WO2020065080A2 - Long non-coding rna h19 for use in treatment of ischemic acute kidney injury - Google Patents

Abstract

The invention relates to a polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or inflammation in the kidney, wherein said polynucleotide agent comprises, or is capable of expressing, a long non-coding H19 RNA sequence.

Description

Long Non-Coding RNA H19 for use in treatment of ischemic acute kidney injury

The present invention relates to the use of H 19 RNA in prevention and treatment of ischemic kidney injury.

This application claims the benefit of European Patent application EP18197675.4 of September 28, 2018, the contents of which are incorporated herewith by reference.

Background

Ischemia-reperfusion (l/R) injury of the kidney is a major cause of acute kidney injury (AKI). Due to its high morbidity and mortality it represents a major socioeconomic health problem. A variety of injurious insults in native kidneys may promote its development (e.g. during cardiac surgery). In addition, it is an unavoidable phenomenon during the kidney transplantation procedure.

The adult kidney is characterized by an ability to respond to acute injury by cellular regeneration, partly through activation of embryonic signaling pathways, including the Wnt, Bone Morphogenetic Protein (BMP) and retinoic acid signaling pathway as well as the paired- box (Pax) gene family, especially Pax2. The present invention identifies a novel mechanism of renal regeneration involving the long non-coding RNA H19, which the inventors found to be highly induced in ischemic kidneys as a pro-proliferative, pro-regenerative, anti-inflammatory and anti-apoptotic factor. Under physiological conditions H19 shows a high expression in embryonic kidneys and is undetectable in adult life (see results).

The H19 gene is located downstream of the insulin-like growth factor 2 (Igf2) gene on chromosome 7 in mice and 1 1 p15.5 in humans. H19 has been shown to be re-activated during liver regeneration following two-thirds partial hepatectomy (Yamamoto et al., J Hepatol. 2004; 40: 808-814) and in rat vascular smooth muscle cells upon injury (Kim et al., J Clin Invest. 1994; 93: 355-360). H19 has previously been reported to be induced in hypoxic endothelial cells, possibly via regulation of Hypoxia Response Elements (Zhang et al., Sci Rep. 2016; 6: 20559). In a mouse model of hindlimb ischemia H19 was found to be induced (Voellenkle et al., Sci Rep. 2016; 6: 24141 ). H19 inhibition decreased human umbilical vein endothelial cell (HUVEC) growth as well as diminished HUVEC ability to form capillary like structures. In myocardial ischemia/reperfusion-injury H19 was found to repress miR-103/107, ultimately leading to increased Fas-associated protein with death domain (FADD) expression regulating programmed necrosis by interacting with receptor-interacting serine/threonine-protein kinase (RIPK) 1 and 3. These studies indicate that H19 might be part of a general mechanism of regeneration following injury. In kidney injury the role of H19 has never been investigated. Therapies aimed at restoring kidney function preventing inflammation and cell death are an area of intense research initiatives in order to ameliorate l/R-injury.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods for prevention or treatment of ischemic kidney injury. This objective is attained by the subject-matter of the independent claims of the present specification.

Summary of the invention

Hypoxia-induced long-non-coding RNA H19, which shows high embryonic expression and is silenced in adults, was shown to be upregulated in renal l/R injury. H19 was found to be upregulated in kidney biopsies of patients with AKI, in murine ischemic kidney tissue as well as cultured and ex vivo sorted hypoxic endothelial cells (EC), tubular epithelial cells (TEC) and pericytes. H19 was found to be transcriptionally activated in EC and TEC by Hypoxia-inducible factor 1 -alpha as well as LHX8 and SR11. H19 overexpression promotes angiogenesis in vitro and in vivo. In vivo, transient AAV2-mediated H19 overexpression resulted in a significant improvement of kidney function, a reduction of apoptosis, inflammation as well as a preservation of capillary density and tubular epithelial injury. Effects are mediated by sponging of miR-30a-5p, which in turn leads to target regulation of DII4, ATG5 and Snail . These results demonstrate that H19 promotes recovery from renal ischemic injury by stimulating pro- angiogenic signalling. H19 overexpression is a promising future therapeutic option in the treatment of patients with ischemic AKI.

A first aspect of the invention relates to a polynucleotide agent for use in treatment or prevention of a disease or of a symptom associated with an ischemic state or with an inflammation in the kidney. The polynucleotide agent according to this aspect of the invention comprises or consists of, or is capable of expressing, in a mammalian cell, a long non-coding H19 RNA sequence.

In particular embodiments, the long non-coding H19 RNA sequence is characterized by SEQ ID NO 01 , 02 or 03 or a sequence similar thereto having the same biological effect.

A second aspect of the invention relates to a polynucleotide agent capable of hybridizing to miR-30a-5p for use in treatment or prevention of a disease or symptom associated with an ischemic state or inflammation in the kidney. This can be an antisense oligonucleotide made synthetically, or an RNA oligomer expressed from an expression cassette administered to the patient. Particular medical conditions in which the polynucleotide agent according to the invention are of advantage include ischemia-reperfusion injury, or for prevention of renal damage as a result of an operative cardiac intervention associated with reduced cardiac output, particularly aortic surgery with cross-clamping of the aorta, and kidney transplantation.

Another aspect of the invention relates to an adeno-associated virus, particularly AAV serotype 2, comprising a polynucleotide expression cassette encoding the polynucleotide agent according to the above aspects of the invention.

Terms and definitions

The term “AAV vector’’ in the context of the present specification relates to a viral vector composed of 60 AAV capsid proteins and an encapsidated AAV nucleic acid. For medical use, an AAV vector can be derived from an AAV virion, but the AAV vector is engineered to be replication-incompetent by removing the rep and cap genes from the AAV genome. The encapsidated AAV nucleic acid may comprise a transgene which is to be delivered into a target cell.

“Capable of forming a hybrid” in the context of the present specification 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.

“Nucleotides” in the context of the present specification are nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with RNA or DNA oligomers or longer RNA sequences (specifically with miR-30a-5p) on the basis of base pairing. The term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphotioates, 2Ό- 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; 2Ό, 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 term hybridizing sequence or polynucleotide agent capable of hybridizing encompasses a polynucleotide sequence comprising or essentially consisting of RNA (ribonucleotides), DNA (deoxyribonucleotides), phosphothioate deoxyribonucleotides, 2’-0-methyl-modified phosphothioate ribonucleotides, LNA and/or PNA nucleotide analogues, or any mixture thereof.

In certain embodiments, the hybridizing sequence according to the invention comprises 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

In certain embodiments, the hybridizing sequence is at least 80% identical, more preferred 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reverse complimentary sequence of SEQ ID NO 07 (miR-30a-5p). The rev comp sequence of is miR-30a-5p; it is shown as SEQ ID NO 08. The sequence that hybridizes to miR-30a-5p can be SEQ ID NO 08 or a derivative thereof, for example a shorter sequence capable of binding to miR-30a-5p. In certain embodiments, the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.

In the context of the present specifications 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 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 stated otherwise, 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.

The term“polyadenylation sequence” or “polyadenylation signal” in the context of the present specification relates to a sequence leading to the addition of a poly(A) tail to the transcribed RNA. A common polyadenylation sequence in eukaryotes is A2UA₃.

In a first aspect, the invention relates to a polynucleotide agent for use in treatment or prevention of a disease or of a symptom associated with an ischemic state or inflammation in the kidney. The polynucleotide agent of the invention comprises, or consists of, a long non- coding H 19 RNA sequence. Alternatively, the polynucleotide agent of the invention is capable of expressing the H19 RNA sequence in a mammalian cell, particularly a human cell. In certain embodiments, the H19 RNA sequence is characterized by SEQ ID NO 01 , 02 or 03, particularly by SEQ ID NO 01 .

In certain embodiments, the H 19 RNA sequence is characterized by a sequence at least (>) 90%, > 92%, > 94%, > 96%, or even > 98% identical to a reference sequence selected from SEQ ID NO 01 , SEQ ID NO 02 and SEQ ID NO 03, wherein the H19 RNA sequence has biological activity essentially similar to that of SEQ ID NO 01.

In certain embodiments, the polynucleotide agent is an expression construct encoding and capable of expressing said long non-coding H19 RNA sequence under control of an RNA polymerase promoter operable in a human cell.

In particular embodiments, the promoter driving transcription of the RNA in the expression construct is a polymerase II (pol II) promoter, particularly a constitutive pol II promoter, more particularly a CMV promoter (CMV immediate early) or a CAG promoter. In certain other embodiments, the promoter is a promoter that can be induced, for example by small molecule drugs such as tetracycline.

In certain embodiments, the sequence encoding the non-translated RNA in the expression construct is followed by a sequence leading to polyadenylation in the cell. In particular embodiments, this sequence can be derived of SV40.

In certain particular embodiments, the expression construct is part of the genome of an adeno- associated virus [AAV] In certain particular embodiments thereof, the expression construct comprises inverted terminal repeat (ITR) sequences positioned 5’ and 3’ of a sequence encoding the long non-coding H19 RNA sequence.

In certain embodiments, the polynucleotide agent as an RNA or as an expression construct for the transcription of H19 RNA, comprises a sequence capable of hybridizing to a miR122 polynucleotide in a position 3’ of the H19 sequence (or the sequence encoding the long non- coding H19 RNA sequence). In the expression sequence, the sequence will be positioned 5’ of the polyadenylation sequence. In certain particular embodiments, the miR122 polynucleotide is selected from SEQ ID 04, 05 and 06.

In certain embodiments, the miR122 polynucleotide is characterized by a sequence at least (>) 90%, > 92%, > 94%, > 96%, or even > 98% identical to a reference sequence selected from SEQ ID NO 04, SEQ ID NO 05 and SEQ ID NO 06, wherein the H19 RNA sequence has biological activity essentially similar to that of SEQ ID NO 04.

In certain embodiments, the polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or inflammation in the kidney of the invention is comprised in a circular DNA plasmid, optionally packaged by lipid vesicle (liposome, exosome); or in a virus, particularly a virus selected from adeno-associated virus (AAV), adenovirus (AV), lentivirus, more particularly an AAV-2 vector.

According to another aspect of the invention, the polynucleotide agent of the invention is capable of hybridizing to miR-30a-5p. Without wishing to be bound by theory, the inventors believe that the capability of H19 RNA to bind and neutralize miR-30a-5p, which is pro- inflammatory and may have an important role in the mechanisms leading to damage the following ischemia in the kidney. MiR-30a-5p has been shown to modulate angiogenesis. It targets ATG5 and Snail . In certain embodiments of this aspect, the polynucleotide agent is a short antisense oligonucleotide, particularly in oligonucleotide comprising synthetic analogs of the natural DNA or RNA nucleotides, such as locked nucleic acid or similar analogues, or mixtures of natural and synthetic nucleotide building blocks.

In certain embodiments of this aspect, the polynucleotide agent is an oligonucleotide 8 to 25 nucleotides long. In certain embodiments, the polynucleotide agent is an oligonucleotide 8 to 25 nucleotides long capable of hybridizing to and abrogating the biological activity of miR-30a- 5p. In certain particular embodiments, this polynucleotide agent comprises thioate modifications at the 5’ and 3’ termini. In certain embodiments, for which any particular sequence capable of of hybridizing to and abrogating the biological activity of miR-30a-5p is considered, the polynucleotide agent comprises between 2 and 5, particularly 3 or 4 thioate modifications at each the 5’ and 3’ terminus. In certain particular embodiments, the polynucleotide agent comprises LNA moieties instead of“classical” ribonucleoside moieties in addition to the phosphothioate moieties.

In certain embodiments of any aspect of the invention, the treatment for which the polynucleotide agent is employed is for ischemia-reperfusion injury, or for prevention of renal damage as a result of an operative cardiac intervention associated with reduced cardiac output, particularly aortic surgery with cross-clamping of the aorta, or kidney transplantation.

Another aspect of the invention relates to the expression construct as developed by the inventors. Such polynucleotide expression vector according to the invention comprises an expression cassette comprising or essentially consisting of, in the order from 5’ to 3’,

a polymerase II promoter operable in a mammalian cell, particularly in a human cell; a sequence encoding a long non-coding H19 RNA sequence, particularly sequence characterized by SEQ ID NO 01 , 02 or 03, more particularly by SEQ ID NO 01 ;

a sequence element encoding a sequence capable of binding to miR122; and a polyadenylation sequence.

In certain embodiments of this aspect, the pol II promoter is a constitutive promoter, particularly a promoter selected from immediate early CMV promoter-enhancer, SV40 promoter, elongation factor 1 alpha (EF1 a) promoter, ubiquitin C promoter, human beta actin promoter. In particular embodiments, the expression cassette comprises two adenovirus associated virus inverted terminal repeat (ITR) sequences, one positioned 5’ of the pol II promoter sequence and one positioned 3’ of the polyadenylation sequence.

In certain particular embodiments, the expression cassette is part of the genome of an AAV serotype 2. The administration of the polynucleotide agent comprising an expression cassette for H19 delivered by AAV serotype 2 has led to protection of renal tissue and enhanced renal function.

Similarly within the scope of the present invention is a method or preventing or treating ischemic kidney injury in a patient in need thereof, comprising administering to the patient a polynucleotide agent according to the above description.

Similarly, a dosage form for the prevention or treatment of ischemic kidney injury is provided, comprising a polynucleotide agent according to one of the above aspects of the invention.

Wherever alternatives for single separable features are laid out herein as“embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

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.

Description of the figures

Fig. 1 shows the expression of LncRNA H 19 in cells, humans and mice under ischemic/hypoxic conditions. LncRNA H 19 expression was determined in kidney transplant patients with short compared to long cold ischemia times (CIT, A, n=5 patients per group), in mouse embryonic (E13.5), adult, and ischemia/reperfusion (l/R) kidneys (B, n=8 mice per group). FACS-based sorting of mouse kidney homogenates following ischemia/reperfusion-injury exposed to rat antimouse-CD31-PE antibody for endothelial cells (C) and injured proximal tubular cells positive for Lotus teragonolobus lectin for proximal epithelium and anti-mouse Tim 1 -biotin antibody followed by streptavidin-APC (D). Expression of IncRNA H19 in cultured endothelial cells (HUVEC, E) and proximal tubular cells (RPTEC, F). Each bar represents the mean ± SEM of duplicate cultures and the data are from a representative experiment of 4 independent experiments that were performed. CIT = cold ischemia time, l/R = ischemia/reperfusion, TPL = kidney transplantation; ^* = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001.

Fig. 2 shows transcriptional regulation of IncRNA H19 under hypoxia. Expression of IncRNA H19 (A), SPI 1 (B) and LHX8 (C) following siRNA knockdown of HIFI ot and EPAS at 24, 48 and 72h of hypoxia compared to normoxia (A). Expression of IncRNA H19 following siRNA knockdown of SPI1 and LHX8 under hypoxia of 72h (D). Protein expression of immunoprecipitated SPI1 and LHX8 in HUVECs in hypoxia (E, F). Schematic representation of SPI1 and LHX8 binding sites in the IncRNA H19 promoter region (G). Amplification by qPCR following Chromatin immunoprecipitation (ChIP) using specific primers to verify the binding of SPI1 and LHX8 to the promoter region of IncRNA H19 (H, I). Each bar represents the mean ± SEM of duplicate cultures and the data are from a representative experiment of 3 independent experiments that were performed. ChIP = Chromatin immunoprecipitation, IP = immunoprecipitation, Lnc H19 = IncRNA H19, siC = siRNA control, TSS = transcriptional start site. ^* = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001.

Fig. 3 shows the effect LncRNA H19 overexpression on migration, proliferation, apoptosis and angiogenesis in endothelial cells. HUVECs overexpressing IncRNA H19 were assayed for cellular migration (A) in response to VEGF (50ng/ml_) using modified Boyden chamber (8 pm pore size). Cellular proliferation (B) was determined using EdU cell proliferation system and flow cytometry in HUVEC over expressing IncRNA H19 and treated with VEGF (50 ng/mL). The effect of IncRNA H19 overexpression on angiogenic potential of endothelial cells (C & D) was determined using HUVEC coated on cytodex beads and grown in fibrin gel in the presence of VEGF (50 ng/mL). Cellular apoptosis (E) was determined by cleaved caspase3/7 in HUVEC overexpressing IncRNA H19 in response to the apoptotic stimulus TNFa (10 ng/mL) and cycloheximide (25 pg/mL). Each bar represents the mean ± SEM of duplicate cultures and the data are from a representative experiment of 3 independent experiments that were performed. . EV = empty vector, CHX/TNFa = Cycloheximide/tumor necrosis factor-a; LV-H19 = lentivirus targeting IncRNA H 19, NT = no treatment, VEGF = vascular endothelial growth factor. ^* = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001.

Fig. 4 shows signaling pathways modulated by IncRNA H19 overexpression in HUVECs. (A) Shows representative Western blots of HUVECs transduced with a lentivirus to overexpress IncRNA H19 as compared to cells transduced with an empty vector control. In both settings cells were treated with VEGF (25 ng/mL) at the indicated times. (B) shows quantification of Akt phosphorylation, (C) ERK phosphorylation and (D) p38 phosphorylation. Black bars represent cells following control virus transduction, white bars H19 lentivirus transduction. Each bar represents the mean ± SEM of duplicate cultures and the data are from a representative experiment of 3 independent experiments that were performed. LV-H19 = lentivirus targeting IncRNA H19, NT = no treatment. ^* = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001. Fig. 5 shows the interaction between IncRNA H19 and miR-30a-5p. (A) Shows sequence alignment of miR-30a-5p and a putative binding region within exon 5 of IncRNA H19. In addition, mutant exon 5 sequence is shown, in which 5 nucleotides -TGTTT- were deleted. The interaction between miR-30a-5p and IncRNA H19 was validated by luciferase assay (B). The entire exon 5 of IncRNA H19 was cloned into a 3’UTR renilla luciferase expression plasmid. HUVEC were transfected with the luciferase plasmid and either with a miR-30a-5p mimic, inhibitor, or miR control sequence. LncRNA H19 exon 5-luciferase plasmid was mutated using site-directed mutagenesis to delete 5 nucleotides in exon 5. The overexpression of IncRNA H19 suppresses the expression of miR-30a-5p in HUVECs (C). D & E show the role of miR-30a-5p in angiogenesis. The overexpression of miR-30a-5p by the mimic sequence reduces sprouting of endothelial cells in a 3D fibrin gel. (D) Quality control qPCR to demonstrate the increased expression of miR-30a-5p using a miR-mimic and reduced expression by the specific antimiR (or inhibitor). Each bar represents the mean ± SEM of duplicate cultures and the data are from a representative experiment of 3 independent experiments that were performed. EV = empty vector, LV-H19 = lentivirus targeting IncRNA H19, miR-C = miR control. ^* = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001. Sequences: H19 exon 5: SEQ ID NO 09; miR- 30a-5p SEQ ID NO 7; H19 mutant: SEQ ID NO 10.

Fig. 6 shows miR-30a-5p target regulation. Representative Western blots (A) as well as quantification (B) of HUVECs transfected with a miR-30a-5p mimic or antimiR and probed for DII4, ATG5, and SNAI1. C shows the alignment of the miR-30a-5p sequence and a putative binding site in ATG5 and SNAI1 3’UTR regions. The nucleotides sequence TGTTT was deleted by site-directed mutagenesis from ATG5 and SNAI1 3’UTR to generate the mutant plasmids. HUVEC were transfected with the luciferase plasmids (ATG5 or SNAI1 ) and either with miR-30a-5p mimic, inhibitor, or miR control sequences (D). Each bar represents the mean ± SEM of duplicate cultures and the data are from a representative experiment of 3 independent experiments that were performed. * = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001. Sequences: miR-30a-5p SEQ ID NO 7; H19 mutant: SEQ ID NO 10. H19 exon 5: SEQ ID NO 09;

Fig. 7 shows AAV2-mediated IncRNA H19 overexpression in vivo protects against l/R injury.

Expression of miR-30a-5p in vivo was determined after unilateral kidney clamping in mice subjected to 30 min ischemia and the kidney was reperfused for 1 or 7 day(s). miR- 30a-5p expression in l/R-injury in uninjected mice at 1 and 7 days of reperfusion (A). The overexpression of IncRNA H19 in vivo was achieved through renal vein injection and validated by qPCR (B). Kidney injury after 24 h reperfusion was evaluated by qPCR and the expression of KIM1 (C), NGAL (D) and the inflammatory markers TNFa (E) and IL- 1 b (F) assessed. Injury in outer medulla was assessed after unilateral clamping and 24h of reperfusion in PAS-stained kidney sections (G), H shows quantification of results. AV- GFP = Adeno-associated viral vector 2 targeting a control sequence, AV-H19 = Adeno- associated viral vector 2 targeting IncRNA H19, I L-1 b = Interleukin 1 b; KIM1 = kidney injury molecule 1 , NGAL = Neutrophil gelatinase-associated lipocalin, TNFa = tumor necrosis factor-a, Lnc-H19 = IncRNA H19. Each bar represents the mean ± SEM of 6 mice used per experiment. ^* = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001 , NS = non- significant.

Fig.8 shows LncRNA H19 overexpression in mouse kidneys by use of an adeno-associated virus type 2 (AAV2) confers protection against ischemia/reperfusion injury in bilateral l/R- injury. Both renal veins were injected with AAV2 and mice were followed up for 7 days. Injury score in outer medulla of mice receiving AAV2-H19 or AAV2-CTL as well as quantification or results (A). Renal KIM-1 and NGAL expression in mice receiving AAV2- H19 or AAV2-CTL (B). Blood urea nitrogen concentration in mice receiving AAV2-H19 or AAV2-CTL (C) Endomucin expression visualizing capillary density as well as quantification of results in mice receiving AAV2-H19 or AAV2-CTL (D). Proliferation in outer medulla as shown be Ki67 expression as well as quantification of results in mice receiving AAV2-H19 or AAV2-CTL (E). TUNEL staining as well as quantification of results in mice receiving AAV2-H19 or AAV2-CTL (F) Each bar represents the mean ± SEM of 6 mice used per experiment. AV-GFP = Adeno-associated viral vector 2 targeting a control sequence, AV-H19 = Adeno-associated viral vector 2 targeting IncRNA H19. * = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001.

Fig. 9 shows results of the analysis of inflammatory markers and apoptosis following ischemia/reperfusion injury. Adeno-associated virus 2 carrying IncRNA H19 or control (GFP) were delivered through the renal vein and the left kidney was subjected to unilateral clamping for 30 min followed by 24h of reperfusion. Five urn paraffin-embedded sections were stained for T cells (CD3, A) and macrophages (F4/80, B). In addition, the kidney sections were stained for apoptotic cells using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (C). The fluorescently labeled kidney sections were visualized using a confocal microscope. Each bar represents the mean ± SEM of 6 mice used per experiment. ^* = p < 0.05, ^*** = p < 0.001.

Fig. 10 shows the pro-inflammatory response of bone marrow-derived murine macrophages (BMDM) overexpressing IncRNA H19 or control (GFP) virus. BMDM were transduced with adeno-associated virus 2 carrying either IncRNA H19 or control sequences. The macrophages were sensitized with interferon g (100 U/mL) and activated with lipopolysaccharide (1 mg/mL). The activated macrophages were assayed for nitric oxide production using modified Griess reagent, and the supernatants were analyzed for TNFa and IL-6 release by ELISA. Each bar represents the mean ± SEM of duplicate cultures and the data are from a representative experiment of 3 independent experiments that were performed. ^* = p < 0.05, ^** = p <0.01 , ^*** = p < 0.001.

Example 1: LncRNA H19 is dysregulated in hypoxic/ischemic kidney injury in humans, mice and cells.

We first assessed the expression level of IncRNA H19 in kidney biopsies of transplant patients with long compared to short cold ischemia time and found it to be highly increased, suggesting a possible induction by ischemia (Fig. 1 A). In murine kidney tissue IncRNA H19 was found to be highly expressed in embryonic kidneys (E13.5), almost entirely repressed in adult kidneys and re-activated in ischemic kidneys of mice (30 minutes of ischemia, 24h of reperfusion; see Fig. 1 B). In order to identify the cellular origin of IncRNA H19 in ischemic AKI we isolated CD31 ⁺ endothelial cells (Fig. 1 C), healthy LTA7KIM1 (data not shown) as well as injured LTA7KIM1 ⁺ proximal tubular epithelial cells (Fig. 1 D) by fluorescence-associated cell sorting using specific antibodies and/or lectins from mouse kidneys following l/R-injury. Intriguingly, IncRNA H19 was found to be enriched in ischemic CD31 ⁺ - endothelial cells and injured LTA7KIM1 ⁺ - proximal tubular epithelial cells, while its expression was unchanged in healthy LTA7KIM1 - proximal tubular epithelial cells. In line with this finding IncRNA /-/^ was found to be induced in cultured endothelial cells (HUVECs, at 72h of hypoxia) and proximal tubular epithelial cells (RPTEC, at 24h of hypoxia) cells by hypoxia/reoxygenation (Fig. 1 E & F). We performed a subcellular fractionation in endothelial cells and proximal tubular epithelial cells and found that IncRNA H19 was mostly localized in the cytoplasm (data not shown).

Given the aforementioned data, the role of IncRNA H19 in endothelial and tubular epithelial cells was analyzed in detail by modulation of its expression. LncRNA H19 was lentivirally overexpressed and silenced by use of antisense oligonucleotides.

Transcriptional regulation of IncRNA H19

When investigating the transcriptional regulators of IncRNA H19 under hypoxic conditions, the hypoxia-responsive transcription factors HIF1 a and Endothelial PAS domain protein 1 (EPAS or HIF2a) were found to be induced with sustained expression up to 24h of hypoxia in HUVECs. Moreover, HIFI a and EPAS (HIF2a) were significantly silenced by use of siRNA at 24, 48 and 72h of hypoxia.

Given the induction of expression of IncRNA H19 in HUVECs at 72h of hypoxia (see Fig. 1 G) other transcription factors were searched for using the Matinspector Genomatix transcription factor search site (www.genomatix.de) by assessing potential binding in a region 1000bp upstream of the transcriptional start site of IncRNA H19. Several interesting transcription factors were assessed and the list was narrowed down to four transcription factors, which show regulation at 72h of hypoxia, including SPI 1 or PU.1 , LIM homeobox 8 (LHX8), Krueppel-like factors 8 and 15 (KLF8, KLF15).

HIF1 a and EPAS (HIF2a) were silenced by siRNA for 24, 48 and 72h and the expression levels of IncRNA H19 assessed. Interestingly, silencing of both transcription factors resulted in blunting of H19 expression at 72h of hypoxia (see Fig. 2A). In addition, silencing of HIF1 a and EPAS (HIF2a) also resulted in suppression of increased expression levels of SPI 1 , and LHX8 at 72h of hypoxia (see Fig. 2B & C), suggesting that there may be a link between hypoxia- inducible factors and our newly identified transcription factors.

Importantly, the siRNA knockdown of SPI 1 and LHX8 resulted in suppressed expression levels of IncRNA H19 at 72h of hypoxia (see Fig. 2D). Double knockdown of SPI 1 and LHX8 resulted in an even more pronounced reduction of IncRNA H19 expression level at 72h of hypoxia (see Fig. 2D). The expression of SPI 1 and LHX8 in HUVECs was validated by Western blot of immunoprecipitated SPI 1 and LHX8 at 72h of hypoxia (see Fig. 2 E).

Fig. 2G shows a schematic representation of binding sites in the region upstream of the IncRNA H19 transcriptional start site (TSS). Three putative SPI1 (site 1 : -370, site 2: -403, and site 3: -596) and 2 LHX8 binding sites (site 1 : -47, and site 2: -898) were identified upstream of IncRNA H19 transcriptional start site (TSS). To validate the functionality of SPI 1 and LHX8 binding sites chromatin immunoprecipitation (ChIP) was performed using antibodies specific to LHX8 and SPI 1. Subsequently a qPCR-based amplification was performed using primers flanking the aforementioned regions. Here, we were able to show specific activation of SPI1 and LHX8 (see Fig. 2H, I). Two sites for LHX8 and 3 sites for SPI 1 were identified (see Fig. 2G). Site 1 (at 47bp upstream of TSS for LHX8 and 370bp upstream of TSS for SPI1 ) was found to be associated with the most pronounced expression of IncRNA H19 (see Fig. 2G).

The LncRNA H19 promoter was cloned into firefly luciferase promoter plasmid and transfected into HUVECs and RPTEC. Cells were placed in a hypoxia chamber for the indicated times. The cell lysates were analyzed for luminescence activity. Analysis of the activity of the IncRNA H19 promoter luminescence readout at 24, 48 and 72h of hypoxia in HUVECs and tubular epithelial cells, indicates that the IncRNA H19 promoter is indeed activated under hypoxia.

In contrast, HIFI a was induced at 24h of hypoxia in RPTECs, which coincides with expression changes of IncRNA H19 in RPTECs. Silencing of HIF1 a by siRNA resulted in suppression of IncRNA H19 induction in RPTECs. Functional role of IncRN A H19 in endothelial cell biology

To investigate the functional role of IncRNA H19 in vitro, antisense oligonucleotides (ASO) were used to silence IncRNA H19 expression. Overexpression was achieved by use of lentiviral vectors.

The effect of IncRNA H19 overexpression or inhibition on cellular migration in HUVECs was evaluated using a modified Boyden chamber assay. Silencing of IncRNA H19 slightly decreased the capacity to migrate (data not shown), while lentiviral overexpression significantly and highly promoted VEGF-induced cellular migration (see Fig. 3A).

Similarly, endothelial cell proliferation in response to VEGF was significantly induced by IncRNA H19 lentiviral overexpression (see Fig. 3B), while IncRNA H19 silencing had minimal effects (data not shown).

The induction of angiogenic markers under hypoxia was evaluated by qPCR. Delta-like 4 (DII4), Delta-like 1 (Dili ), apelin, Notch 1 , Notch 4, were induced in HUVECs under hypoxic conditions as expected while Kinase insert domain receptor (KDR) remained unchanged. Coincidently, lentiviral overexpression of IncRNA H19 significantly promoted VEGF-induced sprouting angiogenesis as compared to empty vector control treated cells in a 3D angiogenesis assay (see Fig. 3C & D).

To reveal an effect of IncRNA H19 on apoptosis induction in HUVECs, a cleaved caspase 3/7 assay was performed. Interestingly, overexpression of IncRNA H19 protected from TNFa/cycloheximide-induced apoptosis (see Fig. 3E). Again, silencing of IncRNA H19 did not result in changes regarding apoptosis induction (data not shown).

The effect of IncRNA H19 overexpression on signaling pathways in response to VEGF was assessed in HUVECs. Overexpression of IncRNA H19 as compared to an empty vector control enhanced AKT (see Fig. 4 A & B), and ERK (see Fig. 4 A & C) phosphorylation after 5 and 10 min of VEGF treatment. Most notably, the p38 pathway was aberrantly deregulated as the p38 pathway was induced after 5 min after VEGF treatment and remained active even after one hour, whereas compared to control cells the p38 pathway was induced after 10 min of VEGF treatment and returned to basal levels by 30 min. Interestingly, it appears that both p38 subunits alpha and beta are deregulated (see Fig. 4A & D).

Downstream interaction partners of IncRNA H19 in endothelial cells

LncRNAs are known to function as microRNA sponges in their role as competing endogenous RNA. In order to identify altered microRNA expression following IncRNA H19, the inventors searched for overexpression microRNA binding sites in the IncRNA H19 sequence bioinformatically. Consequently, miR-30a-5p was identified to have a putative binding site in exon 5 of IncRNA H19 (see Fig. 5A). The function of miR-30a-5p in the setting of hypoxia/ischemia in the kidney has not been described yet. Exon 5 of IncRNA H19 (wildtype as well as mutated) was cloned into the plS2 renilla luciferase vector to demonstrate the interaction between IncRNA H19 and miR-30a-5p. Intriguingly, the wildtype sequence was significantly suppressed by miR-30a-5p overexpression (miR-mimic), while mutation by deletion of -TGTTT- sequence of the exon 5 binding site resulted in unchanged luciferase activity, which was comparable to all controls (see Fig. 5B). Additionally, the lentiviral overexpression of IncRNA H19 resulted in a suppression of miR-30a-5p expression in endothelial cells compared to empty vector control and non-treated cells (see Fig. 5C).

Since IncRNA H19 seems to play a major role in endothelial cell biology, a 3D angiogenesis assay was performed to study the effect of miR-30a-5p overexpression on endothelial cell sprouting activity. As a result, overexpression of miR-30a-5p significantly reduced the number of angiogenic sprouts of HUVECs. However, the inhibition of miR-30a-5p did not result in increased sprouting (see Fig. 5D and E). Overexpression of miR-30a-5p mimic was validated by quantitative PCR using a miR-30a-5p specific probe (see Fig. 5F).

To understand the mechanism of miR-30a-5p in angiogenesis regulation, target genes that are involved in blood vessel formation and are putatitive miR-30a-5p targets were bioinformatically screened for. The bioinformatic scan revealed two potential miR-30a-5p targets that are reportedly involved in angiogenesis 1 ) the Autophagy related 5 (ATG5) and 2) the snail family transcriptional repressor 1 (Snail ) (see Fig. 6). Western blot analysis revealed that all three targets were significantly reduced by miR-30a-5p overexpression in HUVECs whereas miR- 30a-5p inhibition significantly increased DII4 expression while a modest increase of ATG5 and SNAI 1 was observed (see Fig. 6A & B). To validate the interaction between miR-30a-5p and its target genes ATG5 and SNAI 1 , the 3’UTR (wildtype or mutant) of both ATG5 and SNAI 1 was cloned into the plS2 renilla luciferase vector and the assay was performed in HUVECs transfected with the 3’UTR SNAI 1 and ATG5 vectors. miR-30a-5p was overexpressed using miR-mimics, inhibition was achieved by antimiR use. Interestingly, the wildtype sequences were significantly down regulated in cells overexpressing miR-30a-5p (mimic), however neither the miR-30a-5p inhibitory sequence nor the mutated ATG5 and SNAI 3’UTR sequences (deletion of TGTTT) had an effect on luciferase activity (see Fig. 6D & E) indicating that ATG5 and SNAI 1 regulation are specific to miR-30a-5p.

Functional role of IncRNA H19 in tubular epithelial cell biology

The effect of IncRNA H19 modulation on renal proximal tubule epithelial cells (RPTEC) is in stark contrast to its function in endothelial cells. The migratory capacity of RPTECs in response to complete medium (with 10 % FBS) as a chemoattractant did not differ between cells overexpressing IncRNA H19 or cells that were transduced with an empty virus control in a modified Boyden chamber migration assay. Similarly, in a scratch wound healing assay IncRNA H19 overexpression did not enhance wound closure as compared to RPTECs that received the empty vector treatment. In addition, the cellular proliferation in RPTECs did not differ between the groups. However, there was a modest but significant protection against an apoptotic stimulus in RPTECs overexpressing IncRNA H19 as compared to control.

In vivo role of IncRNA H19 in tubular epithelial cell injury, proliferation and capillary density Since IncRNA H19 was found to be upregulated in mice upon renal l/R Injury, the aim of the study was to elucidate the contribution of IncRNA H19 to renal l/R injury in vivo.

First, IncRNA H19 constitutive knockout mice were assessed. As expected, given the loss of IncRNA H19 in adult kidney tissue under basal conditions, IncRNA H19 knockout mice were comparable in all respects to wildtype mice subjected to renal l/R-injury. Outer medullary injury, capillary density, injury marker expression did not differ between wildtype and knockout mice (data not shown).

The mouse IncRNA H19 sequence was cloned into an adeno-associated virus 2 vector (AAV2- H19) and pharmacologically overexpressed in vivo by injection of AAV2 -H19 or control virus (AAV2-GFP or AAV2-CTL) directly into the renal vein at the time of clamping.

AAV2 -H19 uptake following renal vein injection was evaluated in various organs by immunofluorescence. A widespread distribution was detected, including the kidney, liver, the heart and the lungs, except for brain. Despite this widespread distribution, side effects in distant organs were not detected in mice. The distribution of IncRNA H19 following AAV2 -H19 injection within the kidney was determined by RNA FISH using a specific mouse H19 fluorescently labeled probe combined with either tomato lectin staining (endothelial cells) or lotus teragonolobus lectin (proximal tubules). The expression of IncRNA H19 was uniform across the renal vasculature whereas IncRNA H19 expression was limited to proximal tubules.

At 24h of reperfusion miR-30a-5p expression was increased in l/R-kidneys of mice, while it was suppressed at day 7 of reperfusion (see Fig. 7A). These results likely indicate that IncRNA H19, which starts to increase on day 1 of reperfusion, sponges and thereby downregulates miR-30a-5p expression subsequently.

AAV2 -H19 delivery via the renal vein resulted in a robust and consistent overexpression of IncRNA H19 in the kidney (see Fig. 7B). A protective effect was detected in mice, which were subjected to injection of AAV2 targeting IncRNA H19 as compared to control AAV2 directly into the renal vein at the time of unilateral renal l/R-injury. Mice were injected immediately before a vascular clamp was applied to the renal pedicle (30 minutes of ischemia and 24h of reperfusion). This resulted in a blunting of kidney damage. Expression levels of KIM-1 and NGAL increased greatly, as expected, in mice receiving control virus (AAV2-CTL), while in mice receiving AAV2 targeting IncRNA H19 for overexpression purposes KIM-1 and NGAL remained similar to control mice (see Fig. 7C & D). Similarly, inflammatory gene expression including tumor-necrosis-factor a (TNFa, see Fig. 7E) and interleukin-1 b (IL-1 b, see Fig. 7F) was highly suppressed in mice following renal l/R-injury and concomitant AAV2-H19 treatment, underlining that inflammatory processes are blunted by IncRNA H19 overexpression.

Additionally, outer medullary tubular epithelial injury amounted to 70-80% in mice receiving AAV2-CTL (AV-GFP), while in mice with AAV2-/-/19 the percentage of injury was 40-50% (see Fig. 7G - H). Moreover, inflammatory cell influx (CD3⁺-T cells and F4/80⁺-macrophages,) was highly suppressed in mice following renal l/R-injury and concomitant AAV2-H19 treatment, indicating that the inflammatory response is reduced by IncRNA H19 (see Fig. 9A & B). Likewise, AAV2 -H19 treatment resulted in significantly reduced levels of outer medullary apoptosis as assessed by TUNEL staining compared to AAV2-CTL (control) treated mice (see Fig. 9C)

The reduction of immune cell infiltration was accompanied by a significant reduction of adhesion molecules such as vascular cell adhesion protein 1 (VCAM-1 ), and endothelial selectin (E-Selectin) at the mRNA level in l/R kidneys of mice that received AAV2-H19 as compared to AAV2-CTL (data not shown). The mRNA expression levels of intercellular adhesion molecule 1 (ICAM-1 ) and vascular endothelial cadherin (VE-Cadherin) were slightly suppressed in mice with IncRNA H19 overexpression albeit not to a statistically significant level (data not shown).

To further understand the role of IncRNA H19 in the inflammatory response regulation, bone marrow-derived macrophages (BMDM) were exposed to AAV2 -H19 or AAV2-CTL, stimulated with lipopolysaccharide (LPS) and assayed for nitric oxide, TNFa, and IL-6 release. Interestingly and surprisingly IncRNA H19 overexpression in BMDM enhanced their immune response as recorded by significantly increased levels of nitric oxide, TNFa, and IL-6 (see Fig. 10A - C) suggesting a pro-inflammatory function of IncRNA H19 in immune cells. The aforementioned protection from inflammatory cell influx in the kidney is therefore most likely related to a suppression of adhesion molecule expression.

In vivo role of IncRNA H19 regarding kidney function, capillary density and apoptosis

AAV2-mediated in vivo overexpression of IncRNA H19 prior to bilateral clamping of the renal pedicles resulted in a preservation of outer medullary tubular epithelial cell integrity (see Fig. 8A & B) as well as a highly significant suppression of injury markers including KIM1 (see Fig. 8C) and NGAL (see Fig. 8D) on day 7 following l/R-injury. Moreover, mice following IncRNA H19 overexpression showed significantly reduced levels of blood urea nitrogen (BUN), indicating a preservation of kidney function (see Fig. 8E). Capillary density as assessed by Endomucin staining (see Fig. 8F) was significantly improved on day 7 after l/R-injury, while proliferation in outer medulla was increased (see Fig. 8G) and level of apoptosis (see Fig. 8H) concomitantly reduced by AAV2-mediated IncRNA H19 overexpression on day 7.

These results indicate that IncRNA H19 overexpression is feasible in the kidney and leads to a highly significant protection from renal injury.

Long-term effects of IncRNA H19 overexpression

Four weeks after injection we did not detect any increased levels of IncRNA H19 in the kidney (data not shown). Moreover, mice were observed for 12 months after injection (data not shown). No obvious change in phenotype was noticed. Mice were viable and healthy; no signs of abnormalities or tumor development were detected.

Materials and Methods

Lentivirus -mediated IncRNA H19 overexpression

The complete sequence of human H19 was amplified by PCR and cloned into VVPW lentiviral expression vector (kind gift from Luca Gusella, Mount Sinai Hospital, New York, USA). HEK293T cells grown to 70-80% confluence were transfected with WPW, the packaging plasmid psPAX2, and the envelope plasmid pCMV-VSVG (from Addgene) at a ratio of 3:2:1 respectively, using FuGENE HD transfection reagent according to manufacturer^'s instructions.

Quantitative PCR

Real time PCR was performed using 7500 fast thermocycler (Applied Biosytems). Total RNA was isolated using miRNeasy mini kit and one ug of total RNA was reversed transcribed into cDNA using qScript™ cDNA supermix and the primers were designed using IDT primers designing tool software and purchased from IDT. Primers used were designed according to standard procedures. microRNA analyses

MiR mimics, inhibitors and primers were purchased at Thermofisher Scientific (U.S.A.). For quantitative real time PCR expression analysis of miR-30a-5p expression miR-16 was used as a reference microRNA.

SDS-PAGE and Western Blot

SDS-PAGE was done using 25 ug of cell lysate (cell lysis buffer: 50 mM TRIS-HCI, 150 mM NaCI, 5 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 50mM sodium fluoride, 0.5% T riton X-100, 1 mM PMSF, 10 pg/mL leupeptin, 10 pg/mL aprotinin, and a protease inhibitor cocktail) and resolved using 12 % acrylamide/Bis gels under denaturing conditions at 120 Volts for 90 min. Proteins were transferred onto nitrocellulose membrane (BioRad) at 100 Volts for 60 min and the membranes probed using the primary antibodies at the manufacturer’s recommended concentrations. The membranes were washed 5 times with TTBS and probed with the appropriate HRP labeled anti-species secondary antibodies for 1 hr. The membranes were washed 3 times with PBST and were developed using ECL prime kit (GE Healthcare)

EdU Proliferation Assay

Cellular proliferation was evaluated using Click-iT® EdU flow cytometry assay kit (Invitrogen, C-10420) as per manufacturer’s instructions. Briefly, human umbilical vein endothelial cells (HUVECs) or renal proximal tubular epithelial cells (RPTECs) treated with H 19-expressing lentivirus or empty virus were incubated with 10 uM EdU and incubated at 37 ^°C and 5 % CO2 for 24 h. The cells were washed with wash buffer (PBS+ 1 % BSA) and fixed with Click-iT fixative for 15 min at RT in the dark. The cells were later washed and permeabilized with Click- iT Triton X-100 based permeabilization buffer for 30 min at RT in the dark. The EdU was detected by Alexa-488 antibody provided by the kit and the proliferating cells were analyzed by flow cytometer.

Chemotaxis Assay

The cell migration assay was performed using 24 well transwell plate with 8 urn pores (Corning, 3422). HUVEC or RPTEC cells overexpressing H19 were loaded with the cell tracker CFMDA (Invitrogen) and seeded at a density of 10⁵ cells in the insert in the presence of incomplete medium. Complete medium (with all growth factors and 10% FBS) or with 25 ng/ml_ recombinant human VEGF were placed in the bottom chamber. To eliminate the concentration gradient of the chemotactic factors, in some wells complete medium was placed in the top insert and in the bottom well. The plate was incubated at 37 ^°C and 5 % CO2 for 24 h. The next day the filters were cut out of the insert placed on a microscope slide and visualized using a fluorescent microscope. Five fields of view per filter were counted.

Apoptosis assay

HUVEC and/or RPTEC treated with H19 antisense oligonucleotides (ASO) or overexpressing H19 were subjected to an apoptotic stimulus including cycloheximide (25 ng/ml_) + TNFa (20 ng/ml_) for 3 hours at 37 C 5 % C02. The cells were washed 2X with PBS and apoptosis was determined using Caspase-Glo 3/7® fluorescent assay (Promega) as per manufacturer’s instructions. siRNA Transfection

All siRNA sequences were purchased from Qiagen. The antisense oligos (ASO) were purchased from IDT. The gene knockdown procedure was based on Qiagen HiPerFect (301705) Transfection protocol. The cells were transfected with 10-50 nM siRNA, or with 1 nM ASO at 50-60 % confluent. The gene knockdown was evaluated by Western blot and/or real time PCR after 72 hours.

Luciferase assay

H19 promoter was cloned into pXPG plasmid (a gift from Peter Cockerill, Addgene plasmid # 71248) and H19 exon 5 was cloned into plS2 plasmid (a gift from David Bartel, Addgene plasmid # 12177). HUVECs were transfected with 1 ug of either plasmid using ViaFect (Promega) transfection agent. Luciferase activity was determined after 72 hours using Renilla luciferase assay system (Promega) or firefly luciferase assay system (Promega) as per manufacturer’s instructions.

Three-Dimensional Angiogenesis Assay

The study of sprouting angiogenesis of HUVECs overexpressing H19 in vitro was determined using the protocol described by Nakatsu¹ with some modifications. HUVECs were mixed with collagen coated cytodex 3 microcarrier beads (Sigma-Aldrich, C3275) and incubated at 37 ^°C and 5 % CO2 for 4 h with shaking every 20 min. The coated beads were then transferred to T25 tissue culture flasks and incubated for further 2 h. The coated beads were washed with PBS 2X and mixed with 2 mg/mL fibrinogen (Sigma-Aldrich, F4753) and 0.15 U/mL aprotinin (Sigma-Aldrich, A1 153). Five hundred uL of the HUVECs-coated beads and fibrinogen solution were added to 0.625 U/mL thrombin (Sigma-Aldrich, T4648) in a 24-well tissue culture plate and incubated at 37 ^°C and 5 % CO2 for -15 min until the fibrin gel hardened. One mL of EBM2 was added to each well and sprouts formation were analyzed after 24 h.

ChIP assay

The chromatin immunoprecipitation of the transcription factors SPI 1 and LHX8 was performed using MAGnify™ Chromatin Immunoprecipitation System (ThermoFisher Scientific) As per manufacturer’s instructions.

MicroRNA target prediction

The microRNA databases and target prediction tools miRBase (http://microrna.sanger.ac.uk/), PicTar (http://pictar.mdc-berlin.de/) and TargetScan (http://www.targetscan.org/index.html) were used to identify potential microRNA targets.

Animals

Male C57BL/6 mice were housed under standard conditions. Mice that were 10 to 12 weeks old weighing between 20 and 30 g were used for all experiments. Constitutive H19^tm1Lda knockout mice were obtained from Luisa Dandolo (University Paris Descartes). Ischemia/Reperfusion injury Protocol

Following isoflurane anaesthesia male C57BL/6 mice were subjected to median laparotomy, thereafter renal pedicles were dissected and a vascular clamp was applied for 30 minutes. LncRNA /-/f9 was overexpressed in vivo by injecting adenoviral vectors (AAV serotype 2, H19- AAV2) directly into the renal vein at the time of clamping. For measurement of renal function parameters mice were subjected to bilateral renal IR injury (ischemia time of 15 minutes). Sham operated animals (n = 4) were also included. Blood samples for analysis of renal function parameters (serum-urea) were drawn on days 0, 1 , 3 and 7 (analysed on a Beckman Analyzer, Beckman Instruments GmbH, Munich, Germany). In a second group of mice the renal pedicle was only clamped on the left side (unilateral l/R-injury). In this setting the contralateral kidney serves as an internal control to the injured kidney (l/R-kidney). These animals were sacrificed on day 1 after renal l/R-injury and kidneys were harvested for further examination. In each experiment 6 mice were included per group. In vivo studies conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Animal experiments were approved by local authorities (Kantonales Veterinaramt).

Histology and Immunohistochemical Staining

The kidney from each euthanized mouse was removed and either placed in OCT and snapped frozen in liquid nitrogen or placed in 10 % formalin. The tissues were processed at the Histology Facility at the University of Zurich and stained with periodic acid- Schiff (PAS). For immunofluorescence stainings, 5 urn frozen sections were stained for F4/80 and CD3. Vascular cells were visualized by tomato lectin or endomucin staining, proximal tubular cells by lotus tetragonolobus lectin staining. All tissues were incubated with the primary antibodies overnight and detected with a fluorescently labelled anti-species antibodies. The controls consisted of either no primary antibody or a non-specific IgG. Fluorescent images were obtained using a Leica SP8 upright confocal microscope and LAS AF software at the center of microscopy and image analysis at the University of Zurich.

The severity of morphologic renal damage was assessed in a blinded manner using an arbitrary score based on PAS-stained kidney sections following a modification of a protocol developed by Broekema et al ². Briefly, the extent of four typical l/R-injury- associated damage markers (i.e., dilatation, denudation, intraluminal casts, loss of brush border membrane and cell flattening) was expressed in arbitrary units (AU) in a range of 0 to 4 according to the percentage of damaged tubules: 0, no damage; 1 , less than 25% damage; 2, 25%-50% damage; 3, 50%-75% damage; and 4, more than 75% damage. Construction and production of AAV 2 vectors

Full-length IncRNA H19 was amplified by PCR using mouse genomic DNA, subsequently two miR-122 binding sites (for de-targeting in the liver) were added also by PCR and the whole piece was cloned into the pTREKI plasmid (kindly provided by Roger Hajjar, lcahn School of Medicine at Mount Sinai, New York). For virus production, HEK293-T cells were grown in triple flasks for 24 h (DMEM, 10% fetal bovine serum) before transfection. The transgene plasmid, together with the helper plasmid (pDP2rs, also kindly provided by Roger Hajjar), was transfected into HEK293-T cells using Polyethylenimine (Sigma-Aldrich). 72 h later, cell crude lysates were produced, treated with benzonase and the virus particles were purified over an iodixanol density gradient (Optiprep, Sigma-Aldrich). Viral titers were determined by qPCR (quantitative real-time polymerase chain reaction) on vector genomes using the FastStart Universal SYBR Green Master Mix (Roche). Mice were injected directly into the renal vein at the time of clamping.

RNA Fluorescence in situ hybridization (FISH)

Kidney frozen sections of mice injected with AAV2-H19 were subjected to fluorescence in situ hybridization (FISH) using a Stellaris® RNA FISH IncRNA H19 specific probe labeled with alexa-594 fluorescent tag according to the manufacturer’s instructions. In addition, the kidney sections were either co-stained with tomato lectin to label endothelial cells or with lotus teragonolobus lectin to label proximal tubules epithelial cells.

Murine bone marrow-derived macrophages

The murine bone marrow-derived macrophages (BMDM were prepared as described ³ and cultured in complete DMEM (15 mM HEPES, 1 % non-essential amino acid, 1 % sodium pyruvate, 10 % fetal bovine serum and 100 U/mL penicillin and 100 pg/mL streptomycin), and supplemented with 20 % L929 supernatant (source of M-CSF).

Nitric oxide assay

The nitric oxide assay was performed as described ⁴ and the nitrite accumulation was measured using the modified Griess reagent (Sigma-Aldrich) as per manufacturer’s instructions.

Enzyme linked immunosorbent assay (ELISA)

Supernatant of activated bone marrow-derived macrophages was analyzed for TNFa (ThermoFisher) and IL-6 (Sigma-Aldrich) secretion using cytokine specific ELISA as per manufacturer’s instruction. Ex vivo cell purification

The cellular origin of IncRNA H19 following induction of l/R-injury was investigated by fluorescence-associated cell sorting (FACS) analysis using specific antibodies following a protocol by Chau et al. with modifications ⁵. Following clamping of the right renal pedicle and a reperfusion period of 24 hours both kidneys were extracted, de-capsulated, homogenized, then incubated at 37°C for 30 min with Collagenase II (81 U/ml) and DNase (100U/ml, Roche) in serum free DMEM. After centrifugation, cells were re-suspended in 5ml of PBS/1 % BSA, and filtered (40pm). Cells were separated using the following specific antibodies or lectins: rat anti-mouse-CD31-PE (1 :400 BD Pharmingen) for endothelial cells, Lotus teragonolobus lectin (1 :200, DAKO) for proximal epithelium and anti-mouse Tim1 -biotin (1 :200, E Bioscience) followed by streptavidin-APC (1 :2000, BD Pharmingen) for injured proximal epithelium. Subsequently, RNA was isoltated by Trizol method.

Sequences

The following sequences 01 , 02 and 03 relate to the human H19 sequences as retrieved from the NCBI database; the skilled person is aware that where appropriate, the actual RNA active in the cell comprises uracil (U) rather than T nucleotides in the respective positions.

SEQ ID NO 01 : NCBI Reference Sequence: NR_002196.2

SEQ ID NO 02: NCBI Reference Sequence: NR 31223.1

SEQ ID NO 03 NCBI Reference Sequence: NR_131224.1

SEQ ID NO 04: hsa-miR-122

SEQ ID NO 05: hsa-miR-122-5p: UGGAGUGUGACAAUGGUGUUUG

SEQ ID NO 06: hsa-miR-122-3p: AACGCCAUUAUCACACUAAAUA

SEQ ID NO 07: hsa mR-30a-5p: UGUAAACAUCCUCGACUGGAAG

SEQ ID NO 08: miR-30a-5p antisense: 5'-CUUCCAGUCGAGGAUGUUUACA-3'

Claims

Claims

1. A polynucleotide agent for use in treatment or prevention of a disease or of a symptom, said disease or symptom being associated with an ischemic state or with an inflammation in the kidney, wherein said polynucleotide agent

comprises or consists of,

or is capable of expressing, in a mammalian cell,

a long non-coding H19 RNA sequence.

2. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or with an inflammation in the kidney according to claim 1 , wherein said long non-coding H19 RNA sequence is characterized by SEQ ID NO 01 , 02 or 03, particularly by SEQ ID NO 01 , or a sequence at least (>) 90%, > 92%, > 94%, > 96%, or even > 98% identical to a reference sequence selected from SEQ ID NO 01 , SEQ ID NO 02 and SEQ ID NO 03, wherein the H19 RNA sequence has biological activity essentially similar to that of SEQ ID NO 01.

3. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or with an inflammation in the kidney according to claim 1 or 2, wherein the polynucleotide agent is an expression construct encoding and capable of expressing said long non-coding H19 RNA sequence under control of an RNA polymerase promoter operable in a human cell.

4. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or with an inflammation in the kidney according to claim 3, wherein said promoter is a polymerase II (pol II) promoter, particularly a constitutive pol II promoter, more particularly a CMV promoter (CMV immediate early) or a CAG promoter.

5. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or with an inflammation in the kidney according to claim 3 or 4, wherein said expression construct comprises a polyadenylation signal 3’ of a sequence encoding the long non-coding H19 RNA sequence.

6. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or with an inflammation in the kidney according to claim 3 to 5, wherein said expression construct comprises inverted terminal repeat (ITR) sequences positioned 5’ and 3’ of a sequence encoding the long non-coding H 19 RNA sequence.

7. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or with an inflammation in the kidney according to claim 3 to 6, wherein said expression construct comprises a sequence capable of hybridizing to a miR122 polynucleotide in a position 3’ of the sequence encoding the long non-coding H19 RNA sequence, particularly 5’ of the polyadenylation signal more particularly wherein the miR122 polynucleotide is selected from SEQ ID 04, 05 and 06, or a sequence at least (>) 90%, > 92%, > 94%, > 96%, or even > 98% identical to a reference sequence selected from SEQ ID NO 04, SEQ ID NO 05 and SEQ ID NO 06, wherein the H19 RNA sequence has biological activity essentially similar to that of SEQ ID NO 04.

8. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or inflammation in the kidney according to any one of claims 3 to 7, wherein the expression construct is comprised in

a. a circular DNA plasmid, optionally packaged by lipid vesicle (liposome, exosome);

b. a virus or virus-derived vector, particularly a virus selected from adeno- associated virus (AAV), adenovirus (AV), lentivirus, more particularly AAV-2 vector.

9. A polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or inflammation in the kidney, wherein the polynucleotide agent is capable of hybridizing to miR-30a-5p.

10. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or inflammation in the kidney according to claim 9, wherein the polynucleotide agent is a oligonucleotide 8 to 25 nucleotides long.

11. The polynucleotide agent for use in treatment or prevention of a disease or symptom associated with an ischemic state or inflammation in the kidney according to any one of the preceding claims, wherein the treatment is for ischemia-reperfusion injury, or for prevention of renal damage as a result of an operative cardiac intervention associated with reduced cardiac output, particularly aortic surgery with cross-clamping of the aorta, or kidney transplantation.

12. A polynucleotide expression vector, comprising an expression cassette comprising or essentially consisting of, in the order from 5’ to 3’,

a. a polymerase II promoter operable in a mammalian cell, particularly in a human cell;

b. a sequence encoding a long non-coding H19 RNA sequence,

particularly sequence characterized by SEQ ID NO 01 , 02 or 03, more particularly by SEQ ID NO 01 ,

or a sequence at least (>) 90%, > 92%, > 94%, > 96%, or even > 98% identical to a reference sequence selected from SEQ ID NO 01 , SEQ ID NO 02 and SEQ ID NO 03, wherein the H19 RNA sequence has biological activity essentially similar to that of SEQ ID NO 01 ;

c. a sequence element encoding a sequence capable of binding to miR122; and d. a polyadenylation signal.

13. The polynucleotide expression vector according to claim 12, wherein the pol II promoter is a constitutive promoter, particularly a promoter selected from the group containing the immediate early CMV promoter-enhancer, the SV40 promoter, the elongation factor 1 alpha (EF1 a) promoter, the ubiquitin C promoter and the human beta actin promoter.

14. The polynucleotide expression vector according to claim 12 or 13, further comprising two adenovirus associated virus inverted terminal repeat (ITR) sequences, one positioned 5’ of the pol II promoter sequence and one positioned 3’ of the polyadenylation signal.

15. The polynucleotide expression vector according to any one of claim 12 to 14, wherein the polynucleotide expression vector is an adeno-associated viral vector, particularly AAV serotype 2 vector.