Patent Application
20210261966
The present invention relates to inhibitors targeting stress induced non-coding RNA transcript 1 (SINT1).
Chronic heart failure is characterized by clinical symptoms of cardiac dysfunction and represents the culmination of prolonged left ventricular growth in response to pathologic stressors including ischemia, hypertension, aortic stenosis, and genetic mutations. Although it is a leading cause of hospitalization and mortality worldwide, and in spite of its immense impact on human health and the associated socio-economic implications, therapeutic strategies for chronic heart failure remain limited. Of particular concern is the fact that current treatment modalities (based on heart glycosides, diuretics, vasodilatators or neurohumoral intervention) have limited potential to treat the underlying cause of heart disease. Thus, its prevalence, morbidity and mortality remain high, and with increasing hospitalization and health care costs, heart failure threatens to become a pandemic health issue. Hence, drug discovery should ideally focus on targeting key causative molecular drivers of disease maintenance and progression.
The advent of genomewide transcriptomic and epigenetic analysis has not only deepened the mechanistic understanding of gene regulation but has also served to unveil a novel class of gene modulating non-coding RNAs, termed enhancer-templated RNAs (eRNAs). Enhancers are regulatory DNA elements that bind transcription factors to induce gene transcription through the formation of secondary structures that mediate the interaction of the enhancer with the promoter. Transcription at enhancer elements positively correlates with enhancer activity and is characterised by high histone 3 lysine 4 monomethylation (H3K4me1) and low or absent H3K4 trimethylation (me3). In addition to H3K4 methylation marks, enrichment of H3K27 acetylation and absence of the repressive H3K27me3 signature are other characteristics of active enhancers and correlate positively with the expression of eRNAs.
eRNA transcription has been shown to be regulated by specific transcription factors and their expression serves to induce transcription of either one or both neighbouring 5′ and 3′ genes (De Santa et al., PLoS Biol 8, e1000384, 2010; Li et al., Nature 498, 516, 2013). A number of eRNAs have been identified in the heart but their biological function remains unclear (Ounzain et al., Eur Heart J 36, 353, 2015). Active enhancers during heart development have been identified by genomewide chromatin immunoprecipitation with antibodies against the transcriptional coactivator p300 coupled to parallel sequencing (ChIP-Seq) on mouse embryonic hearts. A report by Ounzain et al. revealed that a subset of active enhancer loci produces eRNAs and that the majority of these transcripts are selectively expressed in the heart. The eRNAs mm67 and mm85 identified by Ounzain et al. positively correlated with the expression of the flanking coding gene myocardin. Recent studies have also identified eRNAs induced by myocardial infarction and transaortic constriction surgery (TAC) in mice (Ounzain et al., J Mol Cell Cardiol 76, 55, 2014). eRNA mm132 expression was shown to be elevated in response to both stressors and correlated positively with the upregulation of its flanking gene endothelin 1, which has been implicated in cardiac hypertrophy and heart failure. Furthermore, eRNA Novlnc6 expression is inhibited in human patients with dilated cardiomyopathy and in a mouse model of myocardial infarction. Thus, the specific modulation of cell-type and stress-responsive eRNAs has the potential to very precisely influence pathophysiologic gene networks.
Hypoxia inducible factors (HIFs) are heterodimeric transcription factors composed of HIF1α and HIF1β subunits that occupy central roles in regulating oxygen homeostasis (Wang et al., Proc Natl Acad Sci USA 92, 5510, 1995) and the pathogenesis of human disease including cancer and cardiovascular disease (Semenza, Cell 148, 399, 2012). They are activated in hypoxic tissue to induce a transcriptional program embracing coding and non-coding RNA transcripts that are entrusted to modulate both the supply and consumption of oxygen. To date however, it is unclear if HIFs also activate transcription of eRNAs to afford cell- and signal-specificity of select HIF output responses.
In the present invention a conserved pathology-induced eRNA, termed stress induced non-coding RNA transcript 1 (SINT1), was identified as a critical determinant of maladaptive cardiac growth, disease-associated metabolic reprogramming and contractile dysfunction. SINT1 expression is restricted to the ventricular myocardium and imposes function through induction of a pro-hypertrophic gene cluster comprising of suppressor of morphogenesis in genitalia 1 (SMG1) and synaptotagmin XVII (SYT17). SINT1 induction correlates with hypertrophic cardiomyopathy in humans and mice, and in vivo inactivation, in particular anti sense oligonucleotide (ASO)-mediated inactivation, of SINT1 surprisingly prevents stress-induced cardiac pathogenesis and reverses pathology and dramatically improves overall survival in diseased mice. Mechanistically, the inventors disclose SINT1 interaction at the promoters of SMG1 and SYT17 and uncouple their impact on gene programs critical for the development of pathologic cardiac hypertrophy and its progression to heart failure.
Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods for treatment of cardiomyopathy. This objective is attained by the subject matter of the claims of the present specification.
The term inhibitor in the context of the present specification relates to oligonucleotide agents that bind specifically to either the transcribed enhancer RNA SINT1 or the genomic region encoding SINT1, thereby decreasing or abolishing the molecular function of SINT1.
“Capable of forming a hybrid” in the context of the present invention relates to sequences that under the conditions existing within the cytosol of a mammalian cell, are able to bind selectively to their target sequence. Such hybridizing sequences may be contiguously reverse-complimentary to the target sequence, or may comprise gaps, mismatches or additional non-matching nucleotides. The minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T/U nucleotides, and the backbone chemistry.
The term oligonucleic acid agent in the context of the present specification refers to an oligonucleotide capable of specifically binding to and leading to a significant reduction of the physiological role of SINT1. Examples of oligonucleic acid agents of the present invention are antisense oligomers made of DNA, DNA having phosphorothioate modified linkages in their backbone, ribonucleotide oligomers, RNA comprising bridged or locked nucleotides, particularly wherein the ribose ring is connected by a methylene bridge between the 2′-O and 4′-C atoms, RNA having phosphorothioate modified linkages in their backbone or any mixture of deoxyribonucleotide and ribonucleotide bases as an oligomer.
The term antisense oligonucleotide or oligonucleotide agent in the context of the present specification refers to any oligonucleotide capable of specifically binding to and leading to a significant reduction of the physiological role of SINT1. Examples of antisense oligonucleotides of the present invention are antisense oligomers made of DNA, DNA having phosphorothioate modified linkages in their backbone, ribonucleotide oligomers, RNA comprising bridged or locked nucleotides, particularly wherein the ribose ring is connected by a methylene bridge between the 2′-O and 4′-C atoms, RNA having phosphorothioate modified linkages in their backbone or any mixture of deoxyribonucleotide and ribonucleotide bases as an oligomer. The terms oligonucleic acid agent and antisense oligonucleotide or oligonucleotide agent are used interchangeably in the present specification.
In certain embodiments, the antisense oligonucleotide of the invention comprises analogues of nucleic acids such as phosphotioates, 2′O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2′O, 4′C methylene bridged RNA building blocks). The antisense sequence may be composed partially of any of the above analogues of nucleic acids, with the rest of the nucleotides being “native” ribonucleotides occurring in nature, or may be mixtures of different analogues, or may be entirely composed of one kind of analogue.
The term gapmer is used in its meaning known in the field of molecular biology and refers to an antisense oligonucleotide complementary to its target sequence, that comprises a central block of a deoxyribonucleotide oligomer flanked by short ribonucleotide oligomers. The flanking ribonucleotide oligomers consist of nuclease and protease resistant ribonucleotides. In certain embodiments, the nuclease and protease resistant ribonucleotides comprise 2′-O modified ribonucleotides, in particular bridged nucleic acids with a bridge between the 2′-O and 4′-C of the ribose moiety.
“Nucleotides” in the context of the present invention are nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with miRNA oligomers on the basis of base pairing. The term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymin), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphotioates, 2′O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2′O, 4′C methylene bridged RNA building blocks). The hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.
In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).
One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear. Unless otherwise stated, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
A first aspect of the invention relates to an inhibitor directed against the enhancer RNA SINT1 (SEQ ID NO 001), for use in a method for the prevention or treatment of heart disease, particularly cardiomyopathy. SEQ ID NO 001 represents the genomic template of the transcribed human SINT1 eRNA.
An alternative of this aspect relates to an oligonucleic acid agent directed at and capable of specifically inhibiting and/or degrading the enhancer RNA SINT1, for use in a method for the prevention or treatment of heart disease, particularly cardiomyopathy.
In certain embodiments, the oligonucleic acid agent comprises a sequence hybridizing to SINT1. The agent sequence is at least 95% identical, particularly 96%, 97%, 98%, 99% or 100% identical to a sequence selected from Table 3 or Table 4. In certain embodiments, the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.
In certain embodiments, the inhibitor or oligonucleic acid agent has a dissociation constant (KD) smaller than 100 nM, in particular smaller than 50 nM, more particular smaller than 10 nM, in relation to its binding to the target SINT1.
In certain embodiments, the interaction of the inhibitor with other non-specifically bound targets has a KD larger than 1 μM, in particular larger than 10 μM, more particular larger than 100 μM.
In certain embodiments, the oligonucleic acid agent of the invention is an antisense oligonucleotide, particularly an antisense gapmer.
In certain embodiments, the inhibitor is an antisense oligonucleotide.
The oligonucleic acid agent of the invention is not only suitable for the prevention of cardiomyopathy, but is also able to reverse pathogenic alterations already established.
Antisense Composed Partially or Entirely of Nucleoside Analogues
In certain embodiments, the oligonucleic acid agent comprises or is essentially composed of LNA moieties and comprises about 20 or fewer nucleotides.
In certain embodiments, the oligonucleic acid agent is essentially composed of LNA moieties and is described by a sequence selected from Table 3. In a particular embodiment, the nucleoside analogues of any sequence of Table 3 are linked by phosphate esters. In a particular embodiment, the nucleoside analogues of any sequence of Table 3 are linked by phosphothioate esters.
In certain embodiments, the oligonucleic acid agent for use in a method of treatment or prevention of heart disease comprises, or essentially consists of one or several peptide nucleic acid (PNA) moieties.
In certain embodiments, the antisense oligonucleotides of the invention are between 8 and 40 bases in length, in particular between 12 and 16 bases in length, more particular between 15 and 16 bases in length.
In certain embodiments, the antisense oligonucleotide comprises ribonucleotides and deoxyribonucleotides, in particular modified ribonucleotides and modified deoxyribonucleotides. A non-limiting example of a modification of deoxyribonucleotides and ribonucleotides are phosphorothioate modified linkages in their backbone. A non-limiting example of a modification of ribonucleotides is a 2′-O to 4′-C bridge.
Antisense Gapmers
In certain embodiments, the oligonucleic acid agent is a gapmer characterized by a central DNA block, the sequence of which is complementary to SINT1, and which is flanked on either side (5′ and 3′) by nuclease-resistant LNA sequences which are also complementary to SINT1. The central DNA block contains the RNase H activating domain, in other words is the part that lead the target DNA to be hydrolyzed. In certain embodiments, the flanking LNA is fully phosphorothioated.
The flanking exonuclease-protected nucleoside analogues impart high binding energy. In certain embodiments, the flanking exonuclease-protected nucleoside analogues are characterized by a ribose unit having a 2′-O/4′-C bridge.
In certain embodiment the 2′-O/4′-C bridge of the flanking region of the gapmer is a five-membered, six-membered or seven-membered bridged structure.
In certain embodiments, the central deoxyribonucleotide oligomer block of the gapmer comprises at least 5 deoxyribonucleotides.
In certain embodiments, the oligonucleic acid agent comprises 12-20 nucleotides. In certain particular embodiments, the oligonucleic acid agent comprises 14-16 nucleotides.
In certain embodiments, the hybridizing sequence of the oligonucleic acid agent according to the invention comprises 14, 15 or 16 nucleotides.
In certain embodiments, the central deoxyribonucleotide oligomer block of the gapmer comprises a phosphate backbone between the deoxyribonucleotides.
In certain embodiments, the oligonucleic acid agent comprises, or essentially consists of, a central block of 5 to 10 deoxyribonucleotides linked by phosphate ester bonds flanked on either side by 2′-O modified ribonucleotides or PNA oligomers. In certain embodiments, the oligonucleic acid agent comprises, or essentially consists of, a central block of 5 to 10 deoxyribonucleosides flanked by LNA nucleoside analogues. In certain particular embodiments, said LNA nucleoside analogues are linked by phosphothioate moieties.
In certain embodiments, the oligonucleic acid agent of the invention comprises or essentially consists of any of the sequences of Table 4, wherein the non-underscore letters signify nucleoside analogues, particularly LNA, more particularly LNA linked by phosphothioate esters, and the central underscored letters signify DNA nucleosides linked by phosphate esters, and the link between a nucleoside analogue and a DNA nucleoside is selected from phosphate ester and thiophosphate.
RNAi/siRNA/shRNA
In certain embodiments, the oligonucleic acid agent of the invention is an RNA interference agent.
An RNA interference (RNAi) agent in the context of the present specification refers to a ribonucleotide oligomer that causes the degradation of its enhancer RNA (eRNA) target sequence.
In certain embodiments, the RNAi agents of the invention comprise, or consist of,
In certain embodiments, the sequence tract complementary to the targeted enhancer RNA molecule is a contiguous sequence tract 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleotides in length.
In certain embodiments, the RNAi agents of the invention include, but are not limited to, small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs and non-coding RNAs or the like, Morpholinos (phosphodiamidate morpholino oligomers) and Dicer substrate siRNAs (DsiRNAs, DsiRNAs are cleaved by the RNAse III class endoribonuclease Dicer into 21-23 base duplexes having 2-base 3′-overhangs), UsiRNAs (UsiRNAs are duplex siRNAs that are modified with non-nucleotide acyclic monomers, termed unlocked nucleobase analogues (UNA), where the bond between two adjacent carbon atoms of ribose is removed), self-delivering RNAs (sdRNAs) including rxRNA™ (RXi Pharmaceuticals, Westborough, Mass., USA).
In some embodiments, the RNAi agents of the invention comprise analogues of nucleic acids such as phosphotioates, 2′O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2′O, 4′C methylene bridged RNA building blocks). The hybridizing sequence may be composed partially of any of the above nucleotides, with the rest of the nucleotides being “native” ribonucleotides occurring in nature, or may be mixtures of different analogues, or may be entirely composed of one kind of analogue.
In certain embodiments, the antisense oligonucleotide comprises or consist of the sequence of SEQ ID NO 060 to SEQ ID NO 202, for use in a method for the prevention or treatment of cardiomyopathy. In certain embodiments, the sequences of SEQ ID NO 060 to SEQ ID NO 202 contain modified nucleotides. In certain embodiments, the SEQ ID NO 060 to SEQ ID NO 202 are the corresponding RNA sequences (T to U) and are used as RNA interference agent.
In certain embodiments, the antisense oligonucleotide comprises or consist of the sequence of SEQ ID NO 060 to SEQ ID NO 069, for use in a method for the prevention or treatment of cardiomyopathy. In certain embodiments, the sequences of SEQ ID NO 060 to SEQ ID NO 069 contain modified nucleotides. In certain embodiments, the SEQ ID NO 060 to SEQ ID NO 069 are the corresponding RNA sequences (T to U) and are used as RNA interference agent.
In certain embodiments, the antisense oligonucleotide is for use in the treatment of cardiac hypertrophy.
In certain embodiments, the antisense oligonucleotide is for use in the treatment of a cardiomyopathy resulting of cardiac overload. Cardiac overload can result from a cardiac pressure overload or a cardiac volume overload. Non-limiting examples of cardiomyopathies resulting from cardiac overload are hypertension-induced pathologic hypertrophy, stenosis(blockage)-induced pathologic hypertrophy, hypertrophic cardiomyopathy (congenital and idiopathic), restrictive pathologic hypertrophy and ischemic heart disease.
In certain embodiments, the antisense oligonucleotide is for use in the treatment of hypertension-induced pathologic hypertrophy, stenosis(blockage)-induced pathologic hypertrophy, hypertrophic cardiomyopathy (congenital and idiopathic), restrictive pathologic hypertrophy or ischemic heart disease.
In some embodiments, the antisense oligonucleotide comprises deoxynucleotides, ribonucleotides, phosphothioate and/or 2′-O-methyl-modified phosphothioate ribonucleotides.
In some embodiments, the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, phosphothioate ribonucleotides and/or 2′-O-methyl-modified phosphothioate ribonucleotides.
Another aspect of the invention relates to an antisense oligonucleotide directed against the enhancer RNA SINT1 (SEQ ID No 001) that comprises or consists of any one of the sequences of SEQ ID NO 060 to SEQ ID NO 202. In certain embodiments, the antisense oligonucleotide directed against the enhancer RNA SINT1 (SEQ ID No 001) comprises or consists of any one of the sequences of SEQ ID NO 060 to SEQ ID NO 069. In certain embodiments, the SEQ ID NO 060 to SEQ ID NO 202 are the corresponding RNA sequences (T to U) and are used as RNA interference agent. In certain embodiments, the SEQ ID NO 060 to SEQ ID NO 069 are the corresponding RNA sequences (T to U) and are used as RNA interference agent.
In certain embodiments, the oligonucleic agent of the invention is conjugated to, or encapsulated by, a nanoparticle, a virus and a lipid complex.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
Table 1 qPCR primers for mouse sequences
Table 2: qPCR primers for human sequences
Table 3: Gapmer sequences directed against humanSINT1
Table 4: Gapmer sequences directed against humanSINT1. Central underscored positions are DNA; flanking sequences without underscore on either side are LNA linked by phosphothioate ester bonds.
SINT1 Identification and Correlation with Pathology
To identify potential HIF-regulated eRNAs, the inventors performed genome-wide epigenetic and transcriptomic analysis of cardiac left ventricular biopsies derived from two distinct mouse models of cardiac hypertrophy: surgery-induced aortic stenosis [transaortic constriction (TAC)] and ventricular-specific deletion of the von Hippel Lindau (Vhl) gene (referred to as Vhl cKO) (Barrick et al., Am J Physiol Heart Circ Physiol 292, H2119 (2007); Krishnan et al., Cell Metab 9, 512 (2009)). The latter genetic-based approach relies on the fact that the product of Vhl, pVhl, acts as a negative regulator of oxygen-sensitive Hifα subunits, and its deletion in the heart leads to constitutive activation of Hif1α and spontaneous cardiac hypertrophy (Krishnan et al., Cell Metab 9, 512 (2009)). Chromatin immunoprecipitation-coupled sequencing (ChIP-seq) was applied on ventricular biopsies of the above-noted models to precisely identify enhancer domains as marked by mono-methylated histone H3 lysine 4 (H3K4me1) signals, and regions of active or poised transcription as indicated by tri-methylated histone H3 lysine 4 (H3K4me3) marks. In parallel, RNA sequencing (RNA-seq) was performed on the same biopsies to detect differentially expressed transcripts. Next the inventors cross-referenced and performed differential signal analysis of the data against ENCODE and other published datasets (De Santa et al., PLoS Biol 8, e1000384 (2010); Blow et al., Nat Genet 42, 806 (2010); Mouse et al., Genome Biol 13, 418 (2012)) to select for RNAs exhibiting enhancer localization, conservation with the human genome and HIF1a dependence (described in
SINT1 is a HIF1α-Dependent Non-Coding RNA with Enhancer Function
As SINT1 transcription correlated with Hif1α activation, the inventors investigated if SINT1 is a direct target of Hif1a. In silico analysis of the SINT1 promoter revealed a conserved hypoxia response element (HRE) at position −121 bp and −195 bp upstream of the transcription start site (TSS) in the mouse and human genome, respectively (
Given that SINT1 expression parallels that of Smg1 and Syt17, the inventors asked if the SINT1 transcript interacts with the promoters of Smg1 and Syt17 to drive transcription. To that end, the inventors quantified SINT1 binding at the Smg1 and Syt17 promoter by Chromatin Isolation by RNA purification (ChIRP) (Chu et al., Mol Cell 44, 667 (2011)), utilizing glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and nuclear paraspeckle assembly transcript 1 (Neat1), established coding and non-coding hypoxia targets (Graven et al., J Biol Chem 269, 24446 (1994); Choudhry et al., Oncogene, (2014), respectively, as promoter controls for the efficiency and specificity of SINT1 interaction. After cross-linking endogenous RNA to its target, the inventors precipitated SINT1 with biotinylated oligonucleotides and performed qPCR on SINT1 ChIRP pull-down products (
SINT1 is Necessary for Pathology-Induced Smg1 and Syt17 Activity
To systematically ascertain the function of the SINT1, the inventors generated short-hairpin RNAs (shRNA) targeting mouse SINT1 (shSINT1), of which two individual clones efficiently inhibited hypoxia-induced SINT1 expression resulting in the concordant upregulation of Smg1 and Syt17 in NMC on the RNA and protein level (
Smg1 and Syt17 Drive Distinct Aspects of Pathologic Transition
As little is known as to Smg1 and Syt17 function in heart cells, the inventors characterized the sub-cellular localization of the respective proteins in NMCs. As shown in
SINT1 Inactivation In Vivo Attenuates Disease Development In Vivo
Enhancers are established modulators of spatio-temporal gene expression and eRNA templated at these regions can potentially exhibit tissue- and context-specific gene expression. Hence, the inventors assessed tissue distribution of SINT1 expression in mice subjected to aortic stenosis-induced hypertrophy (TAC) or hypertension-induced hypertrophy (1K1C). As noted in the qPCR analysis (
Next, the inventors interrogated SINT1 function in mice exhibiting overt indications of pathologic growth, dilatation and contractile dysfunction in order to evaluate the therapeutic implications of SINT1 inhibition. C57Bl/6 mice were randomly assigned into two groups, with the groups subjected to either sham or 1K1C surgery and further subdivided for scrGM or SINT1GM3 treatment upon pathology development (
SINT1 Function can be Uncoupled Via Smg1 and Syt17 In Vivo
In vitro phenotypic analysis and gene expression profiling of Smg1 and Syt17 function revealed a cooperative role of both genes in normalizing dysregulated cardiomyocyte growth and metabolism but a unique capacity of Syt17 in correcting the maladaptive contractility induced by pathologic stress (
SINT1 Correlates with Human Cardiac Hypertrophy and is Necessary for Disease Transition
The in-silico analysis indicated conservation of this gene cluster structure in various species, (
Concepts and Evidence of the Invention
Collectively, this invention discloses a novel mode of hypoxia-dependent gene regulation initiated by HIF1α activation of the SINT1 eRNA and its binding to and stimulation of mRNA synthesis of its neighboring gene-promoters SYT17 and SMG1. This mode of gene regulation (as opposed to direct transcriptional activation of SYT17 and SMG1 by HIF1a), provides an effective means of engendering cell-specific hypoxia transcriptional responses and offers a potential mechanistic explanation of at least some of the contextual effects that HIF1α mediates in different tissues and pathologic settings (Vanharanta et al., Nat Med 19, 50 (2013)).
As a mitochondrial-localized member of the calcium-sensing protein family, SYT17 contributes to the regulation of contractility in response to hypoxic stress through re-normalization of expression of a broad range of cell signaling and transcription networks to maintain normal contractility in the face of pathologic insult (
The PI3K-related kinase SMG1 and its downstream phosphorylation target UPF1 represent central components of cell growth control, attributed, in part, to nonsense-mediated decay (NMD), a process dedicated to the control of both the quality and quantity of a large number of mRNAs (Mcllwain et al., Proc Natl Acad Sci USA 107, 12186 (2010)). Although the inventors were unable to detect dramatic shifts in RNA species containing nonsense mutations or premature termination codon in this setting, the inventors did detect shifts in RNAs implicated in metabolic control of cell growth, and in growth pathways (
Previous studies have attributed the deregulation of cardiac growth and function to the phosphatidylinositol-3 kinase (PI3K) signalling network (Stocker et al., Curr Opin Genet Dev 10, 529 (2000)). PI3Kα activation upon stimulation of receptor tyrosine kinases and G-protein coupled receptors induces pathologic cardiac hypertrophy through induction of protein translation and nucleotide biosynthesis (in part, via mammalian target of rapamycin (mTOR) (Wang et al., Physiology (Bethesda) 21, 362 (2006))), while PI3Kγ activation is linked to contractile dysfunction through inhibited Protein kinase A-cAMP pathway signaling (Crackower et al., Cell 110, 737 (2002); Patrucco et al., Cell 118, 375 (2004)). This intriguing capacity of PI3K signalling to simultaneously regulate two fundamentally critical aspects of disease transition is recapitulated at the phenotypic and gene expression level upon SINT1 inactivation—wherein SINT1-mediated modulation of the SMG1 and SYT17 pro-hypertrophic cluster simultaneously alters the growth and contractile maladaptation associated with disease transition. Strikingly, re-normalisation of SMG1 and SYT17 upregulation induced by TAC, either through Gapmer-mediated SINT1 inactivation, or the dual-targeted inactivation of SMG1 and SYT17 (
Given the correlation between SINT1, SMG1 and SYT17 co-expression in cardiomyopathy in independent human cohorts of HCM and aortic stenosis, it is conceivable to suggest a role of this axis in driving cardiac pathology. Indeed, suppression of stress-induced SINT1 production in vivo in mice resolved established cardiomyopathy through repression of Syt17 and Smg1 transcription, indicating that a tight coupling of enhancer transcription and successive induction of promoters in their vicinity is disease relevant. Both aortic stenosis (as the most prevalent valvular heart disease) and HCM (as the primary cause of sudden cardiac death), represent a large fraction of cardiac disease whose therapy today inefficient to prevent heart failure. Notably, SINT1 (or SMG1 or SYT17) induction was detected neither in human biopsies of dilated cardiomyopathy (DCM) nor in ventricular biopsies of a Muscle LIM protein (Mlp)−/− DCM mouse model (Arber et al., Cell 88, 393 (1997).) (
Materials and Methods
Animal Breeding and Maintenance
Hif1α fl/fl mice were obtained from Randall S. Johnson (University of California, San Diego, USA) and Gregg L. Semenza (Johns Hopkins University School of Medicine, USA), respectively. Vhl fl/fl mice were kindly provided by Rudolf Jaenisch (Massachusetts Institute of Technology, USA). MLC2v-cre/+ line and Mlp+/+ and Mlp−/− hearts were from Ju Chen (University of California, San Diego, USA). The data presented in this disclosure represents studies with male mice aged from 8-24 weeks old of the C57131/6 background. Mice were randomly assigned to groups, and surgery, AAV injections, Gapmer delivery and echocardiography performed blinded. Animal numbers for experiments were chosen based on expected mortality rates, anticipated phenotype and functional changes of hearts in wild-type mice in response to surgery. Animals were excluded from the study in case of death before the experimental endpoint or based on the evaluation of pain using a standardized score sheet, approved by BVET. All mice were maintained in a specific pathogen-free (SPF) facility at RCHCI and EPIC, ETH Zurich and/or Cardiovascular Assessment Facility (CAF), Department of Medicine, University of Lausanne. Maintenance and animal experimentation were in accordance with the Swiss Federal Veterinary Office (BVET) guidelines.
Human and Mouse Ventricular Biopsies
Left-ventricular samples were obtained from patients with hypertrophic cardiomyopathy aortic stenosis or dilated cardiomyopathy and healthy controls. Clinical and demographical data from patients were previously published (Mirtschink et al., Nature 522,444 (2015)). Biopsies were conducted in compliance with the local ethics committee, and written informed consent was received from all subjects prior to inclusion. Myocardial samples were obtained from patients with severe aortic stenosis undergoing aortic valve replacement and a Morrow resection from the hypertrophied left ventricular septum. Only patients without significant aortic valvular regurgitation and with preserved contractile function were included. Myocardium was also obtained from patients with severe HCM during septal myectomie surgery. The myocardial samples were acquired directly in the operating room during the surgery and immediately placed in precooled cardioplegic solution (110 mM NaCl, 16 mM KCl, 16 mM MgCl2, 16 mM NiPSC-hCMO3, 1.2 mM CaCl2), 11 mM glucose). Samples were frozen (−80° C.) immediately in the surgery room.
Transaortic Banding
16-20 week old mice were subjected to transaortic banding (TAC) through constriction of the aortic arch (between the innominate artery and the left carotid artery) as described (Kassiri et al., Circ Res 97, 380 (2005)). The mice were monitored regularly and their heart functions were determined by echocardiography. SINT1-targeting Gapmers (Exiqon) were generated, stored and used as recommended by the manufacturer. Gapmers were used at a dose of 10 mg/kg or 5 mg/kg as indicated in
1 Kidney 1 Clip Surgery
Under inhalation anesthesia by isoflurane a clip was placed around the left renal artery of 6-8 weeks old mice in order to reduce the renal blood flow, whereas the right kidney was removed as previously described (Krishnan et al., Cell Metab 9, 512 (2009)). The sham procedure, including the entire surgery with the exception of artery clipping, was applied in control mice. Postoperative analgesia was provided by subcutaneous application of Temgesic (Buprenorphin 0.1 mg/kg) directly after surgery as well as every 6-8 hours for 2 consecutive days after surgery. Gapmers were used at a dose of 10 mg/kg or 5 mg/kg as indicated in
In Vivo Transthoracic Ultrasound Imaging
Transthoracic echocardiography is performed in 2D- and M mode in the parasternal long-axis view using the MS400 (18-38 MHz) probe from Vevo2100 color Doppler ultrasound machine (VisualSonics) as previously described (Ounzain et al., J Mol Cell Cardiol 76, 55 (2014)).
Direct Arterial Blood Pressure Measurement
By a small incision in the carotid artery a heparinized catheter was introduced under inhalation anesthesia by isoflurane as described previously (Krege et al., Hypertension 25, 1111 (1995)). The free end of the catheter was externalized at the neck of the animal. The supply of anesthetic gas was stopped and the animal was returned to its cage placed on a heating surface for complete wake. 3-4 hours after awakening of the animal, the catheter was connected to a pressure sensor. The blood pressure was monitored for about 1 hour in awaked unconstrained animals.
Isolation and Maintenance of Primary Neonatal Mouse Cardiomyocytes
NMC were isolated as described previously (Krishnan et al., Cell Metab 9, 512 (2009)). Cultured cardiomyocytes were treated with phenylepinephrine at a concentration of 100 μM for 48 h, isoproterenol at 10 μM for 48 h and Dimethyloxaloylglycine (DMOG) at 100 μM for 24 h. Gapmers were added to cardiomyocytes at a concentration of 0.5 μM using the Accell siRNA delivery media (Dharmacon) according to the manufacturer's instructions.
Human Cardiomyocyte Culture
Human iPSC derived cardiomyocytes were thawed and cultured as recommended by the manufacturer (Cellular Dynamics International). Cells were transduced with lentiviruses and/or treated with Gapmers at a concentration of 0.5 μM 7-10 days after thawing and harvested at day 10-12.
Lentivirus Production and Transduction
Lentiviruses were generated in HEK-293T cells, purchased from ATCC and regularly checked for the presence of mycoplasma contaminants using a PCR-based detection kit (Sciencell). NMC were transduced as previously described (Mirtschink et al., Nature 522, 444 (2015)).
Lentiviral and Plasmid Expression Constructs
Lentiviral shRNAs were purchased from Sigma or custom designed using BLOCK-iT™ RNAiDesigner (Life Technologies) software and synthesized by Sigma. The following shRNAs in the lentiviral pLKO.1 vector from Sigma as part of their TRC library were purchased: Smg1 (shSmg1, TRCN0000088685) and Syt17 (shSyt17, TRCN0000173230), Hif1α (shHif1α, TRCN0000232220). SHC002 (Sigma) was used as a non-targeting shRNA. The following custom designed shRNAs in the pLKO.1 construct against SINT1 were used: shSINT1 #1, sense (5′-3′) (SEQ ID NO 002) (the sequences give the coding strand DNA sequences encoding the shRNA; the shRNA actually employed is the reverse complementary strand to SEQ 2/3):
and shSINT1 #3, sense (5′-3′) (SEQ ID NO 003):
The HIF1αΔODD expression construct was generated as described (Huang et al., Proc Natl Acad Sci USA 95, 7987 (1998).) and cloned into pLKO.1-CMV for lentiviral generation (Troilo et al., EMBO Rep 15, 77 (2014)). The pcDNA3 HA-Smg1 expression construct was kindly provided by Oliver Mühlemann (University of Bern, Switzerland).
To generate the AN-SRF fusion protein, the CAGGS promoter followed by AN was subcloned into the pV5-AviC vector between the MluI and SalI restriction sites. Afterwards, SRF was subcloned into the pV5-AN-AviC vector between the XhoI restriction site. SINT1 was amplified from mouse genomic DNA and cloned into the pcDNA3.1 vector between the HindIII and BamHI restriction sites. Subsequently, the 5× BoxB cassette was subcloned from pR6K BoxB vector into pcDNA3.1 vector between the BamHI restriction site. Sequence integrity of the cloned region was verified by sequencing and BLAST alignment (http://www.ncbi.nlm.nih.gov/blast).
Antisense Oligonucleotide Synthesis
Synthesis and purification of Gapmers were performed by Exiqon. As a standard Gapmers were purified and analyzed using anion-exchange HPLC, desalted and lyophilized as a sodium salt. Compound identity was confirmed by ESI-MS at a purity of >85%. The Gapmers contain phosphorothioate backbone modifications and proprietary modifications within the sequence, which differ between the in vivo and in vitro versions of the Gapmers.
Transient Transfection
293T cells grown in 12-well plates were transfected with 0.8 μg of the AN-SRF fusion construct and 0.8 μg of the SINT1-BoxB vector using Lipofectamine 2000 as recommended by the manufacturer. The medium was changed to DMEM containing 0.5% FCS 4 h after transfection and gene expression was analysed 48 h after transfection.
In Vitro Metabolic Measurements
Cellular oxygen consumption and extracellular pH, as readouts for oxidative metabolism and glycolysis, were measured using the Seahorse Bioscience XF24 or XFe96 Flux Analyzer at 37° C. with correction for positional temperature variations adjusted from four empty wells evenly distributed within the plate. NMC were seeded at 5×104 cells/well 3-5 days prior to the analysis.
In Vitro Contractility Assay
Assays were performed on neonatal mouse and human iPSC derived cardiomyocytes and analyzed with the IonOptix Cell Analyzer System. Cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope and perfused with a modified tyrode buffer (137 mM NaCl, 5 mM KCl, 15 mM Glucose, 1.3 mM MgSO4, 1.2 mM NaH2PO4, 20 mM HEPES, 1 mM CaCl2), pH 7.4) and field stimulated at a frequency of 1 Hz. Contractility was recorded and analyzed using the IonWizard software.
Histological Analysis
Hearts were embedded in OCT and sectioned at 10 μm. The sections were fixed for 10 min with 4% PFA/PBS and after 2×2 min PBS washes, the sections were blocked for 1 h with 2% HS/PBS. The sections were permeabilised for 10 min with 0.2% Triton X-100/PBS. After 3×5 min PBS washes, the sections were incubated with the primary antibody diluted in 2% HS/0.025% Triton X-100/overnight in a humidified chamber at 4° C. After 3×10 min PBS washes the secondary antibody was incubated overnight in a humidified chamber at 4° C. After 5×10 min PBS wash, the sections were mounted using ProLong gold Antifade. The sections were stained with laminin (1:200) to visualize the cell outline and imaged using the Leica sp8 confocal microscope. The cell cross-sectional area was quantified using Image J.
Immunocytochemistry and Cell Size Quantification
Immunofluorescent staining was performed as described previously (Krishnan et al., Cell Metab 9, 512 (2009)). Pictures of all channels were taken using a 20× magnification. Using the software Cell Profiler, cardiomyocytes were identified using the cell type-specific antibody and cell area was quantified. Multi-nucleated cells were counted manually and average area per cell was corrected taking into account the number of multi-nucleated cells.
RNA Fish
5×104 NMCs were seeded on poly-L-lysine coated 12 mm cover glasses. RNA FISH was performed using the QuantiGene® ViewRNA ISH Cell Assay Kit as recommended by the manufacturer Affymetrix. The following RNA FISH probes were used: Smg1 (VB4-15594), Syt17 (VB6-15595) and SINT1 (custom synthesis). Samples were imaged using the Leica sp8 confocal microscope.
[3H]Leucine Incorporation Assay
[3H]leucine incorporation was quantified in order to assess protein synthesis in NMC as previously described (Mirtschink et al., Nature 522, 444 (2015)). Radioactive labeled maintenance medium containing 0.5 μCi/ml [3H]leucine was added 3 days after NMC isolation and 4×105 cells per 3 cm dish labeled and incubated for 20 hours. On day three cells were collected by trypsinization. Incorporated radioactivity was normalized to absolute cell number.
Antibodies and Fluorescent Reagents
Antibody against sarcomeric α-actinin (A7811) was from Sigma. The Smg1 antibodies used for immunoblotting (sc-135563) and immunocytochemistry (HPA073972) were from Santa Cruz and Sigma, respectively. The antibody against Syt17 (15413-1-AP) was from Proteintech. Upf1 antibody (07-1014) and phospho-Upf1 (Ser1127, 07-1016) were from Merck Millipore. Hif1α antibody (H1alpha67) used for ChIP and immunoblotting, pan-Cadherin (ab6528), laminin (ab11575) and Atp5a1 (ab14748) antibodies were from Abcam. H3K4me1 (#39297), H3K4me3 (#39159) and H3K27ac (#39133) antibodies used in ChIP were from Active Motif. Myosin binding protein C antibody was kindly provided by Mathias Gautel (King's College London, UK).
SDS-PAGE and Immunoblotting
Dissected hearts were homogenized by freeze slamming and solubilized in a modified SDS sample buffer sonicated and boiled for 5 minutes. Cultured cardiomyocyte lysates were harvested with the modified SDS sample buffer, sonicated and boiled. Protein extracts were resolved on 6-12% polyacrylamide minigels (BioRad) and transferred overnight onto nitrocellulose membrane (GE Healthcare). Immunodetection and visualization of signals by chemiluminescence was carried out as described (Hirschy et al., Dev Biol 289, 430 (2006)).
Luciferase Promoter Assays
1.5 kb of the SINT1 promoter was amplified from mouse BAC genomic DNA and cloned into the pGL3 luciferase reporter vector (Stratagene). The HRE-mutant was generated by recombinant PCR (Casonato et al., J Lab Clin Med 144, 254 (2004); Elion et al., Curr Protoc Mol Biol Chapter 3, Unit 3 17 (2007)). Sequence integrity of the respective wildtype and mutant promoters were verified by sequencing and BLAST alignment (http://www.ncbi.nlm.nih.gov/blast). The reporter assay was performed by transient co-transfection of the appropriate luciferase reporter, pSV-β-galactosidase (Promega), in the presence of HIF1αΔODD. Luciferase and β-galactosidase activity was measured with the Luciferase Assay System kit (Promega) as recommended by the manufacturer and analyzed on the FLUOstar Omega (BMG Labtech). Mouse cardiomyocytes were transfected with Trogene or Lipofectamine 2000 (Life Technologies) as recommended by the manufacturer.
For enhancer activity determination, a 2.9 kb fragment containing SINT1 and additional flanking sequences was amplified from mouse BAC genomic DNA and cloned into the pGL3 luciferase enhancer reporter vector (Stratagene) as previously described (Lam et al., Nature 498, 511 (2013)). A 3 kb control genomic fragment encompassing the peroxisome proliferator activated receptor γ (Pparγ promoter void of enhancer features was amplified and cloned in a similar manner. Sequence integrity of the amplified region was verified by sequencing and BLAST alignment (http://www.ncbi.nlm.nih.gov/blast).
Quantitative RT-PCR
RNA was isolated and genomic DNA removed with the RNeasy Plus Kit (Qiagen) and cDNA generated using Superscript II (Invitrogen) as recommended by the manufacturer. qPCR reactions were setup as recommended by the manufacturer (Biorad) and analyzed on the PikoReal Real-Time PCR System (Thermo Scientific). Ct values were normalized to the housekeeping gene Hprt1. The following qPCR primers were used:
ChIP and ChIRP
ChIP assays were performed using material from NMC and the assay performed using the ChIP-IT kit (Active Motif) as recommended by the manufacturer and analyzed by qPCR. For ChIP-seq analyses (Active Motif) hearts were removed from the mice, snap frozen in liquid N2 and stored at −80° C. For fixation, hearts were cut into small pieces in PBS, 1% formaldehyde and incubated at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125M glycine and tissue pieces were treated with a Tissue-Tearor. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 μg) was pre-cleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 μg of antibody against Hif1α, H3K4me1 and H3K4me3. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65° C., and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. qPCR reactions were carried out in triplicate on specific genomic regions using SYBR Green Supermix (Bio-Rad). The resulting signals were normalized for primer efficiency by carrying out qPCR for each primer pair using Input DNA. ChIP and Input DNAs were prepared for amplification by converting overhangs into phosphorylated blunt ends and adding an adenine to the 3′-ends. Illumina genomic adapters were ligated and the sample was size-fractionated (200-300 bp) on a 2% agarose gel. After a final PCR amplification step (18 cycles), the resulting DNA libraries were quantified and sequenced on HiSeq 2500 (50 nucleotide reads, single-end). Reads were aligned to the mouse genome (mm9) using the BWA algorithm. Duplicate reads were removed and only uniquely mapped reads (mapping quality >=25) were used for further analysis. Alignments were extended in silico at their 3′-ends to a length of 150 bp, which is the average genomic fragment length in the size-selected library and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in BAR and bigWig files. Peak locations were determined using the MACS algorithm (v 1.4.2) with a cutoff of p-value=1e−7. Signal maps and peak locations were used as input data to Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations. In silico promoter analyses and alignments were performed using Matlnspector and DiAlignTF (Genomatix). Primer sequences used for SINT1 in the ChIP were 5′-CCACAGAGCAGGAAGCAGAGA-3′ (SEQ ID NO 058) and 5′-GGTTTGAATGCGAAATGTCCTTAC-3′ (SEQ ID NO 059). ChIRP was performed as previously described (Chu et al., Mol Cell 44, 667 (2011)) using biotinylated oligonucleotides from Microsynth AG or Sigma.
RNA Sequencing
The twelve paired-end libraries (three replicates for each four samples) were processed by using the Trimmomatic v0.36 (Bolger et al., Bioinformatics 30, 2114 (2014)) software. All Illumina standard adapter and primer sequences were trimmed and read length cutoff of 60 bases and a window based quality filtering (window length: 5base; phred quality score cutoff: 20) was applied. Filtered reads were mapped to the genome of Rattus norvegicus (Gen Bank assembly accession: GCA_000001895.4) by using the STAR v2.5.3a (Dobin et al., Bioinformatics 29, 15 (2013)) RNA-seq aligner. The generated binary alignment map (bam) files were processed by using the featureCounts v1.5.0-p3 (Liao et al., Bioinformatics 30, 923 (2014)) software for generating a count matrix. Differential gene expression analysis was carried out by using the DeSeq2 (Love et al., Genome Biol 15, 550 (2014)) software, by importing the count matrix. Differential gene expression analyses were performed by taking normoxic libraries as control to other treated libraries. Differentially expressed genes were filtered by taking a p-value cutoff of 0.05 of FDR (false discovery rate) test. KEGG enrichment of the differentially expressed genes were carried out by using p-value <0.05 by using the clusterProfiler (Yu et al., OMICS 16, 284 (2012)) package in R (https://www.r-project.org/). After performing the trimming and filtering step, on an average 86.44% sequences could be retrieved from the twelve libraries. For generating an abundance count matrix all twelve libraries were mapped, by using STAR aligner, on the Rattus norvegicus genome. STAR mapping showed an average of 88.80% of reads from the twelve libraries could be mapped. The abundance count matrix was imported to the DeSeq2 software for calculating the fold change values with respect to the normoxic libraries. In total three replicates for each sample was used for evaluating the differential gene expression.
Statistical Analysis
For statistical analyses, unpaired, two-tailed Student's t-tests (Excel) or one-way ANOVA analyses followed by a Dunnet's multiple comparison post-test were used as indicated in the respective figure legends. No statistical methods were used to predetermine sample size.
Validation of Antisense Oligonucleotides Directed Against Human SINT1
iPSC-hCM expressing ectopic HIF1αΔODD were treated with the respective gapmers of table 3 and tested for SINT1 knockdown by qPCR (
The positions of any sequence in Table 3 can be chosen from LNA, PNA, DNA. In certain embodiments, central (8 to 12, particularly 10) positions are DNA; flanking sequences (4-2, respectively) on either side are LNA or PNA. In certain embodiments, the LNA sequences are linked by phosphothioate ester bonds.
GCTGGTTTA
TGAACCAAT
ACCAATGCA
AATGCAGTA
ATGCAGTAA
TGCAGTAAT
CTCTCTCTA
GAATTTACT
ATTTACTTT
GAATCAGAA
| Number | Date | Country | Kind |
|---|---|---|---|
| 18180595.3 | Jun 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/067494 | 6/28/2019 | WO | 00 |
