprising a mutated RS domain amino acid sequence and comprising at least one nuclear localization signal (NLS). In a preferred embodiment, the agent specifically binding to the protein comprises a proteinaceous binding domain that is specifically binding to the protein comprising a mutated RS domain amino acid sequence. The binder can be an antibody or protein binding fragment thereof, or an artificially designed peptide binder, fused or otherwise suitably linked to the at least one NLS, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof, or an NLS-RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9 fusion or an RBM20 L-rich region binding fragment thereof, a nanobody - NLS fusion construct as described herein. Nanobodies can be generated via in vivo llama/alpaca immunization, the term shall also include sybodies, i.e. generated via in vitro library screens (phage or ribosome display methods for screening). Other binders include single-domain antibodies, e.g., VHH antibodies with a single variable domain on a heavy chain (see, for example, Henry KA, MacKenzie CR. Editorial: Single-Domain Antibodies-Biology, Engineering and Emerging Applications. Front Immunol. 2018 Jan 23;9:41. doi: 10.3389/fimmu.2018.00041. PMID: 29410670; PMCID: PMC5787064).
Preferred is the agent for use according to the present invention wherein the agent is selected from an agent for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as a genetic integration construct for genetically fusing the at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as, for example, a prime editing construct containing at least one NLS. This provides a single gene editing approach to correct the effect of all possible mislocalization mutants, instead of using multiple guide RNAs for each mutation possible.
Preferred is the agent for use according to the present invention wherein the agent is selected from a compound that improves the binding of the protein comprising a mutated RS domain amino acid sequence to its nuclear transporter protein, such as a small molecule binding to the nuclear transporter protein and/or the protein comprising a mutated RS domain amino acid sequence and thereby improving the nuclear transport of the protein comprising a mutated RS domain amino acid sequence.
Preferred is the agent for use according to the present invention wherein the agent is selected from a genetic construct for expressing or overexpressing the nuclear transporter protein of the protein comprising a mutated RS domain amino acid sequence, such as integrating or other expression construct for expressing a polynucleotide encoding the binder-NLS fusion and/or the nuclear transporter protein.
Further preferred is the agent for use according to the present invention, wherein the at least one NLS sequence is selected from the group consisting of a non-classical or classical NLS, such as, for example a monopartite or bipartite classical NLS, in particular an NLS of SV40, C-myc, nucleoplasmin, EGL-13, or TUS-protein, the acidic M9 domain of hnRNP Al, the sequence KIPIK in yeast transcription repressor Mata2, and the complex signals of U snRNPs (see also Lu, J., Wu, T., Zhang, B. et al. Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Commim Signal 19, 60 (2021). https://doi.org/10. 1186/s 12964-021 -00741-y).
Further preferred is the agent for use according to the present invention, wherein the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNPO3) or an ortholog thereof. Further preferred is the agent for use according to the present invention, wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
Further preferred is the agent for use according to the present invention, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
The inventors show that introducing a SV40 NLS tag restores nuclear localization and splicing activity of RBM20 in HeLa and HEK293 cell lines, as well as in iPSC-CMs. The transfection of HEK293T and iPSC-CMs cells with plasmids encoding for RBM20 RS-domain variants with an NLS tag resulted in significant restoration of splicing activity for all variants in comparison to the non-tagged variants (Fig. 2b, c).
As a control, addition of the same NLS tag to the V914A RBM20 variant, which resides outside the RS-domain and does not lead to cytoplasmic mislocalization had no effect on the splicing activity. These data suggest that RS-domain mutant variants are competent in splicing regulation when their nuclear localization is restored. The inventors validated these results in iPSC-CMs, by overexpressing WT-, R634Q-, or NLS-tagged R634Q-RBM20 in cells with an endogenous RBM20 frameshift mutation (S635FS), where no endogenously active RBM20 is expressed. Overexpressed NLS-R634Q localized to the nucleus, similar to WT-RBM20 (Fig. 2c). Genome-wide RNA-Seq analysis showed that the expression of 1,751 genes was altered in cells expressing R634Q-RBM20, compared to WT (Fig. 2d). The genes are consistent with severe cardiac impairment and similar to the genes altered in expression in cells expressing P633L in the cytoplasm. RBM20 target gene expression was consistently either unchanged or down-regulated. Strikingly, the inventors found only few differentially expressed genes between WT and NLS-tagged-R634Q expressing cells (Fig. 2d). Moreover, splicing of RBM20 target genes was restored in NLS-R634Q expressing iPSC-CMs to similar levels seen in WT (Fig. 2e). These findings demonstrate that tested RS-domain RBM20 mutations do not affect the intrinsic splice regulatory activity of the protein. They affect only its ability to be translocated into the nucleus. These results highlight the importance of identifying factors involved in nuclear transport of RBM20. Based on these results, in an embodiment the inventors establish a new therapeutical approach to deliver the NLS-tag to the mutant RBM20. First, the inventors are testing a nanobody fused to an NLS, which could serve as an alternative nuclear importer to target cytoplasmic RBM20 variants back to the nucleus. The inventors started to test this as proof-of-principle in the inventor’s eGFP-RBM20-WT and -R634Q HeLa reporter cells, with an eGFP specific nanobody. The initial results suggest that the inventors can regulate the localization of RBM20 with such a nanobody-NLS fusion as alternative nuclear transporter. The inventors plan to design and produce now a set of RBM20-specific nanobodies that should recognize all RBM20 variants and/or a set of artificial in silico designed peptide binders. The targeting efficiency of such newly generated RBM20 specific nanobodies and/or binders will be then validated and tested in the inventor’s established HeLa, iPSC-CMs and RBM20-DCM-mouse models.
For the treatment of patients, the inventors supply an RBM20 specific nanobody-NLS fusion and/or peptide binder-fusion either by delivering its cDNA with AAV-based vectors, or its mRNA with lipid bioparticles specifically to cardiomyocytes. These are tested and optimized in iPSC-CMs. Work is focused on optimizing the delivery methods in vivo using the inventor’s mouse models. As an alternative, the inventors are also investigating the possibility to introduce the NLS sequence via prime editing into the endogenous RBM20 locus. The advantage is that this approach can potentially repair the localization of all RBM20 variants, and does not require a personalized gRNA per patient. The exact position and design for this approach is investigated and is further optimized.
The inventors also demonstrate that supplying a full TNP03 cDNA both in vitro and in vivo enhances the probability of mislocalized RBM20 variants to interact with TNP03. Such a TNP03 overexpression rescued the localization and splicing activity of all tested RBM20 variants in iPSC-CMs. Strikingly, the nuclear localization of P633L was completely rescued to the level of the WT protein; similar results were obtained for R634Q-RBM20, although a small fraction of the protein remained in the cytoplasm (Fig. 5a, b). Restoring nuclear localization of the mutant RBM20 variants also up-regulated the splicing activity (Fig. 5c, d). These results indicate that mislocalization of the mutant variants can be rescued by up-regulating the TNP03- RBM20 interaction.
To test whether increasing TNP03 levels could indeed improve the RBM20 mislocalization in vivo, the inventors delivered Tnpo3 cDNA via AAV9 to mouse hearts bearing homozygous P635L-RBM20 (P635L+/+) mutations (P633L in humans). The inventors analyzed the RBM20 localization and splicing function four weeks after AAV9 injection (Fig. 5e). Strikingly, increasing Tnpo3 levels (Fig. 5f) resulted in partial rescue of RBM20 localization (Fig. 5g) and Tin alternative splicing (Fig. 5h, i) independently of differences in Rbm20 expression levels. This preclinical model of RBM20-DCM reveals a novel therapeutic strategy for mislocalizing RBM20 variants for future studies. See also Figure 16.
As can be seen from the above, preferred strategies of the present invention to achieve an effective prevention and/or treatment by improving the mislocalization of RBM20 are at least one of i) increasing TNPO3 levels in a desired cell to rescue the RBM20 mislocalization, for example using a TNPO3 expression construct; ii) using a compound, for example a small molecule, or a co-factor, to mediate, improve or increase the binding of the (mutated) RBM20 protein and/or the RS domain thereof to the TNP03 protein in order to improve the RBM20 transport and RBM20 mislocalization; and/or iii) modifying the mutated RBM20 protein and/or the RS domain thereof in order to improve the RBM20 transport and RBM20 mislocalization by using a genetic construct to fuse at least one NLS to the RBM20 and/or by attaching (tagging) at least one NLS to the RBM20 using a binder that also comprises at least one NLS, for example as a nanobody -NLS format.
In another important aspect of the present invention, the object of the present invention is solved by providing a method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of a cell, comprising the steps of a) providing at least one L-rich domain amino acid sequence of a protein comprising a mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, b) providing at least one candidate compound prospectively binding to the L-rich domain amino acid sequence, and c) detecting the binding of the at least one candidate compound to the L-rich domain amino acid sequence, wherein a binding of the at least one candidate compound to the L-rich domain amino acid sequence indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
In another important aspect of the present invention, the object of the present invention is solved by providing a method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell, comprising the steps of i) providing at least one mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, ii) providing at least one nuclear transporter protein comprising a domain binding to an RS domain, and/or at least one nuclear transporter protein RS domain binding domain amino acid sequence, and iii) detecting the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the absence and presence of at least one candidate compound, wherein an increase of the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the presence of at least one candidate compound indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell.
In a preferred embodiment of this method according to the present invention, the present invention uses the binding of a compound, such as a peptide, to the L-rich region and/or formation and structure of the TNPO3-RBM20 complex in order to screen for compounds, such as small molecules, as prospective therapeutics through further stabilizing the interaction of TNPO3 with RBM20 variants. It was surprisingly found in the context of the present invention that a loss of interaction of RBM20 with TNPO3 results in the formation of undesired RBM20 RNP granules, regardless of the mutation present. Hence, an improvement of the binding of RBM20 and TNPO3 will both reduce granules and improve mislocalization of RBM20.
Preferred is the method according to the present invention, wherein the at least one protein comprising a mutated RS domain amino acid sequence further comprises at least one added NLS sequence, as discussed above and herein. This will revert and rescue the mislocalization further. Preferred is the method according to the present invention, wherein at least one of the amino acid sequences as used, i.e., comprising the RS domain or the nuclear transporter binding sequence is labelled, and/or wherein the at least one candidate compound is labelled. This allows for a detection in the assay, i.e., detecting the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the absence and presence of at least one candidate compound. Labelling can be done with any suitable label that optimally does not substantially interfere with the binding of the components. The label may be attached directly or indirectly, for example directly labelled with a GFP fusion or indirectly labelled using an antibody or fragment thereof that specifically binds to an amino acid sequence of the components, the antibody being suitably labelled.
Preferred is the method according to the present invention, wherein at least one binding cofactor is present in the assay, in particular a suitable protein, such as importin or small GTPase Ran, and/or an RNA molecule. These cofactors can be added to further mimic the situation in vivo.
Preferred is the method according to the present invention, wherein the mutated RS domain amino acid sequence is derived from RBM20 or an ortholog thereof, and/or the domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof. A preferred RS domain amino acid sequence according to the present invention, whether individually or included in the protein, such as RBM20, comprises an amino acid sequence that is to at least 90%, preferably to at least 95%, and most preferred to at least 99% or even to 100% identical to the amino acids 613-673 of the human non-mutated RBM20 protein. Orthologs of the RS domain amino acid sequence according to the present invention may be selected from bovine, dog, equine, cat, chicken, monkey, mouse, and rat.
A preferred domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof, or comprises an amino acid sequence that is to at least 90%, preferably to at least 95% identical to the amino acids around the arginine-rich helix of Tnpo3 HEAT repeat 15 of the human transportin 3 (Tnpo3, Transportin-SR2, UniProt Q9Y5L0) protein (see Maertens GN, et al. Structural basis for nuclear import of splicing factors by human Transportin 3. Proc Natl Acad Sci U S A. 2014 Feb 18; 111(7):2728-33. doi: 10.1073/pnas.l 320755111. Epub 2014 Jan 21. PMID: 24449914; PMCID: PMC3932936), in particular between about amino acid positions 650-675 of the protein. Orthologs of the transportin 3 amino acid sequence according to the present invention may be selected from bovine, dog, equine, cat, chicken, monkey, mouse, and rat.
The method according to the present invention may be performed in vitro or in vivo. While the above binding-assays may be performed in a cell-free model, preferred is a method that is performed in a cellular assay. In a preferred method according to the present invention, the amino acid sequences of a) i) and/or ii) are therefore provided in a cell and are preferably provided as recombinantly expressed amino acid sequences.
In general, any suitable cellular assay may be used in the context of the present invention. Preferred is the method according to the present invention, wherein the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells (see also example, below).
An advantage of the cellular assay is that detecting a change in the biological function of the components as involved can be readily achieved. Preferred is the method according to the present invention, wherein further a change in the biological function of i) the at least one protein comprising a mutated RS domain amino acid sequence, and optionally an increase of the expression and/or a change in the biological function of ii) the at least one nuclear transporter protein comprising a domain binding to an RS domain is detected instead or in addition to the binding of the mutated RS domain amino acid sequence in the absence and presence of the at least one candidate compound, wherein a change in the biological function of a protein of i) and/or an increase of the expression and/or change in the biological function of a protein of ii) indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell.
Preferred is the method according to the present invention, wherein the biological function is tested based on detecting cytoplasmatic mislocalization of the protein comprising the mutated RS domain amino acid sequence, the formation of cytoplasmatic granules of the protein comprising the mutated RS domain amino acid sequence, and/or the splicing activity of the protein comprising the mutated RS domain amino acid sequence. As mentioned above, all these parameters are expected to be suitable to indicate an improvement of the pathological situation, and thus can indicate a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell.
In general, any suitable candidate compound can be used for the method of the present invention. Preferred is the method according to the present invention, wherein the compound is selected from the group consisting of natural compound, plant extract, a peptide, such as an artificial in silico designed peptide binder, a protein, a small molecule (molecular weight of less than about 500 Da), an RNA, an antibody or antigen binding fragment thereof. Furthermore, the screening can be used to identify and/or test an agent for use according to the present invention as described above.
In another important aspect of the present invention, the object of the present invention is solved by providing a method for producing a pharmaceutical composition, comprising performing a method according to the present invention, and admixing the compound or agent as identified with at least one pharmaceutically carrier.
The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
The pharmaceutical compositions according to the invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
The pharmaceutical compositions according to the invention may be in liquid, dry or semi-solid form, such as, for example, in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granulate, sugar-coated tablet, lozenge, pill, ampoule, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, moist compress, gargling solution, plant juice, nasal agent, inhalation mixture, aerosol, mouthwash, mouth spray, nose spray, or room spray.
In another important aspect of the present invention, the object of the present invention is solved by providing a compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention for use in medicine, in particular for use in the prevention or treatment of a disease or condition in a cell of a subject that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising a mutated RS domain amino acid sequence. Preferred is the compound or pharmaceutical composition for use according to the present invention, wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte. Further preferred is the compound or pharmaceutical composition for use according to the present invention, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM). Further preferred details regarding the medical use are described below.
In another important aspect of the present invention, the object of the present invention is solved by a method for increasing the amount of a protein in the nucleus of a cell and/or decreasing its amount in the cytoplasm of said cell, comprising providing to a cell an effective amount of the compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention. An effective amount is sufficient to increase the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell as treated. Preferred is the method according to the present invention, wherein the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
In another important aspect of the present invention, the object of the present invention is solved by providing a method for preventing or treating a disease or condition related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of a protein comprising a mutated RS domain amino acid sequence in a subject in need of said prevention or treatment, comprising administering to the subject an effective amount of the compound as identified according to the method according to the present invention, or a pharmaceutical composition as produced according to the present invention. Preferred is the method according to the present invention, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
In context of the present invention, the term “subject”, as used in certain embodiments, preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human, and a patient. The term “patient” preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, horse, cattle, cow, cat, dog, monkey, or preferably a human, for example a human patient, for whom diagnosis, prognosis, or therapy is desired. The subject of the invention may be at danger of suffering from a disease, such as CM, in particular DCM. A more detailed description of medical indications relevant in context of the invention is provided herein.
The term "treating" or “treatment” as used herein means stabilizing or reducing an adverse symptom associated with a condition; reducing the severity of a disease symptom; slowing the rate of the progression of a disease; inhibiting or stabilizing the progression of a disease condition; or changing a metric that is associated with the disease state in a desirable way. The term "preventing" or “prevention” as used herein means the avoidance of the occurrence of an adverse symptom associated with a condition or disease before they occur.
Regardless of the route of administration selected, the compounds/agents of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound/agent to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 pg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 pg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 pg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; the pharmaceutical composition may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.
The inventors provided the proof-of-principle, that introducing a S V40 NLS tag restores nuclear localization and splicing activity of RBM20 in HeLa and HEK293 cell lines, as well as in iPSC- CMs. The transfection of HEK293T cells with plasmids encoding for RBM20 RS-domain variants with an NLS tag resulted in significant restoration of splicing activity for all variants in comparison to the non-tagged variants (Fig. 2b).
In the context of the present invention, the term “about” shall indicate a deviation of +/- 10% of a given value, if not stated otherwise. Preferred is the value as given, +/- 5%, more preferred is the value as given and determined according to a method according to the state of the art.
DCM-causing variants in the RS-domain of RBM20 have been shown, by the inventor’s group and others, to result in aberrant RBM20 localization and RNP granule formation in the cytoplasm [24-29], These mutations are associated with a more severe disease phenotype than an RBM20 KO in vivo [25, 26] Prior to this study, it was unknown why these single point mutations lead to RBM20 protein mislocalization and whether restoration of nuclear localization could restore the splicing activity in vivo. Here, the inventors demonstrate that all tested mislocalizing RBM20 RS-domain mutants regain their splicing activity upon addition of the SV40 NLS tag, as previously shown for S635A [40], This was done also in iPSCs for at least one RS mutant, and not only in HeLa cells with a reporter gene.
The inventors identify TNPO3 as the main nuclear importer of RBM20 with the genome-wide ICS CRISPR-Cas9 screen. TNPO3 belongs to the 0-karyopherin family of importins, which directly interact with their cargo via amino acid sequence recognition. TNPO3 specifically recognizes arginine-serine (RS) repeats that are present in many splicing factors like SRSF1, ASF/ SF2, and others [34, 37, 38, 41-43], Here the inventors show that mutations in the RS-domain of RBM20 diminish the interaction with TNPO3. The RBM20 RS-domain mutants that abolish the interaction the most showed simultaneously the strongest mislocalization. The applied ICS screen is well suited to be used in a targeted fashion with iPSC-derived cardiomyocytes in the future. This may reveal additional factors involved in nuclear import or in regulating post-translational modifications of RBM20. It has been observed that the target-specific binding of TNP03 to its cargo can be phosphorylation-dependent [34, 37, 38] or independent [42], In the case of RBM20, serine residues in the RS-domain are normally phosphorylated [26, 31, 40, 44], however, phosphomimetic mutations do not rescue the localization phenotype [26], Importantly, both pooled and individual CRISPR KOs of kinases AKT2, CLK1, and SPRK1 - previously shown to phosphorylate RBM20 [33] - did not impact RBM20 localization (Fig. 3d). This result suggests that either the kinases complement each other, or that the RBM20-TNPO3 interaction is potentially phosphorylation-independent.
The inventors further used in vitro and in vivo models to demonstrate that the overexpression of TNPO3 can improve the nuclear import of RBM20 mutants and restore the splicing deficiency. Importantly, the inventors observed that cytosolic granules were reduced after enhancing nuclear transport. Further investigations will be needed to understand the nature and the effect of RBM20 granule formation. The inventor’s data provide the first evidence that cytoplasmic granule formation of RBM20 variants is the result of mislocalization, and not its cause. The inventors show that WT RBM20, when forced to remain in the cytoplasm, also forms granules of the same nature as the mutant variants. These results demonstrate that RS- domain mutations of PBM20 do not confer pro-aggregative qualities.
Mutations in TNPO3 have been linked to impaired myogenesis [43] and myopathies [45, 46], which includes one mutation in TNPO3 that was linked to familial DCM [47], Detailed structural studies will be needed to further decipher the RBM20-TNPO3 interaction and the direct impact of different mutations on both partners.
Altogether, the inventor’s data reveals a new therapeutic avenue for DCM patients with disease causing variants in the RS-rich region of RBM20. Enhancing RBM20 nuclear import could be achieved by endogenous tagging of PBM20 with another NLS to be recognized by other importins. Alternatively, developing a small molecule to stabilize the interaction between the mutant RS-domain variants and TNPO3 could be beneficial too. The inventor’s data demonstrates that these actions can alleviate the known splicing deficiency and abolish cytoplasmic granule formation via enhanced nuclear import.
Mutations in the cardiac-specific alternative splicing regulator PBM20 result in the cytoplasmic mislocalization of the protein and are associated with severe forms of dilated cardiomyopathy. The inventors applied the recently developed image-enabled cell sorting (ICS) technology (Schraivogel et al., High-speed fluorescence image-enabled cell sorting. Science. 2022 Jan 21;375(6578):315-320. doi: 10.1126/science.abj3013. Epub 2022 Jan 20. PMID: 35050652; PMCID: PMC7613231), in combination with functional genomics, proteomics, clinical, and animal studies, to decipher the pathobiological mechanism of mislocalizing RBM20 mutations. The inventors showed that RS-domain mutations do not affect RBM20’s intrinsic splice regulatory activity, as previously believed, but rather its import into the nucleus. By genetically tagging RBM20 variants with the SV40 nuclear localization sequence (NLS), the inventors restored their nuclear localization. The inventors show that they are functionally equivalent to the wild type and that the main factor driving their pathological phenotype is their cellular localization. The inventors identify TNPO3 as the main nuclear importer of RBM20 and demonstrate that their interaction is disrupted by mutations in RBM20’s RS-domain, in proportion to the severity of the mislocalization phenotype. Finally, the inventors show that enhancing the interaction between TNPO3 and RBM20 by increasing the amount of available transporter, rescues the aberrant localization and splicing phenotypes in vitro and in vivo. The inventor’s results open up an undiscovered avenue for developing therapeutic strategies for DCM. Firstly, by enhancing the interaction between RBM20 and TNPO3 to up-regulate the physiological import mechanism. Secondly, by introducing the SV40 NLS-tag to the RBM20 gene via gene therapy, or by employing an RBM20-specific nanobody -NLS fusion protein as an alternative nuclear importer. The inventors show that both of these approaches restore RBM20’s nuclear localization, splicing activity, and resolve cytoplasmic granules.
The present invention relates to the following items:
Item 1. An agent that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence.
Item 2. The agent for use according to Item 1, wherein the agent is selected from the group consisting of an agent specifically binding to the protein comprising a mutated RS domain amino acid sequence and comprising at least one nuclear localization signal (NLS), an agent for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, a compound that improves the binding of the protein comprising a mutated RS domain amino acid sequence to its nuclear transporter protein, and a genetic construct for expressing or overexpressing the nuclear transporter protein of the protein comprising a mutated RS domain amino acid sequence.
Item 3. The agent for use according to Item 2, wherein the agent is selected from the group consisting of a proteinaceous binding domain that is specifically binding to the protein comprising a mutated RS domain amino acid sequence, such as an antibody or binding fragment thereof, or an artificially designed peptide binder, fused or linked to the at least one NLS, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof, or an NLS-RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9 fusion or an RBM20 L-rich region binding fragment thereof, a nanobody-NLS fusion, an expression construct for expressing a polynucleotide encoding the binder-NLS fusion and/or the nuclear transporter protein, a genetic integration construct for genetically fusing the at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as, for example, a prime editing construct containing at least one NLS, and a small molecule binding to the nuclear transporter protein and/or the protein comprising a mutated RS domain amino acid sequence and thereby improving the nuclear transport of the protein comprising a mutated RS domain amino acid sequence.
Item 4. The agent for use according to any one of Items 1 to 3, wherein the at least one NLS sequence is selected from the group consisting of a non-classical or classical NLS, such as, for example a monopartite or bipartite classical NLS, in particular an NLS of SV40, C-myc, nucleoplasmin, EGL-13, or TUS-protein, the acidic M9 domain of hnRNP Al, the sequence KIPIK in yeast transcription repressor Mata2, and the complex signals of U snRNPs.
Item 5. The agent for use according to any one of Items 1 to 4, wherein the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNP03) or an ortholog thereof.
Item 6. The agent for use according to any one of Items 1 to 5, wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte. Item 7. The agent for use according to any one of Items 1 to 6, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
Item 8. A method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of a cell, comprising the steps of a) providing at least one L-rich domain amino acid sequence of a protein comprising a mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, b) providing at least one candidate compound prospectively binding to the L-rich domain amino acid sequence, and c) detecting the binding of the at least one candidate compound to the L-rich domain amino acid sequence, wherein a binding of the at least one candidate compound to the L-rich domain amino acid sequence indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
Item 9. A method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of a cell, comprising the steps of i) providing at least one mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, ii) providing at least one nuclear transporter protein comprising a domain binding to an RS domain, and/or at least one nuclear transporter protein RS domain binding domain amino acid sequence, and iii) detecting the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the absence and presence of at least one candidate compound, wherein an increase of the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the presence of at least one candidate compound indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell. Item 10. The method according to Item 8 or 9, wherein the at least one protein comprising a mutated RS domain amino acid sequence further comprises at least one added NLS sequence and/or wherein at least one of the amino acid sequences of a), i) or ii) is labelled, and/or wherein the at least one candidate compound is labelled, such as, for example, labelled with GFP or a labelled antibody or fragment thereof specifically binding to the amino acid sequence.
Item 11. The method according to any one of Items 8 to 10, wherein at least one binding cofactor is present, in particular a suitable RNA molecule.
Item 12. The method according to any one of Items 8 to 11, wherein the mutated RS domain amino acid sequence is derived from RBM20 or an ortholog thereof, and/or the domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof.
Item 13. The method according to any one of Items 8 to 12, wherein the amino acid sequences of a), i) and/or ii) are provided in a cell, and are preferably provided as recombinantly expressed amino acid sequences.
Item 14. The method according to Item 13, wherein a change in the biological function of i) the at least one protein comprising a mutated RS domain amino acid sequence, and optionally an increase of the expression and/or a change in the biological function of ii) the at least one nuclear transporter protein comprising a domain binding to an RS domain is detected instead or in addition to the binding in the absence and presence of the at least one candidate compound, wherein a change in the biological function of a protein of i) and/or an increase of the expression and/or change in the biological function of a protein of ii) indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
Item 15. The method according to Item 13 or 14, wherein the biological function is tested based on detecting cytoplasmatic mislocalization of the protein comprising the mutated RS domain amino acid sequence, the formation of cytoplasmatic granules of the protein comprising the mutated RS domain amino acid sequence, and/or the splicing activity of the protein comprising the mutated RS domain amino acid sequence. Item 16. The method according to any one of Items 13 to 15, wherein the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
Item 17. The method according to any one of Items 8 or 13 to 16, wherein the compound is selected from the group consisting of natural compound, plant extract, a peptide, such as an artificial in silico designed peptide binder, a protein, a small molecule (less than about 500 Da), an RNA, an antibody or antigen binding fragment thereof, and an agent for use according to any one of claims 1 to 7, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof.
Item 18. A method for producing a pharmaceutical composition, comprising performing a method according to any one of Items 8 to 17, and admixing the compound as identified with at least one pharmaceutically carrier.
Item 19. A compound as identified according to the method according to any one of Items 8 to 17, or a pharmaceutical composition as produced according to Item 18 for use in medicine, in particular for use in the prevention or treatment of a disease or condition in a cell of a subject that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising a mutated RS domain amino acid sequence.
Item 20. The compound or pharmaceutical composition for use according to Item 20, wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
Item 21. The compound or pharmaceutical composition for use according to Item 19 or 20, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
Item 22. A method for increasing the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreasing the amount of the protein in the cytoplasm of said cell, comprising providing to a cell an effective amount of the compound as identified according to the method according to any one of Items 8 to 17, or a pharmaceutical composition as produced according to Item 18, whereby the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell is increased.
Item 23. The method according to Item 22, wherein the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
Item 24. A method for preventing or treating a disease or condition related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of a protein comprising a mutated RS domain amino acid sequence in a subject in need of said prevention or treatment, comprising administering to the subject an effective amount of the compound as identified according to the method according to any one of Items 8 to 17, or a pharmaceutical composition as produced according to Item 18.
Item 25. The method according to Item 24, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
The invention will now be described further in the following examples with reference to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited are incorporated by reference in their entireties.
Figure 1 shows that the splice regulatory activity of RBM20 variants is proportional to their nuclear localization, (a) Representative images of immunofluorescence (IF) staining for endogenous RBM20 and sarcomeric alpha-actinin in iPSC-CMs. (b) Quantification of RBM20 co-localization with DAPI based on confocal microscopy data from panel (a). Each dot represents a Pearson correlation coefficient R for at least five cells, data are combined from images of cells from three independent iPSC-CM differentiations, (c) ICS-based analysis of DRAQ5:RBM20 correlation in iPSC-CMs. (d) qPCR analysis of TTN exon 232 splicing in iPSC-CMs normalized to GAPDH expression and displayed as a mean fold change of the RBM20-WT line (standard errors are indicated). Three independent iPSC-CM differentiations were used as biological replicates, (e) Left ventricular ejection fraction (LVEF) for patients with P633L-RBM20 or with other pathogenic or likely pathogenic (P/LP) mutations in the RSRSP-stretch at the time of diagnosis, (f) Initial LVEF as a function of the age at presentation, (g) Internal Left Ventricular Diastolic Dimension (LVIDd) corrected for body size for analyzed patients, (h) ICS-sorting strategy for P633L-RBM20. Three independent iPSC-CM differentiations were used as biological replicates, (i) Numbers of significantly (absolute value of log2FC > 1 and adjusted p-value < 0.1, Benjamini -Hochberg, DeSeq2) differentially expressed genes in pairwise comparisons WTvsP633L-nuc and WTvsP633L-cyt . (j) PSL values for differentially spliced exons of known RBM20 targets between WT and R634Q cells (delta PSI > 0.1, p-value < 0.05, t-test). (k) qPCR analysis of TTN exon 242 splicing in sorted iPSC-CMs. (1) Significantly enriched RBM20 interactors (FDR < 0.05 (Limma), and absolute value of log2FC > 0.5 vs no bait negative control) in HeLa reporter cell lines expressing WT-, P633L-, or R634Q-RBM20. Three biological replicates were used, (m) Pathway enrichment analysis for common interactors between WT-, P633L-, and R634Q-RBM20 in the nuclear fraction. Ns = not significant, * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, one-way ANOVA with Tukey’s HSD post-test.
Figure 2 shows that restoring nuclear localization of RS-domain RBM20 variants rescues their splicing function, (a) Illustration of the splicing reporter assay, (b) Ratio of Flue to Rluc in empty vector control -transfected or +/- SV40 NLS RBM20 (WT, R634Q, R634L, S635A, R636S, S637A, S637G, S637D, P638L, V914A)-transfected HEK293T cells. Data from one representative biological replicate is shown, a total of four biological replicates were analyzed. Each bar shows a mean of three technical replicates, (c) iPSC-CMs with S635FS mutation transduced with eGFP-WT, eGFP-R634Q, or eGFP-NLS-R634Q, representative images, (d) Numbers of significantly (absolute value of log2FC > 1 and adjusted p-value < 0.1, Benjamini- Hochberg) differentially expressed genes in pairwise comparisons of S635FS iPSC-CMs expressing eGFP-WT vs eGFP-R634Q and eGFP-WT vs eGFP-NLS-R634Q. (e) PSI-values for differentially spliced exons of known RBM20 targets between eGFP-WT and eGFP-R634Q cells (delta PSI > 0.1, p-value < 0.05, t-test). Three independent iPSC-CM differentiations were used as biological replicates, n.s. = not significant, * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, one-way ANOVA with Bonferroni post-test.
Figure 3 shows that genome-wide ICS screen identifies TNPO3 as the main nuclear transporter of RBM20. (a) Schematic outline of the ICS-screen. Six genome-wide lentiviral libraries were applied to HeLa cells expressing eGFP-RBM20-WT and TetO-Cas9, with the coverage of 100 cells per gRNA. Cells were sorted based on the correlation between RBM20 and DRAQ5 into 7% higher and 7% lower fractions at final coverage of 500 cells per gRNA per sorted bin. Unsorted input samples were collected for sequencing as well, (b) Reads from collected samples were combined in silico to one dataset, with an average of 500 cells per gRNA, 6 gRNAs per gene. Hits were called using the software MAUDE [48], Genes are ranked by their statistical significance. The horizontal dashed lines indicate an FDR of 1%. Positive/negative regulators with FDR <1% are marked in red and blue, respectively, (c) Scatter plot of fold changes visualizing gRNA abundance changes in higher (x axis) and lower (y axis) sorted bins compared with the plasmid library. Red and blue dots indicate statistically significant positive and negative regulators, respectively (FDR < 5% according to MAUDE). Labelled are positive regulators selected for future analyses, (d) The impact of single knock-outs of the selected hits from the genome-wide screen (one gRNA per gene; the inventors picked the gRNA that showed the strongest Z-score in the pooled genetic screen) on RBM20 localization tested with ICS. The top row in the heatmap shows the log 10 of FDR value for each candidate from the screen. The phenotype in the second row represents the standardized difference in RBM20 localization signal between the knock-out (KO) and control cell populations (log2 of the ratio between cell fraction with Pearson correlation coefficient R (DRAQ5 :RBM20) > 0.7 in the knockout divided by cell fraction with Pearson correlation coefficient R (DRAQ5:RBM20) > 0.7 in the control), (e) Correlation between RBM20 and DAPI quantified based on fluorescence microscopy analysis shown in Supplementary Fig. 5g, for the single KOs indicated. Each dot represents a Pearson correlation coefficient R for at least five cells, data are combined from three technical replicates from one single-KO experiment, n.s. = not significant, *** - p < 0.001, one-way ANOVA with Tukey’s HSD post-test.
Figure 4 shows that mislocalization of RS-domain RBM20 mutants is caused by loss of interaction with TNPO3. (a) ICS-measured correlations between RBM20 and DRAQ5 in WT or P633L iPSC-CMs transfected with control siRNA or siRNA targeting TNPO3. (b) Correlation between DAPI and RBM20 quantified based on the confocal microscopy images of WT or P633L iPSC-CMs transfected with control siRNA or siRNA targeting TNPO3, shown in Supplementary Fig. 6a. Each dot represents a Pearson correlation coefficient R for at least five cells, data are combined from three biological replicates, (c) qPCR analysis of TIN exon 242 splicing in WT or P633L iPSC-CMs transfected with control siRNA or siRNA targeting TNPO3 normalized to GAPDH expression and displayed as mean fold changes versus the RBM20-WT line transfected with siRNA control with the standard errors, from two biological replicates with two technical replicates for each of them, (d) ICS-measured correlations between eGFP-RBM20 and DRAQ5 for cells expressing eGFP-WT-, -P633L-, -R634Q-, or - RSS-RBM20, transfected with control siRNA or siRNA targeting TNP03. (e) Correlation between DAPI and RBM20 quantified based on confocal microscopy images of cells expressing eGFP-WT-, -P633L-, -R634Q-, or -RSS-RBM20, transfected with control siRNA or siRNA targeting TNP03, shown in Supplementary Fig. 6d. Each dot represents a Pearson correlation coefficient R for at least five cells, data are combined from three biological replicates, (f) Superimposed AlphaFold2 models of the RRM-RS domain of RBM20 (amino acid 511-673) as wild type (grey), or with single point mutations: P633L (light blue) or R634Q (salmon), (g) Representative AlphaFold2 model of RBM20’s RRM-RS wild type domain (blue, amino acid 511-673) in complex with TNPO3 (grey, full length, amino acid 1-923). The known PRSRSP motif from amino acid 633 - 638 in RBM20 is highlighted as red spheres, (h) Predicted changes in binding affinity between TNPO3 and RBM20 after single point mutations in the RS-domain. 20 different AlphaFold models of the wild type RBM20-TNPO3 complex were independently used to calculate the affinity change with a single point mutation (see Material and Methods), (i) Quantification of the mean TNPO3 signal co-immunoprecipitating with RBM20 based on Western blot analysis (representative image is shown in Fig. 4j) from three replicates with standard deviations indicated, (j) Western blot analysis of RBM20, TNPO3, and MOVIO expression in the cytoplasmic fraction of reporter HeLa cells, as well as their levels co- immunoprecipitating with eGFP-RBM20 (Neg = negative control HeLa line with no bait), (k) Quantification of TNPO3 peptides identified by mass-spectrometry in the cytoplasmic fractions of co-immunoprecipitants with negative control, WT-, P633L-, R634Q-, or RSS-RBM20, normalized to the negative control. Ns = not significant, * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001, Student’s t-test for (b), and one-way ANOVA with Tukey’s HSD post-test for (c), (e).
Figure 5 shows that enhancing RBM20-TNPO3 interaction restores nuclear localization and splicing in vitro and in vivo, (a) Representative confocal microscopy images of RBM20 localization upon lentiviral overexpression of eGFP-TNPO3 in iPSC-CMs with WT, P633L, or R634Q-RBM20. Red arrows point at cells transduced with eGFP-TNPO3. (b) Quantification of RBM20 co-localization with DAPI based on the confocal microscopy data shown in panel (a). Each dot represents a Pearson correlation coefficient R for at least five cells, data are combined from three biological replicates. TNPO3 overexpression effect’s adjusted p-value = 0, Two-way ANOVA with Tukey’s HSD post-test, (c) qPCR analysis of TTN exon 232 (TNPO3 overexpression effect’s adjusted p-value = 0.057, Two-way ANOVA with Tukey’s HSD post- test) and (d) IMMToxon 6 splicing (TNP03 overexpression effect’s adjusted p-value = 0.015, Two-way ANOVA with Tukey’s HSD post-test) in WT, P633L, or R634Q-RBM20 iPSC-CMs with or without overexpression of eGFP-TNPO3 normalized to GAPDH expression and displayed as mean fold change versus the WT line without TNPO3 OE with standard errors indicated. Two biological replicates with two technical replicates for each of them were analyzed, (e) Schematic illustration of in vivo Tnpo3 OE experiment, (f) qPCR analysis of Tnpo3 expression in four WT mice, four P635L+/+ mice injected with PBS, or four P635L+/+ mice injected with Tnpo3. Data is normalized to Gapdh expression and displayed as mean fold change versus WT mice with standard errors indicated, (g) Representative confocal microscopy images of RBM20 localization in WT mice, P635L+/+ mice injected with PBS, or P635L+/+ mice injected with Tnpo3. Red arrows point at characteristic nuclear foci formed by RBM20. (h) qPCR analysis of Tin splicing in four WT mice, four P635L+/+ mice injected with PBS, or four P635L+/+ mice injected with Tnpo3. Data is normalized to Gapdh expression and displayed as mean fold change versus the WT mice with standard errors indicated, (i) RT-PCR of Ttn splicing and Gapdh expression in three WT mice, three P635L+/+ mice injected with PBS, and three P635L+/+ mice injected with Tnpo3. Red arrows point at Ttn isoforms expressed in WT mice.
Figure 6 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part). Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top). The bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 1. The interacting amino acids are not all conserved in mice, and therefore this binder may not bind to mouse RBM20.
Figure 7 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part). Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top). The bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 2. The interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20. Figure 8 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part). Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top). The bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 3. The interacting amino acids are not all conserved in mice, and therefore this binder may not bind to mouse RBM20.
Figure 9 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part). Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top). The bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 4. The interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20.
Figure 10 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part). Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top). The bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 5. The interacting amino acids are not all conserved in mice, and therefore this binder may not bind to mouse RBM20.
Figure 11 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part). Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top). The bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 6. The interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20.
Figure 12 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part). Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top). The bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 7. The interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20.
Figure 13 shows a schematic overview over the regions of RBM20 (upper right part), and an alpha fold predicted ribbon model of RBM20 with the same domain color code (upper left part). Below is the amino acid sequence comparison of the L-rich region of RBM20 between human and mouse (thin yellow bar on top). The bold dark green bars indicate the alpha helical parts, red boxes indicate the amino acids that were predicted by the model to interact with binder 8. The interacting amino acids are conserved in mice, and therefore this binder may bind to mouse RBM20.
Figure 14 shows the quantification of RBM20:DAPI co-localization, measured by Pearson R correlation coefficient. Fiji plugin Coloc2 was used to quantify this parameter. Each dot represents at least one cell. At least 10 cells per genotype per treatment were quantified. The higher the RBM20:DAPI correlation is, the more the RBM20 signal is in the nucleus. It can be seen that in cells (both HeLa and HEK293FT) expressing the binder 2 (Figure 7) (as shown by mScarlet expression), localization of RBM20-WT is not changed compared to non-expressing binder cells. However, the RBM20:DAPI correlation for RBM20-R634Q is significantly higher, and as high as observed for RBM20-WT, upon binder expression. **** - P<0,0001, one-way ANOVA with Tukey’s post test. Ns - p > 0.05.
Figure 15 shows splicing activity of RBM20 WT or R634Q in cells with or without binder. IMMT splicing (one of the well-documented RBM20 targets, Koelemen et al., 2021) was used as a readout. IMMT spliced and unspliced isoform expression was measured via qPCR, using primers targeting the alternative exon junctions (published in Kornienko et al., 2023). As can be seen, in HeLa cells, expression of the binder restores the alternative splicing of IMMT to the levels observed in WT cells, in line with the restoration of nuclear localization showed in Figure 14. In HEK cells an increase in IMMT spliced isoform expression was seen, although the effect was not significant. Importantly, no negative impact of the binder on RBM20 WT splicing activity could be found, indicating that the L-rich region binder 2 (Figure 7) does not affect RBM20s native splicing activity. These data are based on one biological replicate with three technical replicates (outliers are removed). **** - P<0,0001, one-way ANOVA with Tukey’s post test. Ns - p > 0.05.
Figure 16 shows qPCR analysis of in vivo Tnpo3 (panel a) and Tin spliced isoform (panel b) expression in the left ventricles of RBM20-WT, -P635L +/+, or -R636Q +/+ and +/- mice treated with either AAV9- pCAG-iCre-T2A-eGFP (Ctr) or AAV9-Tnpo3 (Tnpo3\ Data is normalized to Gapdh and displayed as fold change versus one of the RBM20-WT Ctr mice. Each dot represents a mean of two technical replicates per mouse, (a) demonstrates an elevated level of Tnpo3 expression in the hearts of Tnpo3 -treated mice, (b) demonstrates that these elevated levels of Tnpo3 expression are sufficient to restore the alternative splicing of Ttn in P635L +/+ and R636Q +/- mice to the levels that are not statistically different from those observed in WT mice.
Figure 17 shows the nuclear localization of (mutated) RMB20 and the effect of a binder in iPSC-CMs. a) images of stainings of wild type and mutated RBM20. b) box plot of colocalization of DAPI and RBM20 stainings. The binder 2 (Figure 7) causes RBM20 (mutated) to be localized in the nucleus.
SEQ ID NO: 1 shows the sequence of the NLS-sequence as used, PKKKRKVGGGS (SEQ ID NO: 1).
SEQ ID NOs 2 to 9 show the amino acid sequences of the L-rich region binding peptides.
Examples
Introduction/Summary
In the context of the present invention, the inventors showed that pathogenic RS-domain variants do not affect the splice regulatory activity, and that the splicing defect is only due to mislocalization of RBM20. The inventors uncovered the molecular basis of RBM20’s nuclear transport, and demonstrate how RS-domain mutations disrupt this process. The inventor’s findings have implications for the development of therapeutic strategies targeted at improving the nuclear import of mislocalized RBM20.
As a first exemplary embodiment, the inventors use an NLS nanobody (here an SV40 NLS tag) to stimulate the nuclear import of mutant RPM20 and to also restore proper splicing. For a validation in HeLa cells, an eGFP-NLS nanobody is used to test nuclear import and restored splicing of an eGFP-R634Q and eGFP-P633L-RBM20. For this, RBM20 specific nanobodies are generated and the reliability of such nanobody -NLS fusions in iPSC-CMs with R634Q or P633L mutations is tested.
Furthermore, the RBM20-NLS delivery method in vivo is optimized by testing AAV9; AAVMAYO (cDNA delivery) or lipid nanoparticles (mRNA delivery). Also an optimization of the treatment dosage and duration in vivo is performed.
Finally, an analysis of potential side effects and risks related to unspecific binding, half-life of the nanobody, its metabolism and catabolism is performed.
As a specific project, gene editing to introduce the endogenous NLS-tag is performed as follows The gRNA sequence and genome positioning of the tag is optimized with prime editing in iPSC- CMs. For testing in vivo AAVMAYO or AAV9 are used. Again, an optimization of the treatment dosage and duration in vivo is performed.
Finally, an analysis of potential side effects and risks related to off-target effects and genome editing is performed.
As a second exemplary embodiment, the inventors target the TNPO3-RBM20 interaction as identified as unique in the context of the present invention.
For TNP03 gene delivery, optimization of the delivery method is performed, testing AAV9, and AAVMAYO. Then an optimization of the treatment dosage and duration in vivo takes place, followed by an analysis of potential side effects and risks related to unwanted upregulation of transport of other TNP03 targets.
For TNPO3-RBM20 complex stabilization, the structure of RBM20 in the complex with TNP03 and its changes upon mutations is analyzed.
Then, a prediction of small molecules that may increase the TNPO3-RBM20 complex stability is done in silico, or a screen for potential small molecules is performed in vitro. Finally, an optimization of the delivery method, dosage and duration, and an analysis of potential side effects is performed.
Materials and methods
Patient data
A kindred with four members carrying the P633L variant in RBM20 was identified through cascade screening. The first identification of this variant of uncertain significance was found in a proband with dilated cardiomyopathy and heart failure in the seventh decade of life. Given ascertainment bias associated with comparing variant carriers identified by family screening to probands who generally present with more severe disease, the inventors compared these four RBM20 P633L relatives to individuals with pathogenic and likely pathogenic variants in the RS-domain identified through family screening only (i.e., non-probands). To assemble this cohort, the inventors identified genotype-positive family members who were diagnosed after family screening from 9 contributing inherited cardiomyopathy centers (Stanford Center for Inherited Cardiovascular Disease, the University of Heidelberg, the University Hospital Zurich, Johns Hopkins University, Brigham and Women’s Hospital at Harvard Medical School, the University of Pennsylvania, the University of Michigan, the University of British Columbia, and Children’s Hospital Atlanta). Data on age at presentation and initial echocardiogram was collected retrospectively by chart review. The study was approved by the independent internal review board (IRB) at each site, and patient consent was obtained as required by each individual institution.
Cell culture
Induced pluripotent stem cells (iPSCs)
The iPSC line used in this study was obtained from the Stanford Cardiovascular Institute Biobank, and single point mutations were engineered as described in [22] . iPSCs were cultured in monolayer in cell culture dishes coated for 1 h at room temperature with Vitronectin (VTN- N) Recombinant Human Protein, Truncated (Gibco, A14700). Cells were cultivated in the E8- Flex medium (Gibco A2858501) and split twice per week using Versene solution (Gibco, 15040066). For splitting, cells were washed once with PBS and incubated with Versene for 5- 10 minutes at room temperature. After that, Versene was aspirated, and cells were resuspended in fresh E8-Flex medium, and re-plated at the desired concentration. Freezing and thawing of cells was done in the presence of RevitaCell (1 : 100) supplement (Gibco, A2644501). Cry opreservation of cells was done in culture media supplemented with 1% RevitaCell and 10% DMSO. The EMBL Ethics committee approved the study protocol for iPSCs.
Cardiomyocyte differentiation
For cardiomyocyte differentiation, iPSCs were cultured as monolayer, and Wnt signaling was modulated as previously described50. Briefly, iPSCs were plated at low confluency on vitronectin-coated plates, to reach 70-80% 4 days post plating. On Day 0, the media was changed to RPMI 1640 (Gibco 21875034) + B27-insulin supplement (Gibco, A1895601) (RPMI+B27-ins) with addition of 4pM CHIR 99021 (LC Laboratories C-6556) in DMSO. On day 1, the medium from the day before was diluted by addition of the equal volume of RPMI+B27-ins. On day 3, medium was changed to RPMI+B27-ins with addition of 2 pM Wnt- C59 inhibitor (Tocris 5148) in DMSO. On Day 5 and Day 7, the medium was changed to RPMI+B27-ins without any additions. On Day 9, the medium was changed to RPMI + B27 supplement (Gibco, 17504044) (RPMI+B27). On Day 11, the medium was changed to RPMI 1640 with no glucose (Gibco, 11879020) with addition of 0.1 % Sodium DL-lactate (L4263- 100ml, Sigma). On Day 14, the medium was changed back to RPMI+B27. After day 16, the cells were passaged every two weeks on VTN-N coated plates until harvested for downstream analyses. For passaging, cells were washed 1 time with PBS and incubated in TrypLE Select Enzyme (10X) (Gibco A1217701) at 37°C for 10-15 minutes. After that, cells were resuspended in 4 times volume of Passaging media, composed of RPMI+B27 supplemented with 10% knockout serum replacement (Gibco 10828010) and 1,6 pM Thiazovivin (Stem Cell technologies, 72252). Cells were pelleted at 350g for 5 minutes and plated in fresh medium on pre-coated VTN-N plates. Medium was changed on the next day to RPMI+B27 and was then changed twice per week.
HeLa and HEK293FT culture
HeLa Tet-Cas9 [51] and HEK293FT (Thermo Fisher Scientific) were maintained in DMEM, high glucose (Gibco, 11965084) supplemented with 10% FBS Supreme (Pan Biotech, P30- 3031), 1% Sodium Pyruvate (Gibco, 11360070), and 1% Penicillin-Streptomycin (Gibco, 15140122). For splitting, cells were washed 1 time with PBS, and incubated with Trypsin- EDTA (0.25%) (Gibco 25200056) for 5 minutes at 37°C. After that, cells were resuspended in fresh media, diluted to the desired concentration, and plated. Cry opreservation of cells was done in the culturing media supplemented with 10% DMSO. Lentivirus production
HEK293FT cells were grown to 80-90% confluency and transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific) with two lentiviral packaging plasmids (pMD2.G and psPAX2), and a plasmid carrying the gene of interest mixed at 1 :1 : 1 ratio to obtain in total 2.5 ug of DNA per well of a 6-well plate. Six hours later, cells were split in approximately 1 :6 ratio (one well of a 6-well plate into a 10 cm tissue culture dish), and cultured at 37°C. Three days later, the supernatant was collected, and remaining cell debris was filtered through the 0.45pm filter. Filtered supernatant was then incubated with the addition of 1 :3 of its volume of LentiX concentrator (Takara/Clontech) at 4°C for at least Ih, to a maximum of overnight, followed by centrifugation at 4°C, 4000g, for 45 minutes. Supernatant was aspirated, and the viral pellets were resuspended in PBS in 1 :200 of the initial supernatant volume. Virus was aliquoted and stored at -80°C until usage.
Engineering of HeLa reporter cell lines
TetO-eGFP-GGSG-NLS-Flag-RBM20 plasmid was cloned via site directed mutagenesis-based insertion of the SV40 NLS sequence into the TetO-eGFP-GGSG-FLAG-RBM20 plasmid using GeneArt Site Directed Mutagenesis Kit (Thermo Fisher Scientific). The latter was cloned via Gibson assembly of the eGFP-GSSG cDNA, Flag-RBM20 cDNA (GenScript), as well as the fragment of TetO-lenti backbone (gift from Dr. Moritz Mall lab, DKFZ, Germany), amplified, purified via gel extraction kit (Qiagen), and incubated with Gibson assembly master mix (NEB) at 50°C for Ih, followed by transformation into 5-alpha E. coli (NEB), plasmid isolation, and sequence confirmation by Sanger sequencing. The single point mutations were introduced into RBM20 cDNA sequence using GeneArt Site Directed Mutagenesis Kit (Thermo Fisher Scientific) and suitable oligos.
For the NLS-rescuing experiment, HeLa Kyoto cells were co-transduced with TetO-eGFP- FLAG-NLS-RBM20-WT, -P633L, or -R634Q, as well as rtTA (Addgene 20342), cultured in the presence of 2 ug/ml of Doxycycline (Sigma) for at least 7 days, single cell sorted for eGFP fluorescence with FACS, and used for the experiment at least two weeks after the sort. pEFa-eGFP-GGSG-Flag-RBM20 plasmids were cloned via Gibson assembly (NEB) of the amplified TetO-backbone (see above), eGFP-GSSG-Flag-RBM20 cDNA from the TetO- plasmids (described above), and pEFa promoter sequence from the Addgene #125592 plasmid. HeLa Tet-Cas9 [51] were transduced with lentivirus delivering pEFa-eGFP-Flag-RBM20-WT, P633L, R634Q, or R634Q-S635E-S637E in 6-well plates by adding 20 pl of lOOx concentrated virus per well, and single-cell derived colonies were obtained by FACS. Two weeks after single cell sorting, the established lines were further analyzed for their purity with FACS and ICS, and the most stable and pure clonal lines were used for downstream applications.
Cell treatments siRNA transfection
HeLa Tet-Cas9 [51] cells were cultured until they reached a confluency of 20% in medium without Penicillin Streptomycin, and transfected using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) and 20 nM of either non-targeting control siRNA (D-001810-02), or siRNA targeting TNPO3 (L-019949-01) (Dharmacon, Horizon Discovery Group). Medium was replaced with the normal HeLa culture medium described above 24 hours later, and cells were harvested for downstream analyses 72 hours post transfection as described above.
Differentiated iPSC-CMs were cultured at 70-90% confluency in RPMI+B27 media and transfected using LipofectamineStem reagent (Thermo Fisher Scientific) and 100 nM of either control siRNA or siRNA targeting TNPO3. Media was changed to fresh RPMI+B27 media 24 hours later. Four days post transfection, cells were transfected again in the same way, changing medium 24 hours afterwards. After 72 hours post the second transfection, cells were harvested for downstream analyses as described above.
TNPO3 overexpression in iPSC-CMs eGFP-TNPO3 cDNA was purchased from Addgene (167590), and lentivirus was produced as described above. iPSC-CMs were cultured at 70-90% confluency in RPMI+B27 media, and transduced with 1 : 1000 volume of the virus. Media was changed to a fresh RPMI+B2724 hours after transfection, and again, 3 days later. Seven days post transduction, cells were either fixed for microscopy analysis (see below), or FACS sorted in bulk into 1.5 ml reaction tubes for further RNA extraction (see below), to directly compare eGFP-TNPO3 positive and negative cells.
Overexpression ofRBM20 variants in iPSC-CMs
TetO-eGFP-FLAG-RBM20-WT, -R634Q, or NLS-R634Q were cloned and packaged into lentiviral particles, as described above. iPSC-CMs with a frameshift mutation in RBM2(Ps RS- domain22 were cultivated at 70-90% confluency in RPMI+B27 medium, and co-transduced with 1 : 1000 volume of the virus delivering TetO-RBM20, as well as rtTA (Addgene 20342). 24 hours later, the media was changed to a fresh RPMI+B27 with addition of 2 pg/ml of Doxycycline (Sigma). Medium was changed again 3 days later to the RPMI+B27 with Doxycycline, and after a total of 7 days post infection, cells were harvested for downstream analyses as described above.
Cell staining
For viability staining, cell suspensions in PBS were stained with 50 pg/ml final concentration of DAPI (Thermo Fisher Scientific). For staining of nuclei of live cells, 100 mM of DRAQ5 (Biostatus) was added to the cell suspension in PBS at room temperature. Cells were analyzed with FACS or ICS not earlier than 5 min after addition of DAPI or DRAQ5.
Antibody staining for ICS
For ICS measurement of endogenous RBM20 localization, iPSC-CMs were harvested with TrypLE Select Enzyme (10X) (Gibco A1217701) (see above), resuspended in the Passaging media (see above), washed once with PBS, and fixed with 4% PFA in PBS at room temperature (RT) for 10 minutes. Then, cells were washed once with PBS, and permeabilized with 0.1% Triton-XlOO (Merck) in 1% BSA (Sigma) in PBS for 5 minutes at RT. Then, cells were incubated with 1 : 100 dilution of anti-RBM20 (ab233147, Abeam) antibody in 1% BSA in PBS for 1 hour at RT, followed by a wash in PBS. Cells were then incubated with 1 :500 dilution of AlexaFluor488 goat anti rabbit antibody (Invitrogen) in 1% BSA for 30 minutes at 4°C in the dark. After this, cells were washed once with PBS, and resuspended in PBS containing DAPI and DRAQ5 at the concentrations derived above.
Antibody staining for microscopy
Cells were cultured either in glass bottom plates, or on coverslips, for microscopy analysis, following cell culture conditions described above. Cells were washed once with PBS, fixed with 4% PFA in PBS for 10 minutes at RT, washed once with PBS, and permeabilized with 0.5% Triton-XlOO (Merck) in PBS for 5 minutes at RT. Then, the potential nonspecific antibody binding sites were blocked by incubation with 2% BSA (Sigma) in PBS for 1 hour at RT. Then, cells were incubated with 1 :250 dilution of primary antibodies (anti-RBM20 for human cells - ab233147, Abeam; anti-RBM20 for mouse cell staining - PA5-58068, Invitrogen; anti-sarcomeric alpha-actinin - ab9465, Abeam; anti-MOVIO - PLA0195, Sigma) in 1% BSA in PBS at 4°C overnight (or Ih RT for MOVIO staining). Cells were then washed 3 times with 2% BSA in PBS at RT, and incubated with goat-anti -rabbit AlexaFluor568 (Invitrogen) and goat-anti-mouse AlexaFluor488 (Invitrogen) antibodies at 1 :500 dilution in 1% BSA in PBS for 1 hour at RT in the dark. Cells were then washed 2 times with 2% BSA in PBS at RT in the dark, and incubated in 2 pg/ml Hoechst 33258 (Invitrogen) in PBS for 10 minutes at RT in the dark for cells cultured on the microscopy plates, or with just PBS for 10 minutes at RT in the dark for coverslips. Microscopy plates were then washed once with PBS and stored at 4°C in the dark until imaged. Coverslip were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and stored at 4°C in the dark until imaged.
FACS and ICS
For all FACS and ICS applications, after being stained, cells were filtered through a 35 pM cell strainer to avoid clumping, and kept on ice until analyzed. For single-cell FACS sorting, cells were sorted based on the desired fluorophore expression into 96-well plates containing culture media, one cell per well. For bulk FACS sorting, cells were sorted into 1.5 ml microcentrifuge tubes containing DMEM with 10% FBS, based on the desired fluorophore expression. For all sterile sorts, BD FACS Aria™ Fusion was used, using a 100 pm sort nozzle. Viability staining with DAPI was used to sort out dying cells. For routine checking of transfection/transduction efficacies, as well as for optimizing experimental conditions, BD LSRFortessa™ was used. Image-enabled cell sorting (ICS) used the BD CellView™ Imaging Technology as previously described5. ICS experiments were performed with a 100 pm sort nozzle, with the piezoelectric transducer driven at 34 kHz, automated stream setup by BD FACSChorus™ Software, and a system pressure of 20 psi. All sorts were performed in purity mode.
For correlation-based sorts and measurements, cells were stained with DRAQ5, and for each cell, a Pearson correlation coefficient R was calculated based on the overlap between RBM20 and DRAQ5. Flow cytometry and ICS data were analyzed using FlowJo_vl0.7.1_CL software.
Microscopy
Widefield fluorescence microscopy analysis was performed using Zeiss Cellobserver microscope equipped with an AxioCam camera, using Plan-APOCHROMAT 20x NA0.8 Air DIC2 or LD Plan-NEOFLUAR 40x NA 0.6Air Ph2 correction collar 0-1.5 objectives. Confocal microscopy analysis was performed using Zeiss LSM900 microscope equipped with 405 nm - 5 mW, 488 nm - 10 mW, 561 nm - 10 mW, 640 nm - 5 mW lasers, using Objective Plan-Apochromat 40x/0.95 Corr M27 air (FWD=0.25mm) objective, and 3 Gallium Arsenide Phosphid-PMT (GaAsP-PMT) for fluorescence detection, standard PMT as transmission detector.
Co-localization analysis
For colocalization analysis of RBM20 and DAPI, Fiji (v.2.1.0/1.53c) plugin Coloc2 was used to reflect Pearson correlation coefficient R. For confocal images, Z-stack images were max projected, and fluorescent channels were split. Area covering at least five cells was selected in RBM20 channel, and was used as ROI/mask for quantification of its correlation with DAPI channel.
RNA extraction
Live cells
Cells cultured in tissue culture dishes were washed 3 times with PBS, lysed in TRIzol (Invitrogen), and transferred to 1.5ml reaction tubes. RNA extraction and DNAsel treatment was performed using the Direct-zol RNA Miniprep Plus Kit (Zymo Research), according to the manufacturer's instructions.
Fixed cells
Fixed iPSC-CMs (at least 5000 cells per sample) were pelleted by centrifugation at 500g for 3 minutes and resuspended in 16 pl of a 1 : 16 proteinase K in PKD buffer (Qiagen), incubated at RT for 5 minutes, briefly spun down, and incubated at 56°C for one hour. The solution was then resuspended in 100 pl of TRIzol LS Reagent (Thermo Fisher Scientific). Then, 20 pl of chloroform was added to the TRIzol-sample, and phase separation was achieved at RT by vigorous shaking and centrifugation at 12,500 r.p.m. for 5 min. From each sample, 40 pl of the aqueous phase were collected, transported to a new eppendorf tube, and mixed with 75.5 pl of isopropanol, and 1 : 150 of glycoblue (Invitrogen). The samples were then left at -80°C for 24- 36 hours to dehydrate. RNA was then precipitated by centrifugation at 4°C at maximum speed for 15 minutes. The supernatant was removed, and the pellet was washed once with 70% ethanol. The pellet was then air dried, and resuspended in 8 pl of nuclease-free water.
Tissue A piece of left ventricle was homogenized in PBS, spun down, and the pellet was then resuspended in TRIzol (Invitrogen). This was followed by RNA extraction and DNAse I treatment using the Direct-zol RNA Miniprep Plus Kit (Zymo Research), according to the manufacturer's instructions. RNA concentration was measured using Qubit High Sensitivity RNA kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
Quantitative RT-PCR
For cDNA synthesis, the SuperScript IV (Thermo Fisher Scientific) kit was used according to the manufacturer's instruction, with addition of 0.5 mM of each dNTP (NEB), 2.5 pM oligo-dT (Thermo Fisher Scientific), 1.25 pM random hexamer primers (Invitrogen), 5 mM DTT (Thermo Fisher Scientific), 2 u/pl RNAse inhibitor (Invitrogen), IX SSIV buffer (Thermo Fisher Scientific), and 10 u/pl SSIV RT (Thermo Fisher Scientific), per each reaction. At least 10 ng of total RNA was used per reaction, but not more than 1 pg.
A one-step qPCR reaction (95°C for 10 minutes, 40 cycles of [95°C 15 seconds, 60°C 1 minute]) was performed using SYBR Green Master Mix (Thermo Fisher Scientific), and primers listed in Supplementary Table 2, using Applied Biosystems StepOnePlus Real-Time PCR System (272006365), and StepOne Software v2.3. Delta-delta cT values were quantified versus GAPDH as a housekeeping gene, and versus a control sample for each experiment.
RNA sequencing
Library preparation and sequencing
Prior to library preparation, RNA quality was checked using the 2100 RNA pico Bioanalyzer (Agilent) kit, and 1-10 ng of RNA were used as input. To prepare RNA sequencing libraries from ultra low-input and highly degraded RNA (RIN 1- 7) extracted from fixed samples (see above), SMARTer RNA-Seq Kit v3 - pico (Takara Bio) was used, according to the manufacturer's instructions. The fragmentation step was omitted. Prepared libraries with unique dual index barcodes for each sample were double checked on Bioanalyzer (Agilent), and pooled together at equimolar concentrations, with six libraries per pool. Each pool was sequenced individually, with final concentrations of 8-10ng/ml for each pool. For each pool, a 2.1 pM solution was loaded on the Illumina sequencer NextSeq 500 and sequenced bi-directionally, generating -500 million paired-end reads, each 75 bases long. Obtained reads were then demultiplexed based on the unique dual barcodes into separate fastq files. Data analysis
Quality control of the sequencing data was done using FASTQC (vO.l 1.5). Reads were aligned to GRCh38.101 using STAR (v2.7.5c), and bam files were sorted by coordinate using samtools (v.1.9). Read count files were generated with featureCounts [52] vl.6.4 for each exon. For differential gene expression analysis, DeSeq2 [53] (v. 1.36.0) was used, and pairwise comparisons between genotypes were performed (expression ~ genotype). Adjusted p values were calculated using the Benjamin & Hochberg method. A gene was considered differentially expressed if the log2 of its expression fold change was greater than 0.5, and if the adjusted p- value was less than 0.1. Pathway enrichment analysis was performed using Metascape [54],
Analysis of differential splicing events was performed using rMATS turbo [55] (v4.1.1). An alternative splicing event was considered significant if the absolute value of inclusion level difference was greater than 0.05, and if the false discovery rate was less than 0.01. The list of RBM20 target genes was taken from [13],
To calculate the percentage of spliced-in values (PSI-values), first, for each exon, inclusive and exclusive reads were identified. A read is considered inclusive, if it includes the exon of interest. A read is considered exclusive, if it spans both the upstream and downstream exons but does not align to the exon of interest. For each exon, inclusive and exclusive reads were counted directly from the BAM files as described in [56], The PSI-values are then calculated as the ratio of inclusive reads to the sum of inclusive and exclusive reads per each exon. For selecting the most differentially spliced exons for a plot, a Student's T-test was used to determine exons of RBM20 target genes with p-value < 0.05, and with absolute value of differences between means of PSI-values greater than 0.05, only between WT and R634Q samples.
Cell-based Luciferase TTN splicing reporter assay
HEK293 cells were seeded on 96-well plates and transfected at 50% confluence with PEI40 at a 1 :3 ratio (DNA: PEI40) and a total of 200 ng of plasmid DNA (1 ng splice reporter TTN-IG Ex241-243 and a 20x molar excess of the RBM20 expression plasmids (compare [57] for WT, mutations were introduced by site-directed mutagenesis in a two-step cycle PCR approach) or control plasmid pcDNA3.1 (Invitrogen, Cat# V79520).
Plasmids and PEI40 in FBS-free medium were incubated for 15 min before the transfection mixture was added to the cells. Each transfection experiment was repeated ten times and cell viability was measured 60 hours post-transfection using PrestoBlue (Thermo Fisher Scientific, cat# Al 3261). Luciferase activity was measured 60 hours post-transfection using the DualLuciferase® Reporter Assay System (Promega) on an Infinite® M200 Pro (TECAN) plate reader. Ratios of firefly to renilla luciferase activity were normalized to the WT RBM20 expressing cells. All data are expressed as the mean of biological replicates (n = 10) ± SEM. Group comparisons were analyzed by one-way ANOVA and Bonferroni posttest. P values were considered statistically significant as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
CRISPR/Cas9 gRNA library design and cloning
The genome-wide CRISPR/Cas9 gRNA libraries were designed as described in [5], Briefly, the library targets 18,408 protein-coding genes listed in the Consensus Coding Sequence Database [58], It consists of six independent sub-libraries, each containing one gRNA per gene [5], Each of these sub-libraries contains the same 118 targeting and 487 nontargeting controls. The library was cloned into the CROPSeq-guide(F+E)-Puro backbone (see sequences), as described in [5], gRNA representation of the genome-wide library at the plasmid stage was checked previously [5], and used as a plasmid stock for the lentivirus generation for this work. These data were also used as a reference for gRNA representation at the plasmid stage for the downstream data processing (see below).
Cloning of individual gRNAs
For individual knockout experiments, gRNAs were synthesized as two short oligos with flanking sequences resembling the Esp3I sticky ends of CROPSeq-guide(F+E)-Puro vector. The oligos (10 mM each) were phosphorylated and annealed using 1 U/pl T4 PNK (NEB), and IX T4-ligase buffer (NEB) in a thermocycler with the following program: 37°C 30 min, 65°C 20 min, 95°C 5 min, ramp down to 25°C at 5°C/min. The phosphorylated and annealed oligos (1 pl) were then ligated with 25 ng of Esp3I-digested (NEB) and gel-extracted (Qiagen) CROPSeq-guide(F+E)-Puro backbone using 1 pl of T4 ligase (NEB), and IX T4 ligase buffer (NEB) in total volume of 10 pl, for 10 minutes at RT, inactivated for 10 min at 65 °C followed by transformation into the NEB Stable competent E. coli (NEB), according to the manufacturer's instructions. Lentivirus for cell transductions was produced as described above.
Pooled and individual CRISPR perturbations
For the pooled screening experiment, HeLa Tet-Cas9 pEFa-eGFP-RBM20-WT were plated with a density of 750 000 cells per 15 cm tissue culture dish, and cultured for three days until a confluency of 40% was reached (6,000,000 cells per 15 cm dish). To achieve >500X gRNA coverage, 60xl06 of cells were transduced per each genome-wide library (ten 15-cm dishes per library). Cells were infected with lentivirus delivering the genome-wide library, with 25 l of 100X concentrated virus per plate, at a low infectivity rate to allow only single qRNA integrations per cell. Twenty -four hours later, cells were trypsinized, resuspended in the culture medium containing 2 ng/pl of Puromycin (Thermo Fisher Scientific), and plated back to the same dishes. Next day, the medium was changed to fresh Puromycin-containing culture medium, to wash away dead cells. Around 20% of cells got Puromycin resistance and were further kept in culture. Three days later, and for the next seven days, cells were split every three days and cultured in the presence of 2 ng/pl Puromycin (Thermo Fisher Scientific) and 2 pg/ml Doxycycline (Sigma) to activate Cas9. After seven days of being cultured in the presence of Doxycycline, the culturing medium containing only Puromycin was used until cells were harvested for ICS. At each splitting, cells infected with the same library from all plates were pooled together after trypsinization, and 1,500,000 of cells were plated per each new of seven 15-cm dishes, keeping the coverage at >500X for each individual genome-wide library. Three days prior to harvesting for ICS, 1,500,000 of cells were plated to fifteen 15-cm dishes for each library, and all of them were used for sorting.
For individual CRISPR perturbations, cells were cultured in 6-well plates until 40% confluency (150,000 cells per well), and 10 pl of 50X concentrated virus were added per each well. On the next day, cells were trypsinized, resuspended in the medium containing 2 ng/pl Puromycin (Thermo Fisher Scientific), and plated back to the same wells. The next day, the medium was changed to a fresh Puromycin-containing medium. Once cells reached 90-100% confluency, they were split and cultured in medium containing 2 ng/pl Puromycin (Thermo Fisher Scientific) and 2 pg/ml Doxycycline (Sigma) for the first seven days, followed by seven days of only Puromycin-containing medium. Cells were split twice per week. At least 16 days post transduction, cells were harvested for downstream analyses as described above.
ICS-based CRISPR screen
Cells were prepared as described above using lentiviral transduction. Samples were kept at 4°C at all times between harvest and genomic DNA isolation after sorting. Sorting was performed as described before5 with the following modifications. Cells were sorted in batches of 100,000 cells in the collection fractions and the input samples were refreshed regularly by the addition of concentrated cell suspension to a total volume of 1 ml. For the selection of the populations from the eGFP-DRAQ5 correlation parameter, ranged gates were drawn comprising the 7% of cells with the lowest or highest correlation index. From each batch of cells used for sorting, an input sample containing the same number of cells as present in the sorted upper and lower sample was collected. Sorted samples and input samples were collected by centrifugation for 5 min at 500g at 4 °C, and pellets were either frozen at -20 °C or stored on ice until gDNA preparation. One million cells were collected per library and pooled into a single tube for gDNA preparation.
Genomic DNA isolation, library preparation, and sequencing
Genomic DNA was isolated from the sorted cells using NEB Monarch genomic DNA purification kit (New England Biolabs), including the RNase treatment and elution in 50 pl elution buffer. DNA concentration was measured using Qubit High sensitivity dsDNA kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
PCR1 was done with 125-525 ng of gDNA (per reaction, 6 reactions per library), 1.5 pl of 10 pM pU6 fwd, 1.5 pl of 10 pM pLTR-CROP-rev, and 25 pl KAPA HiFi Hotstart Readymix (Roche) in 50 pl total volume. For each gRNA sublibrary, six 50 pl reactions were set up using the total amount of gDNA recovered from sorted cells and the input samples. Cycling conditions for PCR1 were one cycle at 95 °C for 3 min; 24 cycles at [98 °C for 20 s, 67 °C for 15 s, 72 °C for 15 s]; one cycle at 72 °C for 1 min; and cooling to 4 °C. PCR reactions of the same template (same sublibrary) were pooled (six PCR products into one of total volume 300 pl) and the product was purified with 0.8X volume of AMPure XP (Beckman) with two 80 % ethanol washes, and elution in 40 pl water. Concentrations were then measured with Qubit High sensitivity dsDNA kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
PCR2 was done with 10 ng PCR1 product (per reaction, 6 reactions per library), 5 pl of 3 pM CROPseq_libQC_i5_s:n staggered primer59 (different primer for each reaction for one sample), 5 pl of 3 pM CROPseq_i7:n barcoded primer (same for all reactions for one sample, but unique to every sample), and 25 pl KAPA HiFi Hotstart Readymix (Roche) in 50 pl total volume. Same primers were used as in5, and are shown in Supplementary Table 4. Cycling conditions for PCR2 were one cycle at 95 °C for 3 min; 8 cycles at [98 °C for 20 s, 67 °C for 15 s, 72 °C for 15 s]; one cycle at 72 °C for 1 min; and cooling to 4 °C. Product was purified as above and eluted in 40 pl H2O. Concentrations were then measured with Qubit High sensitivity dsDNA kit (Thermo Fisher Scientific), and ready libraries were checked using DNA 1000 Bioanalyzer (Agilent) to yield a single product around 300bp.
For Illumina sequencing, libraries were pooled in equimolar ratio (nine libraries per pool) and sequenced using an Illumina NextSeq 500 (75bp, single end mode) in high output mode with 8 reads to read out the i7 barcode, and 67 reads on Readl to read through the stagger sequence and identify the gRNA. PhiX spike-in was used to diversify the libraries.
CRISPR screen data analysis
Abundance of gRNA was quantified from the sequencing reads using MAGeCK (v 0.5.9) tool with default parameters [60], To account for differences in sequencing depth, raw gRNA counts were normalized to the median count of the targeting control gRNAs in the corresponding sample. Scaling of the normalized counts was done by multiplication with the median count of targeting controls across all samples. The evaluation of screen quality was done as described in [5] based on the dropout of essential genes in input cell populations compared to the plasmid library (sequenced previously in [5]) with reference core- and non-essential gene lists described previously in [49], All precision-recall-curves were generated using the R package “ROCR” [61], The MAUDE (v. 0.99.4) [48] package for R was used for hit calling, using targeting controls as a reference for MAUDE analysis. False discovery rates for each plot are indicated in the main text.
Whole cell extract preparation, cell fractionation and western blotting
For whole cell extracts, cells pellets were lysed in NP-40 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% (v/v) NP-40. All protein extraction buffers contain PhosSTOP (Sigma-Aldrich, 04906837001) and Protease Inhibitor Cocktail (Sigma-Aldrich, 05056489001). Cell fractionation was performed as follows. First, cells were resuspended in 2 pellet volumes of hypotonic buffer (10 mM HEPES pH 7.5, 10 mM KC1, 1.5 mM MgCU) incubated on ice for 15 min and homogenized with 20 strokes using a loose pestle. Nuclei and insoluble cellular compartments were pelleted at 3,900 rpm for 15 min and supernatant collected as soluble cytoplasmic fraction, which was corrected to 10% (v/v) glycerol, 3 mM EDTA, 0.05% (v/v) NP-40 and 150 mM NaCl final concentration. The remaining pellet was resuspended in chromatin digestion buffer (20 mM HEPES pH 7.9, 1.5 mM MgCh, 10% (v/v) glycerol, 150 mM NaCl, 0.1% (v/v) NP-40 and 125 U benzonase (MerckMillipore, 70746-4) and incubated for 1 h at 4°C. To ensure extraction of all nuclear soluble and insoluble proteins, as well as of insoluble cytoplasmic components, NaCl concentration was then increased to 500 mM and samples were incubated on ice for 30 minutes. Prior to centrifugation at 20,000 g for 20 min at 4°C, the salt concentration was diluted back to 150 mM NaCl by addition of high salt dilution buffer (20 mM HEPES pH 7.9, 3 mM EDTA, 1.5 mM MgCl2, 10% (v/v) glycerol, 500 mM NaCl and 0.1% (v/v) NP-40) and the supernatant was kept as nuclear and insoluble fraction. 30-100 pg protein/lane was separated on 4%-12% or 3-8% NuPage gels (Invitrogen) and transferred to Trans-Blot Turbo Mini 0.2 pm Nitrocellulose (biorad 1704158) using TransBlot Turbo Transfer System. Membranes were blocked in 5% (w/v) skimmed milk in PBS-T (PBS, 0.1% (v/v) Tween20) for 1 h at room temperature and incubated with primary antibody (in 5% (w/v) skimmed milk in PBS-T) overnight at 4°C. Antibody against GAPDH were used to control loading when necessary. Membranes were washed several times in PBS-T, incubated with HRP-conjugated secondary antibody in 5% (w/v) skimmed milk in PBS-T and visualized using SuperSignal West Dura Chemiluminescent Substrate ECL reagent (Thermo Fisher Scientific, 34075) and visualize using Bio-Rad ChemiDoc Touch (Software v. 2.3.0.07). The limitation of this method seems that, together with the nuclear fraction, the insoluble cytoplasmic fraction gets extracted into the same reaction tube, which explains why some cytoplasmic proteins can be found in the nuclear fraction.
Co-immunoprecipitation
For GFP immunoprecipitations, 1 mg of the whole cell extracts, cytoplasmic fraction or nuclear and insoluble fractions were incubated with 30 pl of GFP-Trap® Magnetic Particles M-270 (ChromoTek) for at 4°C for 3 h. Beads were washed 5 times in IP wash buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.5, 1.5 mM MgCl2, 3mM EDTA, 10% (v/v) glycerol, 0.1% (v/v) NP-40, phosphatase inhibitors (PhosSTOP, Sigma-Aldrich, 04906837001) and protease inhibitor cocktail (Sigma-Aldrich, 05056489001) and eluted in 30 pl of laemmli buffer with 100 pM DTT.
Mass spectrometry
LC-MS/MS analysis.
Samples were subjected to an in-solution tryptic digest using a modified version of the SinglePot Solid-Phase-enhanced Sample Preparation (SP3) protocol (PMID: 25358341, PMID: 29565595). Eluates were added to Sera-Mag Beads (Thermo Scientific, #4515-2105-050250, 6515-2105-050250) in 10 pl 15% formic acid and 30 pl of ethanol. Binding of proteins was achieved by shaking for 15 min at room temperature. SDS was removed by four subsequent washes with 200 pl of 70% ethanol. Proteins were digested overnight at room temperature with 0.4 pg of sequencing grade modified trypsin (Promega, #V5111) in 40 pl Hepes/NaOH, pH 8.4 in the presence of 1.25 mM TCEP and 5 mM chloroacetamide (Sigma- Aldrich, #C0267). Beads were separated, washed with 10 pl of an aqueous solution of 2% DMSO and the combined eluates were dried down.
Peptides were reconstituted in 10 pl of H2O and reacted for 1 h at room temperature with 80 pg of TMT6plex (For inputs, Thermo Scientific, #90066) or with 40 pg of TMTpro (For eluates, Thermo Scientific, #A44522) label reagent dissolved in 4 pl of acetonitrile. Excess TMT reagent was quenched by the addition of 4 pl of an aqueous 5% hydroxylamine solution (Sigma, 438227). Peptides were reconstituted in 0.1 % formic acid, mixed to achieve a 1 : 1 ratio across all TMT-channels and purified by a reverse phase clean-up step (OASIS HLB 96-well pElution Plate, Waters #186001828BA).
Pull downs were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer (Thermo Scentific) as previously described (PMID:30858367). To this end, peptides were separated using an Ultimate 3000 nano RSLC system (Dionex) equipped with a trapping cartridge (Precolumn Cl 8 PepMaplOO, 5 mm, 300 pm i.d., 5 pm, 100 A) and an analytical column (Acclaim PepMap 100. 75 x 50 cm C18, 3 mm, 100 A) connected to a nanospray-Flex ion source. The peptides were loaded onto the trap column at 30 pl per min using solvent A (0.1% formic acid) and eluted using a gradient from 2 to 40% Solvent B (0.1% formic acid in acetonitrile) over 2 h at 0.3 pl per min (all solvents were of LC-MS grade). The Orbitrap Fusion Lumos was operated in positive ion mode with a spray voltage of 2.4 kV and capillary temperature of 275 °C. Full scan MS spectra with a mass range of 375-1500 m/z were acquired in profile mode using a resolution of 120,000 (maximum fill time of 50 ms or a maximum of 4e5 ions (AGC) and a RF lens setting of 30%. Fragmentation was triggered for 3 s cycle time for peptide like features with charge states of 2-7 on the MS scan (data-dependent acquisition). Precursors were isolated using the quadrupole with a window of 0.7 m/z and fragmented with a normalized collision energy of 38. Fragment mass spectra were acquired in profile mode and a resolution of 30,000 in profile mode. Maximum fill time was set to 64 ms or an AGC target of le5 ions). The dynamic exclusion was set to 45 s.
For Inputs: Peptides were subjected to an off-line fractionation under high pH conditions (PMID: 25358341). The resulting 12 fractions were then analyzed on a QExactive plus. Peptides were separated on an UltiMate 3000 RSLC nano LC system (Dionex) fitted with a trapping cartridge (p-Precolumn C18 PepMap 100, 5pm, 300 pm i.d. x 5 mm, 100 A) and an analytical column (nanoEase™ M/Z HSS T3 column 75 pm x 250 mm C18, 1.8 pm, 100 A, Waters). Trapping was carried out with a constant flow of trapping solution (0.05% trifluoroacetic acid in water) at 30 pL/min onto the trapping column for 6 minutes. Subsequently, peptides were eluted via the analytical column running solvent A (3% DMSO, 0.1% formic acid in water) with a constant flow of 0.3 pL/min, with increasing percentage of solvent B (3%DMSO, 0.1% formic acid in acetonitrile). The outlet of the analytical column was coupled directly to an Orbitrap QExactive™ plus Mass Spectrometer (Thermo Fisher Scientific) using the Nanospray Flex™ ion source in positive ion mode.
The peptides were introduced into the QExactive plus via a Pico-Tip Emitter 360 pm OD x 20 pm ID; 10 pm tip (CoAnn Technologies) and an applied spray voltage of 2.2 kV. The capillary temperature was set at 275°C. Full mass scan was acquired with mass range 375-1200 m/z in profile mode with resolution of 70000. The filling time was set at maximum of 100 ms with a limitation of 3x106 ions. Data dependent acquisition (DDA) was performed with the resolution of the Orbitrap set to 17500, with a fill time of 50 ms and a limitation of 2xl05 ions. A normalized collision energy of 32 was applied. Dynamic exclusion time of 20 s was used. The peptide match algorithm was set to ‘preferred’ and charge exclusion ‘unassigned’, charge states 1, 5 - 8 were excluded. MS2 data was acquired in profile mode
MS Data analysis.
Acquired data were analyzed using IsobarQuant (PMID: 26379230) and Mascot V2.4 (Matrix Science) using a reverse UniProt FASTA Homo sapiens database (UP000005640) including common contaminants and the expressed bait sp|P2147_GFPflagRBM20WT|P2147_GFPflagRBM20WT.
The following modifications were taken into account: Carbamidomethyl (C, fixed), TMTIOplex (K, fixed), Acetyl (N-term, variable), Oxidation (M, variable) and TMTIOplex (N- term, variable). The mass error tolerance for full scan MS spectra was set to 10 ppm and for MS/MS spectra to 0.02 Da. A maximum of 2 missed cleavages were allowed. A minimum of 2 unique peptides with a peptide length of at least seven amino acids and a false discovery rate below 0.01 were required on the peptide and protein level (PMID: 25987413). Raw data processing
For RBM20 co-IP in siRNA control vs. siRNA TNP03. The raw output files of IsobarQuant (protein.txt - files) were processed using the R programming language (ISBN 3-900051-07-0). Only proteins that were quantified with at least two unique peptides were considered for the analysis. 159 proteins passed the quality control filters. Raw TMT reporter ion intensities (‘signal sum’ columns) were first cleaned for batch effects using limma (PMID: 25605792) and further normalized using vsn (variance stabilization normalization - PMID: 12169536). Proteins were tested for differential expression using the limma package. The replicate information was added as a factor in the design matrix given as an argument to the ‘ImFit’ function of limma. Also, imputed values were given a weight of 0.05 in the ‘ImFit’ function. A protein was annotated as a hit with a false discovery rate (fdr) smaller 0.05 and a fold-change of at least 100 % and as a candidate with an fdr below 0.02 and a fold-change of at least 50 %.
For RBM20 co-IP in WT, P633L, R634Q and RSS. The raw output files of IsobarQuant (protein.txt - files) were processed using the R programming language (ISBN 3-900051-07-0). Only proteins that were quantified with at least two unique peptides were considered for the analysis. Moreover, only proteins which were identified in two out of three mass spec runs were kept. 771 proteins passed the quality control filters. Data for nuclear and cytoplasmic fractions were analyzed separately. Raw TMT reporter ion intensities (‘signal_sum’ columns) were first cleaned for batch effects using limma (PMID: 25605792) and further normalized using vsn (variance stabilization normalization - PMID: 12169536). Missing values were imputed with ‘knn’ method using the Msnbase package (PMID: 22113085). Proteins were tested for differential expression using the limma package. The replicate information was added as a factor in the design matrix given as an argument to the ‘ImFit’ function of limma. Also, imputed values were given a weight of 0.05 in the ‘ImFit’ function. A protein was annotated as a hit with a false discovery rate (fdr) smaller 0.05 and a fold-change of at least 100 % and as a candidate with an fdr below 0.02 and a fold-change of at least 50 %, compared to the no-bait control. All proteins, classified as hits or candidates in comparison to the negative no bait control, were considered to be interactors with RBM20 in a given sample. Common between WT and the mutant variants, as well as unique for mutant variants only, interactors were then analysed for pathway enrichment with Metascape [54], For Inputs. The raw output files of IsobarQuant (protein.txt - files) were processed using the R programming language (ISBN 3-900051-07-0). Only proteins that were quantified with at least two unique peptides were considered for the analysis. 4553 proteins passed the quality control filters. Raw TMT reporter ion intensities (‘signal sum’ columns) were first cleaned for batch effects using limma (PMID: 25605792) and further normalized using vsn (variance stabilization normalization - PMID: 12169536). Proteins were tested for differential expression using the limma package. The replicate information was added as a factor in the design matrix given as an argument to the ‘ImFit’ function of limma. Also, imputed values were given a weight of 0.05 in the ‘ImFit’ function. A protein was annotated as a hit with a false discovery rate (fdr) smaller 0.05 and a fold-change of at least 100 % and as a candidate with an fdr below 0.02 and a foldchange of at least 50 %.
AlphaFold and MutaSeq predictions
The inventors employed AlphaFold2 (AF2) [35, 36] within the JupyterHub on the EMBL Hamburg HYDE cluster. The default settings of AF2 in Multimer mode (v2.2.2) were used with three recycling rounds per model, with enabled amber relaxation and a total of 5 predictions per model. This resulted in a total of twenty-five predictions per AlphaFold2 run. The predictions were performed with the full-length amino acid sequence of the canonical TNPO3 sequence (amino acid 1-923 of Q9Y5L0, NM_012470.4) and with full-length RBM20 (amino acid 1- 1227 of Q5T481, NM_001134363.3) or only with amino acid sequence 511-673 to predict the RRM-RS domain. All obtained predictions for RBM20’s RS-domain had low pLLDT scores, which indicated the intrinsically disordered nature of this region. The obtained Predicted Aligned Error (PAE) plots of TNPO3 in complex with the RRM-RS domain of RBM20 suggests that both proteins are presumably incorrectly located relative to each other and should not be interpreted. However, based on prior knowledge from the literature that the RS-domain is the right binding site of TNPO3 lead to the idea to use the top twenty models of the complex to account for the uncertainty of domain orientation. The inventors used the Mutabind2 server [39] with all 20 predictions to test the effect of two single amino acid exchanges (P633L or R634Q) within RBM20 on the binding affinity with TNPO3.
Mouse handling and treatments
RBM20-WT or -P635L-Hom strains in a C57BL/6J genetic background were used for experimental procedures. The animals were maintained in individually ventilated plastic cages (Tecniplast) in an air-conditioned (temperature 22 °C ± 2 °C, humidity 50% ± 10%) and light- controlled room (illuminated from 07:00 to 19:00 h). Mice were fed 1318 P autoclavable diet (Altromin, Germany) ad libitum. All animal care and procedures performed in this study conformed to the EMBL Guidelines for the Use of Animals in Experiments and were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
AAV9 production pCMV-Tnpo3 was cloned into the AAV9 packaging backbone (derived from Addgene 137177, gift from the Genetic and Viral core facility, EMBL Rome, Italy) by digesting the backbone with Bglll and Agel (NEB), and amplifying the murine Tnpo3 cDNA (GenScript), followed by Gibson assembly of the two fragments (NEB).
The serotype 9 rAAV containing pCMV-Tnpo3 cDNA was produced in HEK-293T/17 cells using the triple-transfection method with linear PEI (25 kDa) in a Corning Hyperflask. After 72 hours, the cells were lysed and DNA was degraded by adding Triton X-100 (final concentration of 1%) and 19 pl Bensonase (25-35 U/pl) for Ihr at 37°C with 200 rpm shaking62. The cell debris/virus mix was removed and the Hyperflask was washed with 200 ml, sterile IxPBS. The washing solution and the cell suspension were centrifuged at 4000 xg for 20 min. The supernatant was filtered with a 0.45 pm PES filter and then concentrated to a total volume of 30 ml using tangential flow filtration [62], The concentrated virus was then purified by standard methods with an iodixanol gradient. The 200pl final volume of virus in PBS with 0.001% pluronic F-68 was aliquoted and the titer (3.3xl013) was determined by qPCR using primers within the CMV promoter.
Isolation of primary mouse cardiomyocytes for imaging
Microscopy slides were coated with 10 pl/ml of laminin (Gibco, 23017015) in PBS overnight.
For CM isolation, the inventors adapted the protocol described in [63], Briefly, for heart perfusions, the following buffers were used: EDTA buffer (130 mM NaCl, 5 mM KC1, 0.5 mM NaH2PC>4, 10 mM HEPES, 10 mM Glucose, 10 mM 2,3 Butanedione monoxime, 10 mM Taurine, 5 mM EDTA), Perfusion buffer (130 mM NaCl, 5 mM KC1, 0.5 mM NaH2PO4, 10 mM HEPES, 10 mM Glucose, 10 mM 2,3 Butanedione monoxime, 10 mM Taurine, 1 mM MgCl2), Collagenase buffer (Perfusion buffer, 1,5 mg/ml Collagenase II (Gibco, 17101015), 1,5 mg/ml Collagenase IV (Gibco, 17104019), 0,15 mg/ml Protease Type XIV (Sigma, P5147) and stop solution (Perfusion buffer, 5% FBS Supreme (Pan Biotech, P30-3031)). The Collagenase buffer was pre-warmed to 37°C prior usage. Mice were anesthetized in a CO2 chamber, opened up, and the descending aorta and vena cava were both cut. Then, 7 ml of the EDTA buffer were injected steadily for about one minute into the basis of the right ventricle, after which the ascending aorta was clamped. The heart was removed and transferred to a 60- mm dish containing 10 ml of the EDTA buffer. A syringe was used to push 10 ml of EDTA buffer through the left ventricle steadily for approximately 2 minutes. The heart was then transferred to a 60-mm dish containing 10 ml of the Perfusion buffer, and the left ventricle was steadily injected with 10 ml of the Perfusion buffer to flush the remaining EDTA. Next, the heart was transferred to a 60-mm dish containing 10 ml of the Collagenase buffer, and the left ventricle was steadily perfused five to six times with 10 ml of Collagenase buffer. Afterwards, the heart was cut into the desired regions. After a small piece of the left ventricle was saved and snap frozen to be further used for RNA extraction (see above), the rest of the left ventricle was transferred to the dish containing 3 ml Collagenase buffer for CM isolation. The tissue was teared apart into 1 mm x 1 mm pieces and pipetted up and down for about 5 minutes to dissociate the cells, after which the collagenase reaction was stopped by adding 5 ml of the Stop solution. The cells were filtered through a 100 pm filter and pelleted by gravity for about 20 minutes, after which the pellet was gently resuspended in Perfusion buffer and the filtering procedure was repeated one more time. The pellet was then resuspended in pre-warmed DMEM media containing 10% FBS Supreme (Pan Biotech, P30-303), plated onto the pre-coated microscopy slides, and let in the cell culture incubator for three hours. Slides were then washed twice with PBS, followed by fixation and staining protocols described above.
Statistical analysis
All experiments were performed in at least three biological replicates, unless otherwise specified in the figure legends. Statistical significance was quantified either with Welch Two Sample t-test for pairwise comparisons, or with ANOVA with Tukey’s HSD or Bonferroni post-tests for multiple comparisons, unless otherwise specified in the figure legends.
Splice regulatory activity of RBM20 variants is proportional to their nuclear localization The inventors analyzed the localization of P633L- and R634Q-RBM20 variants that the inventors engineered before [22] in iPSC-CMs by endogenous immunofluorescence (IF) followed by confocal microscopy (Fig. 1 a, b) or ICS (Fig. 1c). The R634Q mutation resulted in severe cytoplasmic mislocalization and granule formation of RBM20, as shown for other described RBM20 mutations [24-29], In contrast, the P633L mutation resulted in only partial mislocalization of RBM20 to the cytoplasm. The partially correct localization of P633L correlated with a less severe splice phenotype as determined by the analysis of TTN isoform expression (Fig. Id), and global splicing activity in comparison to R634Q [13], These differences were independent of RBM20 expression levels. Similar localization patterns were observed in HeLa cells stably expressing eGFP -tagged WT or mutated RBM20. These data demonstrate that P633L-RBM20 causes milder mislocalization and mis-splicing compared to other RS-domain variants.
To verify the clinical relevance of this finding, the inventors collected data from patients with pathogenic or likely pathogenic (P/LP) variants in the RSRSP-stretch of RBM20 who were identified by cascade family screening. The inventors compared patients with the P633L variant to the rest of the cohort (Fig. le-g). In patients with the P633L mutation ventricular remodeling, a characteristic of RBM20-DCM, was less severe [14], Left ventricular ejection fraction (LVEF) for patients with RBM20 P633L was normal to mildly decreased and was in the top 50% of other P/LP variant patients at the time of diagnosis (Fig. le). This preserved function was not explained by younger age compared to other cases (Fig. If). Internal Left Ventricular Diastolic Dimension (LVIDd) normalized to body surface area (BSA) was normal or borderline normal (<3 cm/m2) for P633L patients (Fig. 1g). These data offer clinical corroboration of a milder effect of the P633L variant on the underlying mechanism of RBM20-DCM as compared to other pathogenic variants in the RS-rich domain.
The mixed phenotype (nuclear and cytoplasmic) of the P633L-RBM20 variant allowed the inventors to differentiate between the consequences of nuclear and cytoplasmic RBM20 localization in the same genetic background. To that end, the inventors compared gene expression, alternative splicing, and protein interactor changes between differentially-localized P633L-RBM20, WT-RBM20, and R634Q-RBM20.
Using ICS, the inventors sorted iPSC-CMs with differentially-localized RBM20 based on correlation with nuclear staining (Fig. Ih). This was followed by RNA-sequencing of the sorted populations. The inventors identified 1,415 differentially expressed genes in P633L- cytoplasmic (P633L-cyt) compared to WT (Fig. li). Surprisingly, there were only 50 differentially expressed genes between P633L-nuclear (P633L-nuc) and WT (Fig. li). Down- regulated genes in both P633L-cyt and R634Q impacted the cardiac-related processes. Similarly, gene expression of RBM20 targets was either unchanged or down-regulated in P633L-cyt unlike P633L-nuc. This indicates that mislocalization of RBM20 to the cytoplasm may down-regulate genes important for cardiomyocyte function. Overall, nuclear-localized P633L-RBM20 does not alter gene expression, while cytoplasmic P633L affects genes similarly to R634Q.
The inventors tested splicing changes between the sorted populations of iPSC-CMs. For all exons of RBM20 targets [13], the inventors assessed the percentage of spliced-in (PSI) values. PSI is defined as the proportion of all sequenced reads that include the given exon to total reads for this exon (spliced-in and -out, see methods). In cells with R634Q and P633L-cyt, the majority of alternatively spliced exons were spliced-in. In contrast, in WT and P633L-nuc cells, they were spliced-out (Fig. Ij). The inventors confirmed the finding by qPCR analysis of TTN splicing (Fig. Ik). These results demonstrate that P633L-nuc is functional, similar to WT- RBM20.
Next, the inventors investigated whether the protein binding partners of nuclear or cytoplasmic P633L-RBM20 match the nuclear WT or the cytoplasmic R634Q variant respectively. The inventors performed mass-spectrometry analysis of the interactors that co-immunoprecipitated with WT-, P633L-, and R634Q-RBM20 in the nuclear or cytoplasmic fraction in HeLa cells (see methods). The inventors found that the majority of interactors are common for WT and variant RBM20 in the nucleus (Fig. 11). Only 17 proteins mildly lost their ability to bind the mutated protein, and only one of them (RBMX) was a component of the spliceosome [30], Pathway enrichment analysis of common interactors revealed enrichment of categories related to protein folding, mRNA metabolism, and splicing (Fig. Im). These results indicate that RS- domain variants exhibit mainly unaltered interactors if located to the nucleus.
In agreement with published studies [31], the inventors identified MOVIO and PUM1 as interactors of mutant RBM20 in the cytoplasm. Additional proteins involved in mRNA metabolism and protein folding were enriched for binding to cytoplasmic RBM20 variants compared to WT (Fig. 11), suggesting their presence in RNP-granule-related interactors. Notably, since WT-RBM20 presents solely nuclear localization, the amount of WT protein in the cytoplasm is relatively low (Fig. la). This could result in underestimation of its cytoplasmic interactors. Nevertheless, the inventors found two factors to be specifically interacting with WT, and not with the mutants in the cytoplasm, namely TNPO3 and CHD1 (Fig. 11). This provides a potential insight into the nuclear import mechanism that the inventors investigate in detail further. Restoring nuclear localization of RS-domain RBM20 variants rescues their splicing function To investigate whether mislocalizing RS-domain variants of RBM20 are functional after relocalization to the nucleus, the inventors applied a splicing reporter assay [18] in HEK293T cells (Fig, 2a). The inventors analyzed multiple variants of RBM20 with or without the nuclear localization sequence (NLS) of simian virus SV40 (Fig. 2b). Addition of the NLS resulted in significant restoration of WT splicing activity for all tested RS-domain mutations (Fig. 2b). The inventors also observed restored splicing of endogenous IMMT, an RBM20 target gene, in cells expressing NLS-tagged P633L and R634Q. As a control, addition of an NLS to the V914A variant, which resides outside the RS-domain and does not mislocalize to the cytoplasm [29], had no effect on splicing. These data suggest that RS-domain variants are splice competent, once their nuclear localization is restored.
The inventors validated these results in iPSC-CMs by lentiviral overexpressing of WT-, R634Q-, or NLS-tagged R634Q-RBM20 in splice deficient cells carrying the homozygous frameshift mutation (S635FS) [22], Unlike R634Q-RBM20, overexpressed NLS-R634Q localized to the nucleus, similar to WT-RBM20 (Fig. 2c). Genome-wide RNA-Seq analysis showed that the expression of 1,751 genes was altered in cells expressing R634Q-RBM20 compared to WT (Fig. 2d). The genes were consistent with severe cardiac impairment (Supplementary Fig. 4d) and similar to the genes altered in expression in cells with cytoplasmic P633L. RBM20 target gene expression was consistently either unchanged or down -regulated, and never up-regulated. Strikingly, the inventors found only few differentially expressed genes between WT and NLS-tagged-R634Q expressing cells (Fig. 2d). Moreover, splicing of RBM20 target genes was restored in NLS-R634Q expressing cells to similar levels seen in WT (Fig. 2e). These findings demonstrate that all tested RS-domain RBM20 mutations do not affect the intrinsic splice regulatory function of the protein. They affect only its ability to be translocated into the nucleus. These results highlight the importance of identifying factors involved in nuclear transport of RBM20.
Genome-wide ICS screen identifies TNP03 as the major transporter of RBM20
To identify factors that regulate RBM20 subcellular localization, the inventors performed a genome-wide CRISPR-Cas9 screen applying ICS technology [5] (Fig. 3a). The inventors transfected HeLa cells expressing Tet-Cas9 and eGFP-RBM20-WT with a guide RNA library targeting 18,408 protein-coding genes with six gRNAs per gene. The inventors collected the 7% of cells exhibiting the most cytoplasmic (lower fraction) or the most nuclear (higher fraction) eGFP-RBM20 signal, as well as the unsorted input sample (input). Notably, the inventors identified only one gene enriched in the lower fraction (TNPO3, positive regulator of RBM20 import), while 56 genes were enriched in the upper fraction (negative regulators) (FDR < 0.01) (Fig. 3b, c). Since WT-RBM20 localization is solely nuclear, the inventors assumed that negative regulators - gene knockouts that induce a stronger nuclear translocation of RBM20 - are not of direct relevance for the scope of this study. In addition, based on previous experiments [5], cellular shape amongst other parameters can influence the sorting decision of ICS. Therefore, the inventors discarded all negative regulators for further validation. In addition to TNPO3, the inventors selected other hits with less significant FDR scores (CLDN14, GALE, ADAMTS16, SLC29A2, CEBPB, UBQLNL, TRIM33, PMM2, TRIM24, IPPK, XPO6) and based on prior knowledge about their relevance for RBM20 biogenesis [18, 32, 33] (TTN, AKT2, SPRK1, CLK1, LMNA). The inventors tested these potential positive hits by constructing single gene knock-outs in HeLa cells and assessing their impact on WT-RBM20 localization by ICS and fluorescence microscopy. KOs of GALE, CEBPB, TRLM33, TRIM24 mildly impaired nuclear RBM20 localization as measured by ICS (Fig. 3d), and could not be validated by fluorescence microscopy (Fig. 3e). However, KO of TNPO3 (Transportin-3, transportin-SR) resulted in a substantial shift in RBM20 localization and accumulation of the WT protein in the cytoplasm as determined by both ICS and microscopy analysis of the KO cells (Fig. 3d,e). Importantly, TNPO3 was also one of the only two proteins the inventors identified by mass- spectrometry as interacting specifically with WT-RBM20 and losing this interaction with mutants (Fig. 11). Notably, the inventors did not detect CHD1 as a positive regulator in the ICS screen. TNPO3 is known to specifically recognize and transport other RS-domain containing proteins in humans. Together, these results show that TNPO3 is the essential nuclear transporter ofRBM20.
Mislocalization of RS-domain RBM20 variants is caused by loss of interaction with TNP03 To assess the role of TNPO3 in the nuclear transport of RBM20 in iPSC-CMs, the inventors performed siRNA knock-down (KD) of TNPO3 (Fig 4a, b). The inventors found that TNPO3 KD significantly decreased nuclear localization of both WT- and P633L-RBM20. This was accompanied by a decrease of TTN and IMMT alternative splicing upon TNPO3 KD (Fig. 4c). This confirms that TNPO3 is essential for RBM20 nuclear import in iPSC-CMs. The inventors hypothesized that disruption of the direct interaction between RBM20 and TNP03 upon RS-domain mutations may be the main cause of RBM20 mislocalization in DCM. To assess the role of TNPO3 in the nuclear transport of RBM20 variants, the inventors performed siRNA knock-down (KD) of TNP03 in HeLa cells expressing WT-, P633L- R634Q- or R634Q-S635E-S637E-RBM20 (RSS) variants. In the RSS mutant, three out of six residues in the PRSRSP stretch are substituted, and it displayed the most severe defect in RBM20 nuclear localization (Fig. 4d,e). The inventors found that TNP03 KD significantly decreased nuclear localization of WT, P633L, and even R634Q-RBM20, as measured by ICS (Fig. 4d) and confocal microscopy (Fig. 4e). RSS localization did not change. These data show that TNPO3 is responsible for localizing mutant RBM20 protein to the nucleus, and its effectiveness is mutation-dependent.
To gain a deeper understanding of the RBM20-TNPO3 interaction, the inventors used AlphaFold2 [35, 36] to predict the complex. The inventors observed no structural rearrangements within the intrinsically disordered RS-domain in the AlphaFold2 models of the RRM-RS (amino acid 511-673) domain from WT-, P633L- or R634Q-RBM20 proteins (Fig. 4f). The predicted complex of TNPO3 and RBM20’s RRM-RS domain indicated that the PRSRSP region is classified as interaction interface residues in all obtained models (Fig. 4g). This was also the case for the prediction of TNPO3 in complex with full-length RBM20. It has been shown before that the RS-domain of another alternative splicing factor/splicing factor 2 (ASF/SF2) is the major contributor for the interaction with TNP03 and that it can function as a transferable TNP03 -dependent NLS on its own [34, 37, 38], The inventors employed MutaBind2 [39] to calculate the changes in the binding affinity induced by P633L and R634Q. The inventors used the top 20 AlphaFold models of the wild type RRM-RS domain in complex with TNPO3 to account for the uncertainty in the correct placement of the RS domain inside the binding pocket (Fig. 4h). The predicted changes in the binding affinity induced by P633L and R634Q were both positive in comparison to the wild type sequence of RBM20, which indicates a destabilization of the interaction and a decrease in the binding affinity with TNPO3. Mutabind2 generally classifies a single mutation as deleterious if the AAG value is > 1.5 kcal/mol. The R634Q mutation was classified as such in 13 out of 20 models (Fig. 4h). However, the P633L mutation did not impact the AAG value strongly enough in any model, indicating that it should not fully interrupt the interaction with TNPO3. To validate the structural predictions, the inventors measured the level of TNP03 co- immunoprecipitating with WT or mutated RBM20 in HeLa cells (Fig 4i-k). The stability of the interaction with TNP03 decreased in line with the inventor’s predictions and the previously observed severity of RBM20 mislocalization (Fig. 4i). This was observed by Western blot (Fig 4i,j) as well as by mass spectrometry analyses (Fig 4k). TNP03 amount was constant between WT and RBM20 mutant cells, which rules out a potential effect due to differential expression of the transporter (Fig. 4j). The inventor’s results indicate that direct interaction between TNP03 and RBM20 is essential for its nuclear import. The PRSRSP -mutations affect the stability of this interaction thereby resulting in mislocalization and loss of splicing of RBM20 target genes.
Finally, the inventors sought to address whether the granule formation is a consequence or a cause of the protein mislocalization. TNP03 KD resulted in the accumulation of cytoplasmic granules of RBM20-WT in HeLa cells and iPSC-CMs, indicating that granule formation is not specific to the RBM20 mutant proteins. This finding is in agreement with the inventor’s obtained AlphaFold models that showed no structural rearrangements within the RRM-RS domain of WT- or mutant-RBM20 (Fig. 4f). The present study and previous work [31] have identified MOVIO as one of the main interactors of mutant RBM20 in the cytoplasm (Fig. 4j). The inventors analyzed RBM20-WT interaction with MOVIO in the cytoplasm upon TNP03 KD. The inventors observed a significant gain of interaction between WT RBM20 and MOVIO upon loss of TNPO3. Moreover, the inventors observed partial colocalization of MOVIO and RBM20-WT upon TNP03 KD by confocal microscopy, similar to the mutant variants. This concludes that loss of interaction with TNPO3 results in formation of RBM20 RNP granules, regardless of the mutation present.
Enhancing RBM20-TNPO3 interaction restores nuclear localization and splicing in vitro and in vivo
The inventors tested whether enhancing the interaction of TNPO3 and RBM20 might rescue the aberrant localization and splicing deficiency caused by RS-domain mutations. The inventors overexpressed TNPO3 in iPSC-CMs with WT-, P633L-, or R634Q-RBM20. The nuclear localization of P633L was completely rescued to the level of the WT protein; similar results were obtained for R634Q-RBM20, although a small fraction of the protein remained in the cytoplasm (Fig. 5a, b). Restoring nuclear localization of the mutant RBM20 variants also up- regulated the splicing activity (Fig. 5c, d). These results indicate that mislocalization of the mutant variants can be rescued by up-regulating the TNPO3-RBM20 interaction.
To test whether increasing TNPO3 levels could improve RBM20 mislocalization in vivo, the inventors delivered Tnpo3 cDNA via AAV9 to mouse hearts bearing homozygous P63 SERB M20 (P635L+/+) mutations (P633L in humans). The inventors analyzed RBM20 localization and splicing function four weeks after AAV9 injection (Fig. 5e). Strikingly, increasing Tnpo3 levels (Fig. 5f) resulted in partial rescue of RBM20 localization (Fig. 5g) and Ttn alternative splicing (Fig. 5h, i) independently of Rbm20 expression. These findings reveal a novel therapeutic strategy for mislocalizing RBM20 variant proteins for future studies. See also Figure 16.
In conclusion, the inventor’s results show that increasing TNPO3 expression restores splicing and mislocalization of RS-domain mutant RBM20. Moreover, the inventors provide first proof- of-principle that this strategy can serve as a promising therapeutic avenue for developing future therapies for RBM20-mediated DCM.
Design of binding molecules against the amino acid sequence of RBM 20, and the RS domain thereof
As targeting site for the binder design, the L-rich region and an alpha-helix in the RS-rich region of RBM20 were chosen. Since the binders will presumably remain bound to RBM20, binding to a domain being involved in crucial functionalities of RBM20 was avoided. Both the L-rich or the helix within the RS-rich region are furthermore highly conserved in eukaryotes, and therefore good targets to generate a universal binder that can be used both in mouse and human DCM model systems.
Since no post translational modifications are introduced into the L-rich region and the region seems to be easier excisable at the N-terminal end of the protein in comparison to the helix of the RS-rich region, the inventors started with the L-rich region as a target. The structured alpha helical part of the L rich region shows only three amino acids that are not conserved between human and mouse (Figure 6, blue amino acids below dark green bars). The inventors anticipate antigen binders, such as nanobodies, to function with this model, however, artificial binders were pursued as easier to synthetize and to produce, and not requiring animal immunizations or long phage display screenings. The RosettaFold diffusion algorithm (Baek, M., McHugh, R., Anishchenko, I. et al. Accurate prediction of protein-nucleic acid complexes using RoseTTAFoldNA. Nat Methods 21, 117— 121 (2024). https://doi.org/10.1038/s41592-023-02086-5) was used to predict several different binders for the L-rich region (with varying the length of the binder, e.g., between 50-100 / 50- 150 / 50-200 amino acids, and by defining different amino acids within the L-rich region that should be the main target of the designed binder, and by changing the flanking regions around the L rich region that should be included in the target sequence to better represent the native fold and surrounding of the L rich region). In total, 13 binders were selected for follow up experiments based on their prediction binding scores. The algorithm provided the amino acid sequence of the binder and a structure prediction for the binder and the target (L-rich domain).
The amino acid sequence of the 13 selected binders was translated into a DNA sequence for humans (codon optimized) and cloned into a plasmid for transient transfection into human cells. Two NLS sequences were attached to the N-terminus of the binder, SV40 NLS and/or myc NLS. The cloning of 8 binders was successful, and these binders were tested further for their activity to cause RBM20 to be localized in the nucleus.
The amino acid sequences of the binders as identified (without the NLS as shown in the Figures) were:
Binder 1 :
ALLAALAALLAALAAAAAAAAAQAAALERQSLLLKAYDAGKPLEEVVAALVAQAE DAEAARARAAELAAEWAARLAALEAEAAALKARFAALTEAELLALIEEIGKETEAT MAQVDAVFAGAAAALAALYGRDPELVRQLLEQAAALRAQLEAQEAEIAAAAAAAA AAAAAAAA (SEQ ID NO: 2)
Binder 2 (preferred):
AAALLAERLALAQRRRAAAERALALARRSLAEGPAALPELLAAQAEIQAIELEELAL KAKLAQAFQETDPAAAQLLAQLQQLEQLLQQGLAQADALVAQADAATKAAYAAA KKEAEAQAAAAEAAAAQAALAAALAAAAAANPALAALLAELAAAAAAVAANQAA LAALAAQAA (SEQ ID NO: 3)
Binder 3 : APTLEQLAHDAQLAALRAQLAALEAERAALRAAAAAEGDALDEQLLAALEAQLAA LRAQLAALEAAR (SEQ ID NO: 4)
Binder 4:
SALAALAAGTNAQIAALNAQAAALKAAGGPQAEIDTLNEQAAILQQAQADVLSKAA
AAA (SEQ ID NO: 5)
Binder 5:
PVPAAEALAAAAAAQAAALAARLAAQALGQAQQAQLVQQGLLEAAQKVATVALT DTAARLAALLATQLELLADQQQAAAQMAAAAAAAEAAAAAA (SEQ ID NO: 6)
Binder 6:
LVPVTLTTTSLHVHIDPKTGRVTVQRSEETRTLGPLAPADLLLYGAAVETDGESAAAL SSTRWEDGLVRLVLLSPESGGLIALSLALAALLGKDHEAANAYVADLALSREYLDIQ QELAAGSALAQLLVQQQAQQTDTLLTGTLAVLQAVA (SEQ ID NO: 7)
Binder 7:
SAIDELLKKIREKKLENLIFFQLFRAQLEANAAAVAAGGPAARRQAVADLAAALREG AKLLGVDEAVVAPLIEALAAAAAAAEAALAAAGPEAQAALRARAAALHARLLEAV GEAEDLVLGAYEVLELLLKENKETGSMTIEELEATEKSMLETTAKVATERLVAVLAA
EFAG (SEQ ID NO: 8)
Binder 8:
AAAAAAAAAAARAAAWAALTEVLIELLRALGYPDGATIQALAAAATTSDAAQVAL LNLVLSKLDM (SEQ ID NO: 9)
References
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2. Hershberger RE, Morales A, Siegfried JD. Clinical and genetic issues in dilated cardiomyopathy: A review for genetics professionals. Genet Med. 2010; 12(11):655-667. doi:10.1097/GIM.0b013e3181f2481f
3. Verdonschot JAJ, Hazebroek MR, Ware JS, Prasad SK, Heymans SRB. Role of Targeted Therapy in Dilated Cardiomyopathy: The Challenging Road Toward a Personalized Approach. J Am Heart Assoc. 2019;8(11): 1 - 18. doi: 10.1161/JAHA.l 19.012514 Hauh Watkins, Houman Ashrafian CR. Inherited cardiomyopathies. N Engl J Med. 2011 ;364 : 1643 - 1656. doi : 10.1007/978- 1 -4471 -4267-6 3 Jordan E, Peterson L, Ai T, et al. Evidence-Based Assessment of Genes in Dilated
Cardiomyopathy. Circulation. 2021;144(l):7-19. doi: 10.1161/CfRCULATIONAHA.120.053033 Mazzarotto F, Tayal U, Buchan RJ, et al. Reevaluating the Genetic Contribution of Monogenic Dilated Cardiomyopathy. Circulation. 2020;(Dcm):387-398. doi : 10.1161/CIRCULATIONAHA.119.037661 Li D, Morales A, Gonzalez-Quintana J, et al. Identification of novel mutations in RBM20 in patients with dilated cardiomyopathy. Clin Transl Sci. 2010;3(3):90-97. doi: 10.1111/j.1752-8062.2010.00198.x Koelemen J, Gotthardt M, Steinmetz LM, Meder B. Rbm20-related cardiomyopathy: Current understanding and future options. J Clin Med. 2021;10(18). doi: 10.3390/jcml0184101 Parikh VN, Caleshu C, Reuter C, et al. Regional Variation in RBM20 Causes a Highly Penetrant Arrhythmogenic Cardiomyopathy. Circ Hear Fail. 2019;12(3): l-9. doi : 10.1161/CIRCHEARTF AILURE.118.005371 Van Den Hoogenhof MMG, Beqqali A, Amin AS, et al. RBM20 mutations induce an arrhythmogenic dilated cardiomyopathy related to disturbed calcium handling. Circulation. 2018; 138(13): 1330-1342. doi: 10.1161/CIRCULATIONAHA.117.031947 Zeppenfeld K, Tfelt-Hansen J, de Riva M, et al. 2022 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death Developed by the task force for the management of patients with death of the European Society of Cardiology ( ESC ) Endorsed by the. Eur HeartJ. 2022: 1-130. Watanabe T, Kimura A, Kuroyanagi H. Alternative splicing regulator RBM20 and cardiomyopathy. Front Mol Biosci. 2018;5(NOV): 1-11. doi: 10.3389/fmolb.2018.00105 Guo W, Schafer S, Greaser ML, et al. RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat Med. 2012;18(5):766-773. doi: 10.1038/nm.2693 Maatz H, Jens M, Liss M, et al. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing. J Clin Invest. 2014;124(8):3419-3430. doi: 10.1172/JCI74523 Zhu C, Wu J, Sun H, et al. Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes. Nat Commun. 2021 ; 12( 1 ) : 1 -9. doi : 10.1038/s41467-021 -24484-z Brauch KM, Karst ML, Herron KJ, et al. Mutations in Ribonucleic Acid Binding Protein Gene Cause Familial Dilated Cardiomyopathy. J Am Coll Cardiol. 2009;54(10):930- 941. doi: 10.1016/j.jacc.2009.05.038 Briganti F, Sun H, Wei W, et al. iPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy. Cell Rep. 2020;32(10): 108117. doi: 10.1016/j.celrep.2020.108117 Li S, Guo W, Dewey CN, Greaser ML. Rbm20 regulates titin alternative splicing as a splicing repressor. Nucleic Acids Res. 2013;41(4):2659-2672. doi: 10.1093/nar/gksl362 Schneider JW, Oommen S, Qureshi MY, et al. A ribonucleoprotein-granule pathway to heart failure in human RBM20 cardiomyopathy gene-edited pigs. Nat Med. 2020;26: 1788-1800. Ihara K, Sasano T, Hiraoka Y, et al. A missense mutation in the RSRSP stretch of Rbm20 causes dilated cardiomyopathy and atrial fibrillation in mice. Sci Rep. 2020; 10(1): 1-14. doi: 10.1038/s41598-020-74800-8 Zhang Y, Wang C, Sun M, et al. RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis. FASEB J. 2022;36. Fenix AM, Miyaoka Y, Bertero A, et al. Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies. Nat Commun. 2021 ; 12(1): 1 -14. doi: 10.1038/s41467-021-26623-y Wang C, Zhang Y, Methawasin M, et al. RBM20S639G mutation is a high genetic risk factor for premature death through RNA-protein condensates. J Mol Cell Cardiol. 2022;165: 115-129. Gaertner A, Klauke B, Felski E, et al. Cardiomyopathy-associated mutations in the RS domain affect nuclear localization of RBM20. Hum Mutat. 2020;41(l l): 1931-1943. doi: 10.1002/humu.24096

Claims

Claims
1. An agent that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the mutated protein in the cytoplasm of said cell, for use in the prevention or treatment of a disease or condition that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising the mutated RS domain amino acid sequence.
2. The agent for use according to claim 1, wherein the agent is selected from the group consisting of an agent specifically binding to the protein comprising a mutated RS domain amino acid sequence and comprising at least one nuclear localization signal (NLS), an agent for genetically fusing at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, a compound that improves the binding of the protein comprising a mutated RS domain amino acid sequence to its nuclear transporter protein, and a genetic construct for expressing or overexpressing the nuclear transporter protein of the protein comprising a mutated RS domain amino acid sequence.
3. The agent for use according to claim 2, wherein the agent is selected from the group consisting of a proteinaceous binding domain that is specifically binding to the protein comprising a mutated RS domain amino acid sequence, such as an antibody or binding fragment thereof, fused or linked to the at least one NLS, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof, or an NLS-RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9 fusion or an RBM20 L-rich region binding fragment thereof, a nanobody -NLS fusion, an expression construct for expressing a polynucleotide encoding the binder-NLS fusion and/or the nuclear transporter protein, a genetic integration construct for genetically fusing the at least one nuclear localization signal (NLS) to the protein comprising a mutated RS domain amino acid sequence, such as, for example, a prime editing construct containing at least one NLS, and a small molecule binding to the nuclear transporter protein and/or the protein comprising a mutated RS domain amino acid sequence and thereby improving the nuclear transport of the protein comprising a mutated RS domain amino acid sequence.
4. The agent for use according to any one of claims 1 to 3, wherein the at least one NLS sequence is selected from the group consisting of a non-classical or classical NLS, such as, for example a monopartite or bipartite classical NLS, in particular an NLS of SV40, C-myc, nucleoplasmin, EGL-13, or TUS-protein, the acidic M9 domain of hnRNP Al, the sequence KIPIK in yeast transcription repressor Mata2, and the complex signals of U snRNPs.
5. The agent for use according to any one of claims 1 to 4, wherein the protein comprising a mutated RS domain amino acid sequence is RBM20 or an ortholog thereof, and/or the nuclear transporter protein is transportin 3 (TNP03) or an ortholog thereof.
6. The agent for use according to any one of claims 1 to 5, wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
7. The agent for use according to any one of claims 1 to 6, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
8. A method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of a cell, comprising the steps of a) providing at least one L-rich domain amino acid sequence of a protein comprising a mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, b) providing at least one candidate compound prospectively binding to the L-rich domain amino acid sequence, and c) detecting the binding of the at least one candidate compound to the L-rich domain amino acid sequence, wherein a binding of the at least one candidate compound to the L-rich domain amino acid sequence indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
9. A method for identifying a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of a cell, comprising the steps of i) providing at least one mutated RS domain amino acid sequence, and/or at least one protein comprising a mutated RS domain amino acid sequence, ii) providing at least one nuclear transporter protein comprising a domain binding to an RS domain, and/or at least one nuclear transporter protein RS domain binding domain amino acid sequence, and iii) detecting the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the absence and presence of at least one candidate compound, wherein an increase of the binding of the mutated RS domain amino acid sequence of i) to the domain binding to an RS domain of ii) in the presence of at least one candidate compound indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
10. The method according to claim 8 or 9, wherein the at least one protein comprising a mutated RS domain amino acid sequence further comprises at least one added NLS sequence and/or wherein at least one of the amino acid sequences of a), i) or ii) is labelled, and/or wherein the at least one candidate compound is labelled, such as, for example, labelled with GFP or a labelled antibody or fragment thereof specifically binding to the amino acid sequence.
11. The method according to any one of claims 8 to 10, wherein at least one binding cofactor is present, in particular a suitable RNA molecule.
12. The method according to any one of claims 8 to 11, wherein the mutated RS domain amino acid sequence is derived from RBM20 or an ortholog thereof, and/or the domain binding to an RS domain is derived from transportin 3 (TNPO3) or an ortholog thereof.
13. The method according to any one of claims 8 to 12, wherein the amino acid sequences of a), i) and/or ii) are provided in a cell, and are preferably provided as recombinantly expressed amino acid sequences.
14. The method according to claim 13, wherein a change in the biological function of i) the at least one protein comprising a mutated RS domain amino acid sequence, and optionally an increase of the expression and/or a change in the biological function of ii) the at least one nuclear transporter protein comprising a domain binding to an RS domain is detected instead or in addition to the binding in the absence and presence of the at least one candidate compound, wherein a change in the biological function of a protein of i) and/or an increase of the expression and/or change in the biological function of a protein of ii) indicates a compound that increases the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreases the amount of the protein in the cytoplasm of said cell.
15. The method according to claim 13 or 14, wherein the biological function is tested based on detecting cytoplasmatic mislocalization of the protein comprising the mutated RS domain amino acid sequence, the formation of cytoplasmatic granules of the protein comprising the mutated RS domain amino acid sequence, and/or the splicing activity of the protein comprising the mutated RS domain amino acid sequence.
16. The method according to any one of claims 13 to 15, wherein the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
17. The method according to any one of claims 8 or 13 to 16, wherein the compound is selected from the group consisting of natural compound, plant extract, a peptide, such as an artificial in silico designed peptide binder, a protein, a small molecule (less than about 500 Da), an RNA, an antibody or antigen binding fragment thereof, and an agent for use according to any one of claims 1 to 7, in particular an RBM20 binding peptide according to any one of SEQ ID NOs: 2 to 9, or an RBM20 L-rich region binding fragment thereof.
18. A method for producing a pharmaceutical composition, comprising performing a method according to any one of claims 8 to 17, and admixing the compound as identified with at least one pharmaceutically carrier.
19. A compound as identified according to the method according to any one of claims 8 to 17, or a pharmaceutical composition as produced according to claim 18 for use in medicine, in particular for use in the prevention or treatment of a disease or condition in a cell of a subject that is related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of the protein comprising a mutated RS domain amino acid sequence.
20. The compound or pharmaceutical composition for use according to claim 20, wherein the cell is a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte.
21. The compound or pharmaceutical composition for use according to claim 19 or 20, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
22. A method for increasing the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell and/or decreasing the amount of the protein in the cytoplasm of said cell, comprising providing to a cell an effective amount of the compound as identified according to the method according to any one of claims 8 to 17, or a pharmaceutical composition as produced according to claim 18, whereby the amount of a protein comprising a mutated RS domain amino acid sequence in the nucleus of a cell is increased.
23. The method according to claim 22, wherein the cell is selected from the group consisting of a heart muscle cell or cardiomyocyte, such as a mammalian heart muscle cell or cardiomyocyte, in particular a human heart muscle cell or cardiomyocyte, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), HeLa cells, and HEK293 cells.
24. A method for preventing or treating a disease or condition related to the cytoplasmic mislocalization and/or the formation of granules and/or an aberrant splicing activity of a protein comprising a mutated RS domain amino acid sequence in a subject in need of said prevention or treatment, comprising administering to the subject an effective amount of the compound as identified according to the method according to any one of claims 8 to 17, or a pharmaceutical composition as produced according to claim 18.
25. The method according to claim 24, wherein the disease or condition is myopathy, in particular cardiomyopathy (CM), such as hypertrophic (HCM) or dilated cardiomyopathy (DCM).
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