EP4634382A1 - Rnai-based therapies targeting claudin-1 for the treatment and prevention of fibrotic diseases - Google Patents

Abstract

The invention provides RNA interfering agents targeting human Claudin-1 for use in the treatment and/or prevention of fibrotic diseases, including liver fibrosis, lung fibrosis and kidney fibrosis. Also provided are pharmaceutical compositions and kits comprising such RNA interfering agents and methods for administering such RNA interfering agents to patients suffering from fibrotic diseases.

Description

RNAi-based Therapies Targeting Claudin-1 for the Treatment and Prevention of Fibrotic Diseases

Related Patent Application

The present application claims priority to European Patent Application number EP 22214 328, which was filed on December 16, 2022. The European patent application is incorporated herein by reference in its entirety.

Background of the Invention

Fibrotic disease is characterized by excessive deposition of fibrous connective tissue (a process called fibrosis) that can lead to progressive deterioration in the normal structure and function of organs and tissues of the body. Fibrosis is defined by the overgrowth, hardening and/or scarring of a tissue or organ. It is attributed to excessive accumulation of components of the extracellular matrix (ECM), such as collagen and fibronectin (Wynn et al., Nature Medicine, 2012, 18: 1028-1040), in and around inflamed or damaged tissue, which can lead to permanent scarring, organ malfunction and ultimately death. Fibrosis is the final, common pathological outcome of many chronic inflammatory reactions induced by a variety of stimuli including persistent infections, genetic disorders, autoimmune reactions, allergic response, chemical insults, radiations, and tissue injury. Fibrosis can occur in nearly every organ or tissue of the body, more often in the heart, lung, kidney, liver and skin (Rockey et al., N. Engl. J. Med., 2015, 372: 1138-1149) and less frequently in other tissues or organs such as the pancreas, intestine, eye (Wynn, J. Pathol., 2008, 214: 199-210), nerve system (Kawano et al., Cell Tissue Res., 2012, 349: 169-180), mediastinum (Parish and Rosenow, Semin. Respir. Crit. Care Med., 2002, 23: 135-143), retroperitoneum (Caiafa et al., Radiographics, 2013, 33: 535-552), joint and tendon.

Human fibrotic diseases have a poor prognosis comparable with end-stage cancer. They represent an increasing cause of morbidity and mortality worldwide. Since fibrosis is a predominant feature of the pathology of a wide range of diseases across multiple organ systems, fibrotic disorders have been estimated to contribute to about 45% of all-cause mortality in the United States (Wynn, Nature Rev. Immunol., 2004, 4: 583-594). The major health problem associated with fibrotic diseases is also due to our incomplete understanding of the underlying pathogenesis, the marked heterogeneity in the etiologies and clinical manifestations of fibrotic disorders, the absence of appropriate and fully validated biomarkers, and most importantly, the current void of effective disease-modifying therapeutic agents. Indeed, at present, there are only two recently approved drugs specifically indicated for the treatment of fibrotic disease.

In light of the economic burden of patients with fibrotic disease worldwide, and in view of the limited therapeutic arsenal, novel strategies to prevent and/or treat fibrosis are urgently needed.

Summary of the Invention

The present Inventors have uncovered human Claudin-1 (CLDN1), a member of the tight junction protein family known to play a role in pro-carcinogenic and pro-fibrotic signaling and cell-fate, as a mediator and therapeutic target for tissue fibrosis. Using the liver as a model of chronic inflammation-associated fibrogenesis and carcinogenesis, they showed that targeting CLDN1 by GalNac siRNA robustly reduced fibrosis and tumor development in a mouse model of nonalcoholic steatohepatitis (NASH). Beyond the liver, the expression data show that CLDN1 is also a candidate target for kidney and lung fibrosis. Indeed, the present Inventors have performed perturbation studies using liposome-mediated delivery of CLDN1 specific siRNA in cells derived from human adenocarcinoma alveolar basal epithelial cells, wherein treatment with CLDN1 RNAi was found to decrease the expression of key pro- fibrogenic mediators and effectors such as collagen, TGFp, aSMA and epithelial- mesenchymal transition (EMT) markers. They also showed, in primary cells isolated from human kidney, that treatment with CLDN1 RNAis leads to a decrease in CD44 expression, a marker associated with disease progression, an inhibition of ECM component expression (fibronectin, FN1, and C0L1A1) and a reduction of inflammation markers (TNFA), as well as SNAI and TGFB1, two actors mediating EMT and crescent formation.

Consequently, the present invention provides an RNA interfering agent targeting CLDN1 for use in the prevention and/or treatment of a fibrotic disease in a subject.

In certain embodiments, the fibrotic disease affects the liver, lung, kidney, heart, skin, pancreas, intestine, eye, nerve system, mediastinum, retroperitoneum, joint or tendon, in particular the fibrotic disease is hepatic fibrosis, pulmonary fibrosis or renal fibrosis.

In certain embodiments, the fibrotic disease is hepatic fibrosis, wherein the hepatic fibrosis is associated with hepatitis B, C, D or E infection, alcoholic liver disease, primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver disease, obesity, nonalcoholic steatohepatitis, cystic fibrosis, hemochromatosis, or autoimmune hepatitis.

In certain embodiments, the fibrotic disease is pulmonary fibrosis, and the pulmonary fibrosis is one of: idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonitis (NSIP), cryptogenic organizing pneumonia (COP), Hamman-Rich syndrome, lymphocytic interstitial pneumonitis (LIP), respiratory bronchiolitis interstitial lung disease, desquamative interstitial pneumonitis or idiopathic lymphoid interstitial pneumonia, and idiopathic pleuroparenchymal fibroelastosis.

In certain embodiments, the fibrotic disease is renal fibrosis, and the renal fibrosis is renal interstitial fibrosis or glomerulosclerosis, including fibrosis caused by inflammation such as RPGN (rapidly progressive glomerulonephritis) or ANCA (antineutrophil cytoplasmic antibody-associated) vasculitis or caused by metabolic diseases such as diabetes mellitus or vascular diseases such as hypertension.

In certain embodiments, the RNA interfering agent targeting CLDN1 used for preventing and/or treating a fibrotic disease is a single-stranded RNA interfering molecule or a double-stranded RNA interfering molecule. For example, the RNA interfering agent targeting CLDN1 may be a siRNA, a shRNA, a micro-RNA or an aiRNA.

In certain embodiments, the RNA interfering agent targeting CLDN1 used for preventing and/or treating a fibrotic disease is a double-stranded short interfering nucleic acid molecule comprising a first strand comprising a sense region and a second strand comprising an antisense region, wherein the sense region targets a portion of an exon of a CLNDl mRNA sequence, and the antisense region comprises a nucleotide sequence that is complementary to the sense region.

In certain embodiments, the exon of the CLDN1 mRNA sequence is Exon 2 or Exon 4.

In certain embodiments, the sense region of the RNA interfering agent: targets a portion of Exon 2 of the CLNDl mRNA sequence, wherein the portion consists of the sequence set forth in SEQ ID NO: 1: 5 ’ -UGAAGUGUAUGAAGUGCUU-3 ’ ; or targets a portion of Exon 4 of the CLDN1 mRNA sequence, wherein the portion consists of the sequence set forth in SEQ ID NO: 2: 5’-CACCAAGGCCCUAUCCAAA-3’; or targets a portion of Exon 4 of the CLNDl mRNA sequence, wherein the portion consists of the sequence set forth in SEQ ID NO: 3: 5’-UAACAUUAGGACCUUAGAAUU-3’. In certain embodiments, the RNA interfering agent targeting CLDN1 is a doublestranded short interfering nucleic acid molecule comprising a first strand comprising a sense region and a second strand comprising an antisense region, wherein the sense region consists of, or comprises, the sequence set forth in SEQ ID NO: 3 (5’-

UAACAUUAGGACCUUAGAAUU-3’), or a sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity with SEQ ID NO: 3, and the antisense region comprises a nucleotide sequence that is complementary to the sense region.

In certain embodiments, the sense region consists of the sequence set forth in SEQ ID NO: 3, and the antisense region consists of a nucleotide sequence that is perfectly complementary to the sense region.

In certain embodiments, the RNA interfering agent is associated with a delivery system, in particular wherein the RNA interfering agent is conjugated to a A'-acetylgalactosamine (GalNAc) moiety or to a molecule comprising at least one GalNAc moiety. In other embodiments, the RNA interfering agent is encapsulated into a lipid nanoparticle (LNP).

The present invention also provides a pharmaceutical composition comprising an effective amount of an RNA interfering targeting CLDN1, as defined above, and at least one pharmaceutically acceptable carrier or excipient, for the use in the prevention or treatment of a fibrotic disease, as defined above.

In certain embodiments, the pharmaceutical composition may further comprise at least one additional therapeutic agent, wherein the at least one additional therapeutic agent is selected from anti-viral agents, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, kinase inhibitors, signalling inhibitors, antibacterial agents, antibiotics, antioxidants, antiseptic agents, therapeutic agents suitable for the treatment of a fibrotic disease, and combinations thereof.

In another aspect, the present invention provides an RNA interfering agent targeting CLDN1 as defined above.

In yet another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of an RNA interfering agent targeting CLDN1 as defined above and at least one pharmaceutically acceptable carrier or excipient, and optionally at least one additional therapeutic agent as define above. In certain embodiments, said pharmaceutical composition results in modulation of expression or function of epithelial growth factor receptor (EGFR), EPCAM, ECM receptor integrin alpha 5 (ITGA5), and/or ECM component laminin 5 (LAMA5).

It is also contemplated that the present invention be implemented using an antisense oligonucleotide (ASO) targeting CLDN1 instead of an RNA interfering agent targeting CLDN1. Consequently, in all the aspects of the invention described above, the term “RNA interfering agent targeting CLDN1” (or similar) may be replaced with the term “antisense oligonucleotide targeting CLDN1”. Thus, in particular, the present invention provides an antisense oligonucleotide targeting CLDN1 for use in the prevention and/or treatment of a fibrotic disease in a subject. The fibrotic disease to be treated or prevented may affect any organ or tissue of the body, in particular the liver, lung, kidney, heart, skin, pancreas, intestine, eye, nerve system, mediastinum, retroperitoneum, joint or tendon, in particular the fibrotic disease is hepatic fibrosis, pulmonary fibrosis or renal fibrosis, as described above.

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

Brief Description of the Drawing

Figure 1: CLDN1 expression is upregulated in chronic liver disease. (A) CLDN1 upregulation in liver tissues of patients with chronic HCV, HBV infection, or NASH (B) CLDN1 expression in livers of patients with NASH with mild (FO-1) or advanced fibrosis (F3-4) and liver tissues of transplanted HCV-infected patients with stable or progressive fibrotic disease. (C) CLDN1 expression in healthy liver at the single-nucleus level. (p<0.0001, U-test). (D) CLDN1 expression along the cholangiocyte-bipotent progenitor cells- hepatocyte pseudotime trajectory by Slingshot in a combined scRNA-seq and snRNA-seq dataset. (E) CLDN1 expression at the single-cell level in human cirrhotic and healthy liver cells. Differential expression analysis identified CLDN1 as a marker of mesothelial cells, hepatocytes and cholangiocytes (p<0.0001, U-test, respectively).

Figure 2. Validation of cell surface CLDN1 knockdown by GalNac siRNA in Huh7 liver cells. (A) Effect of GalNAc siRNA mediated suppression of CLDN1 expression and validation in Huh7 cells. CLDN1 protein expression following siCLDNl (left panel) or GalNAc-siCLDNl (right panel) mediated knockdown in Huh7 cells was assessed by flow cytometry (n=3 independent experiments with n=2 biological replicates per condition). (B) The graph shows AMFI of CLDN1 mAb compared to Control mAb binding to the respective cells. ** p<0.01, U-test.

Figure 3. Targeting CLDN1 by siRNA delivered in vivo reduces liver fibrosis in a patient-derived mouse model. (A-C) CLDN1 knockdown by GalNAc-siRNA reduces liver fibrosis in humanized liver fibrosis mouse model. (A) Illustration of the experimental approach. (B) CLDN1 expression, fibrosis assessment in humanized areas, and tumor nodule count according to the treatment group (siCTRL n=6, siCLDNl n=5). (C) Representative images of human CLDN1 staining, Sirius red, fibronectin-1 (FN1) immunostaining and macroscopy of GalNAc siCLDNl and siCTRL mice. Scale bar =50 pm, 250 pm, 50pm respectively.

Figure 4. Interaction of CLDN1 with pro-fibrogenic and pro-carcinogenic signal transducers identified by co-immunoprecipitation. (A) Co-immunoprecipitation followed by mass-spectrometry identified CLDN1 interactants. String analysis with MCL clustering is shown. (B) Validation of the main CLDN1 interactants in Huh7 cells by Western Blot analysis. Negative control is the hepatocyte marker ASGR1.

Figure 5. CLDN1 as therapeutic target for RNAi-based therapies in fibrotic kidney and lung diseases. (A) CLDN1 gene expression as RNA levels in membranous glomerulonephritis renal tissues (Moylan et al., Hepatology, 2014, 59(2): 471-482) and fibrotic kidney tissue (Lovisa et al., Nature Med., 2015, 21(9): 998-1009) compared to respective healthy kidneys. (B) CLDN1 gene expression as RNA levels in pulmonary tissues of patients with IPF (Pardo et al, PLoS Med., 2005, 2(9): e251).

Figure 6 Treatment with CLDN1 siRNA in a human lung epithelial cell-based model inhibits the expression of key pathways mediating lung fibrosis. Treatment with CLDN1 siRNA inhibits expression of profibrogenic mediators and EMT markers in a lung cell line model. A549 cells derived from human adenocarcinoma alveolar basal epithelial cells were transfected and treated with non-targeting siRNAs control (siCTRL) or siRNAs targeting CLDN1 expression (siCLDNl) for 48 hours. Gene expression was assessed by qRT-PCR. Graphs show mean ± SD of % mRNA normalized to GAPDH or 18S (3 experiments in triplicate, n=9). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 Mann-Whitney test. Figure 7. Treatment with CLDN1 siRNA inhibits expression of key pathways mediating glomerular kidney disease and kidney fibrosis Human renal epithelia cells were used as a model for PECs. Cells were transfected using liposomes and treated with nontargeting siRNAs control (siCTRL) or siRNAs targeting CLDN1 expression (siCLDNl) for 48 hours. Gene expression was assessed by qRT-PCR. Graphs show mean ± SD of % mRNA normalized to GAPDH or 18S (3 experiments in triplicate, n=9). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 Mann-Whitney test.

Definitions

Throughout the specification, several terms are employed that are defined in the following paragraphs.

As used herein, the term “subject” refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like), that can develop a fibrotic disease, but may or may not be suffering from the disease. Non-human subjects may be transgenic or otherwise modified animals. In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an “individual" or a “patient". The term “individual” does not denote a particular age, and thus encompasses newborns, children, teenagers, and adults. The term “patient” more specifically refers to an individual suffering from a disease. In the practice of the present invention, a patient will generally be diagnosed with a fibrotic disease.

The term “treatment" is used herein to characterize a method or process that is aimed at

(1) delaying or preventing the onset of a disease or condition (here a fibrotic disease);

(2) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease or condition; (3) bringing about amelioration of the symptoms of the disease or condition; or (4) curing the disease or condition. A treatment may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. Alternatively, or additionally, a treatment may be administered after initiation of the disease or condition, for a therapeutic action.

The terms “fibrotic disease” and “fibrotic disorder” are used herein interchangeably and have their art understood meaning. They refer to a clinical condition that is characterized by dysregulated tissue growth and scarring destroying healthy tissue, which can lead to disruption of normal function of virtually any organ of the body, including the heart, lung, kidney, liver and skin. A “pharmaceutical composition” is defined herein as comprising an effective amount of at least one RNA interfering agent targeting CLDN1, and at least one pharmaceutically acceptable carrier or excipient.

As used herein, the term “effective amount” refers to any amount of a compound, agent, or composition that is sufficient to fulfil its intended purpose(s), e.g., a desired biological or medicinal response in a cell, tissue, system or subject. For example, in certain embodiments of the present invention, the purpose(s) may be to prevent the onset of a fibrotic disease, to slow down, alleviate or stop the progression, aggravation or deterioration of the symptoms of the fibrotic disease; to bring about amelioration of the symptoms of the disease, or to cure the disease.

The term “pharmaceutically acceptable carrier or excipient’ refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s), and which is not excessively toxic to the host at the concentration at which it is administered. The term includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see for example “Remington ’s Pharmaceutical Sciences'”, E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

As used herein, the term “complementary” refers to the ability of a nucleic acid to form hydrogen bond(s) by either traditional Watson-Crick base-pairing or other non-traditional type base-pairing. In reference to the nucleic acid molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well-known in the art (see, e.g., Turner et al., CSH Symp. Quant. Biol., 1987, LII, p 123-133; Frier et al., P.N.A.S., 1986, 83, 9373-9377; Turner at al., J. Am. Chem. Soc., 1987, 109, 3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base-pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides, in the first oligonucleotide being base-paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90% and 100% complementarity, respectively). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

As used herein, the term ' identity refers to the percentage of identical nucleotide residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those previously described (Computational Molecular Biology, Lesk Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, A.M. Griffin and H.G. Griffin, Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, Academic Press, 1987; and Sequence Analysis Primer, Gribskov and Devereux, Eds., Stockton Press, New York, 1991). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, BLASTP, BLASTN, and FASTA. The BLAST X program is publicly available from NCBI and other sources. The well-known Smith Waterman algorithm can also be used to determine identity.

The terms approximately " and about " as used herein in reference to a number, generally include numbers that fall within a range of 10% in either direction of the number (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Detailed Description of Certain Preferred Embodiments

As mentioned above, the present invention relates to the use of RNAi-based therapies for the management of fibrotic diseases. More specifically, the present invention provides RNAi agents targeting human Claudin-1 for use in the treatment and/or prevention of fibrotic diseases, which may affect any organ of the body, including, but not limited to, the liver, lung, kidney, skin, heart, brain, pancreas, and eye. Also provided are pharmaceutical compositions, kits and methods for administration of such RNAi agents.

I - RNA Interfering Agents Targeting CLDN1

The RNA interfering agents provided by the present invention target human Claudin-1 (CLDN1). 1. CLDN1

Human Claudin 1 (or CLDN1) is a tight junction protein expressed in various tissues of the human body (Zeisel et al., Gut, 2019, 68(3): 547-561). It is expressed in a junctional and non-junctional form. In the liver, the non-junctional form of CLDN1 serves as a cell entry factor of HCV (Zeisel et al., Gut, 2019, 68(3): 547-561; Evans et al., Nature, 2007, 446(7137): 801-805), a major cause of liver fibrosis and cancer. CLDN1 expression has been reported to be upregulated in liver cirrhosis and hepatocellular carcinoma (HCC) (Holczbauer et al., Pathol. Oncol. Res., 2014, 20(3): 493-502). Furthermore, CLDN1 overexpression is known to induce epithelial-mesenchymal transition in liver cells (Suh et al., Oncogene, 2013, 32(41): 4873-4882), a pathogenic mechanism implicated in fibrosis and cancer. CLDN1 has been shown to be overexpressed in chronic kidney and lung disease (Roehlen et al., Sci. Transl. Med., 2022, 14(676): eabj4221; lida et al, PLoS One, 2022, 17(3): e0265081; Ho Jo et al., Kidney Res. Clin Pract., 2022, 41(3): 275-287, Gaut et al., Hum. Pathol., 2014, 45(3): 628-635). Importantly, CLDN1 expression has been shown to correlate with proteinuria in patients with diabetes, with proteinuria a hallmark of chronic progressive kidney disease (Hasegawa et al., Nature Med., 2013, 19(11): 1496-1504). Similarly, CLDN1 has been shown to be overexpressed in pulmonary fibrosis of different origins (Lappi-Blanco et al., Hum. Pathol., 2013, 44(5): 895-907) including IPF (Roehlen et al., Sci. Transl. Med., 2022, 14: 676).

The terms “human Claudin-l and “CLDN1”, which are used herein interchangeably, refer to a protein having the sequence shown in NCB1 Accession Number NP_066924.1, or any naturally occurring variants found in human populations. The term “CLDN1” refers to the human CLDN1 gene that is located on the long (q) arm of chromosome 3 at position 28 (Gene ID: 9076) and that encodes the human protein, CLDN1. The mRNA sequence of CLDN1 is given by GenBank Accession Number RefSeq(mRNA): AH010563.2, AF115546.1, AF134160, and M_021101.5. One skilled in the art knows that CLDN1 splice variants exist as well as clinical variants.

2. RNA Interfering Agents Targeting CLDN1

The present invention provides RNA interfering agents that specifically target CLDN1 and can be used for inhibiting CLDN1 expression in vivo.

A. RNA Interfering Agents

RNA interference (RNAi) is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target nucleic acid results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Cobum and Cullen, J. Virol., 2002, 76: 9225), thereby inhibiting expression of the nucleic acid. This process has been described in plants, invertebrates, and mammalian cells (Sharp, Nature Struct. Biol., 2001, 8: 746-750; Bernstein et al., Nature, 2001, 409: 363-366; Hannon, Nature, 2002, 418: 244-251). In nature, RNA interference is initiated by ribonuclease III (Dicer), which promotes processive cleavage of long double-stranded RNAs (dsRNAs) into doublestranded fragments termed siRNAs. SiRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs. RNA interference can also be initiated by the hand of man via introduction of nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target nucleic acids. Those skilled in the art are aware that RNA interference can be mediated by a single-stranded or a double-stranded oligonucleotide that includes a sequence complementary or substantially complementary to a target sequence (e.g., in a target mRNA). The advantage of RNA interference lies in its high specificity and potent gene silencing, coupled to the fact that every gene is a potential target and every cell has the necessary machinery. Because RNA interfering molecules are directed to a specific target and thereby silence a specific gene, they have been suggested to be useful in the treatment of diseases as well as for screening new pharmaceuticals and disease mechanisms for pharmaceutical target determination.

The terms interference” , and “RNAi”, which are used herein interchangeably, have their art understood meaning and refer to a biological process in which RNA interfering molecules silence, inhibit or down regulate gene expression by causing the destruction, degradation, and/or cleavage of specific mRNA molecules or by blocking the translation thereof. As used herein, the term ' single-stranded RNA interference” more specifically refers to a method of gene silencing directed, at least in part, by administration of a singlestranded RNA interfering agent to a system (e. , cells, tissues, organs, subjects, etc.) where RNA interference is directed by the agent and which requires the RNA-induced silencing complex (RISC) pathway.

The terms “RNA interfering agent or molecule” and “RNAi agent or molecule” are used herein interchangeably. They refer to any RNA molecule that is capable of specifically inhibiting or down-regulating the expression of a target gene (here the CLDN1 gene). By “silencing, inhibiting or down-regulating expression of a target gene”, it is meant that the expression of the target gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the RNAi agent. In the presence of an RNAi agent, the decrease in the expression of the target gene (here the CLDN1 gene) or the activity or level of the protein (here the CLDN 1 protein) encoded by the target gene, may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% or more as compared to said expression in the absence of the RNAi agent.

As used herein, the term ' RNA interfering agent targeting CLDN1” refers to an RNA interfering molecule that contains a sequence that is substantially homologous to the target gene or genomic sequence, or a fragment thereof, i.e., the CLDN1 gene or mRNA. As used in this context, the term “homologous” is defined as being substantially identical, complementary or substantially complementary, or similar to the target CLDN1 mRNA, or a fragment thereof, to effect RNA interference in the target CLDN1. In addition to native RNA molecules, RNA suitable for inhibiting or interfering with the expression of a target sequence include RNA derivatives and analogs. Preferably, the RNA interfering agent contains a sequence that is identical to the target. Starting from known CLDN1 sequences, in particular CLDN1 mRNA sequences, one skilled in the art knows how to design RNA interfering agents targeting CLDN1.

Thus, in certain embodiments, an RNA interfering agent according to the present invention may target any portion of a CLDN1 transcript (mRNA). The target transcript may be located within a coding sequence of the CLDN1 gene or within a non-coding sequence of the CLDN1 gene. In some embodiments, the CLDN1 target transcript is located within an exon, e.g., within Exon 1, Exon 2, Exon 3, or Exon 4, in particular within Exon 2, Exon 3 or Exon 4, more particularly within Exon 4 (see below). In other embodiments, the CLDN1 target transcript is located within an intron. In yet other embodiments, the CLDN1 target transcript is located within a 5 ’-untranslated region (UTR) or 3’-UTR of the CLDN1 gene. In some embodiments, the CLDN1 target transcript is located within an enhancer region, or within a promoter.

Each of the RNA interfering agent targeting CLDN1 can be screened for potential off- target effects by, for example, expression profiling. Such methods are known to one skilled in the art and are described, for example, in Jackson et al., Nature Biotechnology, 2003, 6: 635- 637. In addition to expression profiling, one can also screen the potential target sequences for similar sequences in the sequence databases to identify potential sequences which may have off-target effects. For example, according to Jackson et al. (Nature Biotechnology, 2003, 6: 635-637), 15, or perhaps as few as 11, contiguous nucleotides of sequence identity are sufficient to direct silencing of non-targeted transcripts. Therefore, one can initially screen the proposed RNA interfering agents to avoid potential off-target silencing using the sequence identity analysis by any known sequence comparison methods (e.g., BLAST). RNAi agents’ sequences are chosen to maximize the uptake of the antisense (guide) strand of the RNAi agents into the RNA-induced silencing complex (RISC) and thereby maximize the ability of RISC to target human GGT mRNA for degradation. This can be accomplished by scanning for sequences that have the lowest free energy of binding at the 5’-terminus of the antisense strand. The lower free energy leads to an enhancement of the unwinding of the 5 ’-end of the antisense strand of a double-stranded RNA interfering agent, thereby ensuring that the antisense strand will be taken up by RISC and direct the sequence-specific cleavage of the human CNLD1 mRNA.

An RNA interfering agent according to the present invention may be any singlestranded RNA (e.g.. mature miRNA, ssRNAi oligonucleotides, ssDNAi oligonucleotides) or double-stranded RNA (i.e., duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the RNA interfering agent is in the same cell as the target gene or sequence. Thus, the terms “RNA interfering agent” and “RNAi agent” refer to the single-stranded RNA that is complementary to a target mRNA sequence (here the CLDN1 mRNA sequence) or to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand. An RNAi agent may have substantial or complete identity to the target gene mRNA sequence (here the CLDN1 mRNA sequence), or may comprise a region of mismatch (/.< ., a mismatch motif). Consequently, the terms “RNA interfering agent” and “RNAi agent” refer to a RNA molecule comprising a strand having a sequence sufficiently complementary to a target mRNA sequence (here the CLDN1 mRNA sequence) to direct target-specific RNA interference (RNAi) thereby inhibiting or down-regulating the expression of the target gene (here CLDNJ).

In certain preferred embodiments of the present invention, an RNAi agent is a siRNA (small interfering RNA), a shRNA (short hairpin RNA), a micro-RNA (micro RNA), or an aiRNA (asymmetric interfering RNA). The development of any type of RNAi agent capable of specifically silencing, inhibiting or down-regulating the expression of a given target gene (here the CLDN1 gene) is within the capabilities of one skilled in the art. The use of RNAi agents, such as siRNAs, shRNAs, micro-RNAs or aiRNAs, to inhibit gene expression is well known in the art.

The terms ' short interfering RNA “small interfering RNA and “siRNA” are used herein interchangeably. They refer to a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may contain a 3’ and/or 5’ overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA). Short RNAi agents typically include a region (the “duplex region”), one strand of which contains an inhibitory region between 15 nucleotides to 29 nucleotides in length that is sufficiently complementary to a portion of the target transcript (the “target portion”), so that a hybrid (the “core region”) can form in vivo between this strand and the target transcript. The core region is understood not to include overhangs.

The terms “asymmetric interfering RNA” and “aiRNA” refer to an siRNA which is characterized by the length asymmetry between the two RNA strands.

The term “short hairpin RNA” (or “shRNA”) refers to a sequence of RNA having one or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises a sequence complementary to a region of the target mRNA. A short hairpin RNA is cleaved by the cellular machinery into siRNA. The stem can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length, for example between 19 and 25 base pairs, or between 19 and 21 base pairs in length. The loop can vary in length. For example, the loop may be 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3’ or 5’ overhang portions. For example, the overhang is a 3’ or a 5’ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

The term “micro-RNA” (or “miRNA” as used herein, has its art understood meaning. miRNAs are a major group of noncoding RNAs that are known to regulate almost a third of all the coding genes. They are small (~ 20-25 nucleotides long) endogenously formed repressors of gene expression. miRNAs usually bind to the 3’ untranslated region (3’UTR) of the target RNA transcripts (mRNAs or circRNAs) and are capable of inducing posttranscriptional gene regulation by blocking translation or by degrading the target RNAs, or by doing both. miRNAs can also be chemically synthesized. In contrast to siRNA, which has perfect complementarity to the target RNA transcripts, miRNA binds imperfectly to the target RNA transcripts.

B. Chemical Modifications

As known in the art, approaches to enhance the efficacy or resistance of RNA interfering molecules include the chemical modification of sugars, phosphate backbone and the bases or the oligoribonucleotides, as well as the modification of the termini and duplex structure. An RNA interfering agent according to the present invention may contain any one of these modifications or a combination thereof.

Thus, an RNAi agent according to the present invention can comprise chemically modified nucleotides and non-nucleotides. RNAi agents suitable for use in the context of the present invention also include molecules wherein a ribose sugar molecule is substituted for another sugar molecule or a molecule which performs a similar function. Moreover, a nonnatural linkage between nucleotide residues can be used, such as a phosphorothioate linkage. The RNA strand can be derivatized with a reactive functional group of a reporter group, such as a fluorophore. Particularly useful derivatives are modified at a terminus or termini of an RNA strand (i.e., at the 5’-end, the 3’-end, or both the 5’ and 3’ends of the strand). For example, the 2’-hydroxyl at the 3’ terminus can be readily and selectively derivatized with a variety of groups, for example a deoxy abasic moiety or glyceryl moiety.

Other useful RNA derivatives incorporate nucleotides having modified carbohydrate moieties, such as 2’-O-alkylated residues or 2’-O-methyl ribosyl derivatives and 2’-O-fluoro ribosyl derivatives. The RNA bases can also be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence may be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated. The bases can also be alkylated, for example, 7-m ethylguanosine can be incorporated in place of a guanosine residue. Non-natural bases that yield successful inhibition can also be incorporated. The most preferred siRNA modifications include 2 ’-deoxy-2’ -fluorouridine or locked nucleic acid (LNA) nucleotides and RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages. Such modifications are known to one skilled in the art and have been described, for example, in Braasch et a , Biochemistry, 2003, 42: 7967-7975. Most of the useful modifications to the siRNA molecules can be introduced using chemistries established for antisense oligonucleotide technology. An RNA interfering molecule may comprise from about 5% to about 100% of modified nucleotides (e.g, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given RNA interfering molecule will depend on the total number of nucleotides present in said molecule.

C. Preparation of RNAi Agents

RNA interfering agents according to the present invention may be generated using any suitable method known in the art. For example, they may be chemically synthesized, produced by in vitro transcription, or produced within a host cell.

For example, an RNA interfering agent can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer (see, e.g., Elbashir, Nature, 2001, 41(l):494-498; Elbashir et al., Genes Dev.,

2001, 15: 188-200; Harborth et al., J. Cell Science, 2001, l(14):4557-4565; Masters et al., Proc. Natl. Acad. Sci., USA, 2001, 98: 8012-8017; and Tuschl et al., Genes Dev., 1999, 13: 3191-3197). Alternatively, several commercial RNA synthesis suppliers are available including, but not limited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL , USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). As such, RNA interfering molecules are not overly difficult to synthesize and are readily provided in a quality suitable for RNAi. In addition, dsRNAs can be expressed as stem loop structures encoded by plasmid vectors, retroviruses and lentiviruses (Paddison et al., Genes Dev., 2002, 16: 948-958; McManus et al., RNA, 2002, 8: 842-850; Paul et al., Nat. Biotechnol., 2002, 20: 505-508; Miyagishi et al., Nat. Biotechnol., 2002, 20: 497-500; Sui et al., Proc. Natl. Acad. Sci. USA, 2002, 99: 5515-5520; Brummelkamp et al., Cancer Cell,

2002, 2: 243; Lee et al., Nat. Biotechnol., 2002, 20: 500-505; Yu et al., Proc. Natl. Acad. Sci., USA, 2002, 99: 6047-6052; Zeng et al., Mol. Cell, 2002, 9: 1327-1333; Rubinson et al., Nat. Genet., 2003, 33: 401-406; Stewart et al., RNA, 2003, 9: 493-501). These vectors generally have a polIII promoter upstream of the dsRNA and can express sense and antisense RNA strands separately and/or as a hairpin structures. Within cells, Dicer processes the short hairpin RNA (shRNA) into effective siRNA.

D. Delivery Systems for RNAi Agents

RNA interfering agents according to the present invention may be administered in free (naked) form or using a delivery system. Indeed, to be effective RNAi molecules need to evade clearance by non-target organs and tissue, have the ability to penetrate the disease target tissues and cells and interact with them without eliciting harmful immune response or other adverse effects. Different delivery systems have been devised to accomplish these objectives and methods for the delivery of nucleic acid molecules are known in the art (see, for example, Akhtar et al., Trends Cell Biol., 1992, 2: 139; “Delivery Strategies for Antisense Oligonucleotide Therapeutics’", Ed. Akhtar, 1995; Maurer et al., Mol. Membr. Biol., 1999, 16; 129-140; Hofland and Huang, Handb. Exp. Pharmacol., 1999, 137: 165-192; and Lee et al., ACS Symp. Ser., 2000, 752: 184-192). As used herein, the term “delivery system” refers to a component or combination of components that, when combined with an RNA interfering agent as described herein, increases the amount of the RNA interfering agent that contacts the intended location in vivo, and/or extends the duration of its presence at the target, e.g., by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or even more as compared to the amount and/or duration in the absence of the delivery system, and/or prevents or reduces interactions that cause side effects.

Examples of such delivery systems that can be used in the context of the present invention include, but are not limited to, vectors (e.g., plasmid or viral vectors), liposomes or other vehicles, such as hydrogels, cyclodextrins (Gonzalez et al., Bioconjugate Chem., 1999, 10: 1068-1074; WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US 2002/130,430), biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (WO 00/53722) or in combination with a cationic peptide (US 2007/275923).

Other delivery systems include delivery of the RNA interfering agents using a basic peptide by conjugating or mixing the RNA interfering agent with a basic peptide, e.g., a fragment of a TAT peptide (Meade and Dowdy, Adv. Drug Deliv. Rev., 2007, 59: 134-140), or mixing with cationic lipids.

RNA interfering agents may be formulated or complexed with poly ethyl enimine (e.g., linear or branched PEI) and/or polyethyleneimine derivatives, including for examples grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see for example Ogris et al., AAPA PharmSci, 2001, 3: 1-11; Furgeson et al., Bioconjug. Chem., 2003, 14: 840-847; Kunath et al., Pharm. Res., 2002, 19: 810-817; Choi et al., Bull. Korean Chem. Soc., 2001, 22: 46-52; Bettinger et al., Bioconjug. Chem., 1999, 10: 558-561; Peterson et al., Bioconjug. Chem., 2002, 13: 845-854; Godbey et al., PNAS USA, 1999, 96: 5177-5181; Godbey et al., J. Contr. Release, 1999, 60: 149-160; Diebold et al., J. Biol. Chem., 1999, 274: 19087-19094; Thomas and Klibanov, PNAS USA, 2002, 99:14640-14645; U.S. Pat. No. 6,586,524). Specific examples include, but are not limited to, polyethyleneimine-polyethyleneglycol-N- acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N- acetylgalactosamine (PEI-PEG-triGAL) derivatives.

In embodiments where the RNA interfering agent according to the present invention is intended to be used in the treatment of liver fibrosis, the RNA interfering agent may be conjugated to the glycoprotein A-acetylgalactosamine (GalNAc) with exhibits high specificity and binding affinity for the asialoglycoprotein receptor (ASGPR), which is specifically and abundantly expressed in hepatocyte membranes (Cedillo et al., Molecules, 2017, 22(8): 1356). Nair et al. (J. Am. Chem. Soc., 2014, 136(49): 16958-16961) first demonstrated that GalNAc-siRNA facilitates targeted delivery of siRNA to hepatocytes in vitro and in vivo (in mice). Since then, the method has been used successfully to deliver several licensed siRNA therapeutics. The exclusive expression and abundance of ASGPRs in hepatocytes and their rapid recycling contributes to enhanced GalNAc-siRNA delivery. The development of this technology has greatly advanced siRNA therapeutics, enabling its use for the systemic delivery targeting proteins synthesized in the liver (Balwani et al., N. Engl. J. Med., 2020, 382: 2289-2301; Ray et al., N. Engl. J. Med., 2020, 382: 1507-1519; Garrelfs et al., N. Engl. J. Med., 2021, 384: 1216-1226). Thus, in certain embodiments, an RNA interfering agent is conjugated to a A-acetylgalactosamine (GalNAc) moiety or to a molecule comprising at least one GalNAc moiety. Other systems that are useful for the delivery of RNA interfering agents in hepatocytes include lipid nanoparticles (LNPs) (Adams et al., N. Engl. J. Med., 2018, 379: 11-21). Thus, in certain embodiments, an RNA interfering agent according to the present invention is delivered using LNP as a delivery vehicle, e.g., the RNAi agent is encapsulated into a lipid nanoparticle (LNP).

Other similar ligands have been developed to target RNA interfering agents to pancreatic beta cells with glucagon-like peptide-1 and to skeletal/cardiac muscle with transferring receptor protein-1. Lipids and cholesterol conjugates help siRNAs interact with the cell membrane and facilitate biodistribution by forming complexes with low-density lipoprotein (LDL) and high density lipoprotein (HDL) particles (Wolfrum et al., Biotechnology, 2007, 25(10): 1149-1157).

One skilled in the art knows how to select the most appropriate delivery system based on the intended use (e.g., fibrotic disease) of the RNA interfering agent. E. Specific RNA Interfering Agent

The present invention provides an RNA interfering agent that specifically targets CLDN1 and can be used for inhibiting CLDN1 expression in vivo, in particular for treating and/or preventing a fibrotic disease.

In certain embodiments, the RNA interfering agent according to the present invention is a double-stranded short interfering nucleic acid molecule comprising a first strand comprising a sense region and a second strand comprising an antisense region, wherein the sense region targets a portion of an exon of a CLND1 mRNA sequence, and the antisense region comprises a nucleotide sequence that is complementary to the sense region. As already mentioned above, the sense region “targets” a given mRNA sequence when it is sufficiently complementary to said target mRNA sequence to direct target-specific RNA interference.

In certain embodiments, the exon of the CLDN1 mRNA sequence is Exon 2 or Exon 4.

In certain embodiments, the sense region of the RNA interfering agent targets a portion of Exon 2 of the CLND1 mRNA sequence, wherein the portion consists of the sequence set forth in SEQ ID NO : 1 : 5 ’ -UGAAGUGUAUGAAGUGCUU-3 ’ .

In other embodiments, the sense region of the RNA interfering agent targets a portion of Exon 4 of the CLND1 mRNA sequence, wherein the portion consists of the sequence set forth in SEQ ID NO: 2: 5’-CACCAAGGCCCUAUCCAAA-3’.

In certain embodiments, the sense region of the RNA interfering agent targets a portion of Exon 4 of the CLND1 mRNA sequence, wherein the portion consists of the sequence set forth in SEQ ID NO: 3: 5’-UAACAUUAGGACCUUAGAAUU-3’. For example, in certain embodiments, the RNA interfering agent according to the present invention is a doublestranded short interfering nucleic acid molecule comprising a first strand comprising a sense region and a second strand comprising an antisense region, wherein the sense region has, or comprises, the sequence set forth in SEQ ID NO: 3 (5’-UAACAUUAGGACCUUAGAAUU- 3’), or a sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity with SEQ ID NO: 3, and the antisense region comprises a nucleotide sequence that is complementary to the sense region. In certain embodiments, the sense region consists of the sequence set forth in SEQ ID NO: 3 and the antisense region consists of a sequence that is perfectly complementary to the sense region.

In certain embodiments, a double- stranded short interfering nucleic acid molecule according to the present invention is such that each strand (sense and antisense) comprises about 15 to about 30 nucleotides, and each strand comprises at least 15 to about 30 nucleotides that are complementary to the nucleotides of the other strand.

In certain embodiments, a double- stranded short interfering nucleic acid molecule comprises a 19 to 23-nucleotide duplex, in particular a 19 to 21-nucleotide duplex.

In certain embodiments, a double-stranded interfering nucleic acid molecule comprises 1 to about 3 overhanging nucleotides at the 3’ end of each strand.

In certain embodiments, a double-stranded interfering nucleic acid molecule comprises blunt end(s).

In certain embodiments, a double-stranded interfering nucleic acid molecule is such that the sense region is connected to the antisense region via a linker molecule.

In certain embodiments, a double-stranded interfering nucleic acid molecule comprises one or more modified nucleotides, in particular one or more locked nucleic acid (LNA) nucleotides.

In certain embodiments, a double-stranded interfering nucleic acid molecule is such that the first strand comprising the sense region includes a terminal cap moiety at the 5’-end and/or 3 ’-end.

In certain embodiments, a double-stranded interfering nucleic acid molecule is such that the second strand comprising the antisense region includes a phosphate group at the 5’-end.

In certain embodiments, a double-stranded interfering nucleic acid molecule is such that it comprises at least one modified internucleotidic linkage.

In certain embodiments, an RNA interfering agent that specifically targets CLDN1, as described above, is conjugated to a GalNAc moiety.

In other embodiments, an RNA interfering agent that specifically targets CLDN1, as described above, is delivered using lipid nanoparticle (LNP) as a delivery vehicle. For example, the RNA interfering agent is encapsulated into a lipid nanoparticle.

3. Antisense Oligonucleotides Targeting CLDN1

The present invention also provides antisense oligonucleotides that specifically target CLDN1 mRNA and can be used for inhibiting CLDN1 expression in vivo, in particular for treating or preventing a fibrotic disease. The terms “antisense oligonucleotide' and “ASO” are used herein interchangeably, and refer to an oligonucleotide capable of modulating expression of a target gene (here the CLDN1 gene) by hybridizing to a target nucleic caid, in particular to a contiguous sequence on a target nucleic acid. The antisense oligonucleotides are not essentially double-stranded and are therefore not siRNAs. Preferably, the antisense oligonucleotides of the present invention are single-stranded. ASOs are capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA.

An antisense oligonucleotide may have a length of 10 to 50 nucleotides, preferably 12 to 30, or more preferably 15 to 25 nucleotides. The antisense oligonucleotide may be a DNA and/or RNA, optionally comprising at least modified nucleosidic building block and/or at least one modified intemucleosidic linkage between two nucleoside building blocks. It may be preferably that an antisense oligonucleotide is not entirely an RNA. However, it may comprise one or more RNA segments.

Starting from known CLDN1 sequences, in particular CLDN1 mRNA sequences, one skilled in the art knows how to design antisense oligonucleotides targeting CLDN1.

All the aspects of the invention described below relate to RNA interfering agents. However, one skilled in the art will understand that, in what follows the term “RNA interfering agent targeting CLDN1” (and similar terms) may be replaced with the term “antisense oligonucleotide targeting CLDN1”.

II - Treatment and/or Prevention of a Fibrotic Disease

A. Indications

The RNA interfering agents according to the present invention may be used in methods to prevent and/or treat fibrotic diseases in a patient. As already mentioned, the fibrotic disease to be prevented and/or treated may affect any organ of the body, including, but not limited to, the liver, lung, kidney, heart, skin, pancreas, intestine, eye, nerve system, mediastinum, retroperitoneum, joint or tendon.

Methods of treatment of the present invention may be accomplished using an RNA interfering agent described herein, or a pharmaceutical composition comprising such an interfering agent (see below). These methods generally comprise administration of an effective amount of an RNA interfering agent, or of a pharmaceutical composition thereof, to a subject in need thereof (i.e., a patient diagnosed with a fibrotic disease or at risk of developing a fibrotic disease). Administration may be performed using any of the administration methods known to one skilled in the art (see below).

Liver Fibrosis. In certain embodiments, the fibrotic disease is liver fibrosis. The terms “liver fibrosis¹' and “hepatic fibrosis , which are used herein interchangeably, refer to the excessive accumulation of extracellular matrix proteins (including collagen), and subsequent scarring process, that occurs in most chronic liver diseases. With time, advanced liver fibrosis results in cirrhosis of the liver. Cirrhosis is the final phase of chronic liver disease and is generally irreversible with a poor long-term prognosis. In the advanced stage, the only option is liver transplant. The risk of liver cancer is significantly increased with cirrhosis and cirrhosis may be viewed as a premalignant condition (hepatocellular carcinoma). Unfortunately, few treatment options are available and most often treatment consists of addressing the causes and/or symptoms of liver cirrhosis. No treatment will cure liver fibrosis subsequent scarring and cirrhosis. Liver transplantation is the only treatment available for patients with advanced stage of fibrosis.

Liver fibrosis that can be treated according to a method of the present invention may be caused by any of a variety of reasons including, but not limited to, viral-induced liver fibrosis such as liver fibrosis caused by hepatitis B, C, E or E; liver fibrosis due to alcohol abuse (alcoholic liver disease), pharmaceutical compounds, oxidative stress, cancer radiotherapy or industrial chemicals; and liver fibrosis caused by diseases such as primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver, obesity, nonalcoholic steatohepatitis, cystic fibrosis, hemochromatosis, and autoimmune hepatitis.

Administration of an RNA interfering agent according to the present invention, or of a pharmaceutical composition thereof, to patients suffering from liver fibrosis may slow, reduce, stop or alleviate the progression of the disease, in particular the development of scarring and cirrhosis, and of hepatocellular carcinoma.

Alternatively, or additionally, administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to a patient suffering from liver fibrosis may result in amelioration of at least one of the symptoms experienced by the individual including, but not limited to, fluid buildup in the legs or stomach, nausea, weakness, and weight loss.

Alternatively, or additionally, administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to a patient suffering from liver fibrosis may help avoiding or, at least delaying, liver transplantation. The effects of a treatment according to the invention may be monitored using any of the assays and tests known in the art for the diagnosis of liver fibrosis affecting the patient. Such assays and tests include, but are not limited to, imaging tests (such as ultrasound elastography and computerized tomography (CT) scan); liver function tests (such as alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT)); or biopsy.

In certain embodiments of a method of prevention or treatment of liver fibrosis according to the present invention, an RNA interfering agent described herein, or a pharmaceutical composition thereof, is administered alone. In other embodiments, the anti- RNA interfering agent, or a pharmaceutical composition thereof, is administered in combination with at least one additional therapeutic agent and/or therapeutic procedure. The RNA interfering agent, or pharmaceutical composition thereof, may be administered prior to administration of the therapeutic agent or therapeutic procedure, concurrently with the therapeutic agent or therapeutic procedure, and/or following administration of the therapeutic agent or therapeutic procedure.

Therapeutic agents that may be administered in combination with an RNA interfering agent, or a pharmaceutical composition thereof, include, but are not limited to, pirfenidone, nintedanib, obeticholic acid, urodeoxycholic acid, emricasan, vitamin E, pioglitazone, liraglutide, pentoxifylline and metformin. The agents pioglitazone, liraglutide, pentoxifylline and metformin are particularly used for treating NASH (non-alcoholic steatohepatitis).

Pulmonary Fibrosis. In certain embodiments, the fibrotic disease to be prevented and/or treated using a method described herein is lung fibrosis. The terms “lung fibrosis ' and “pulmonary fibrosis ' are used herein interchangeably. They refer to a number of conditions, of known or unknown etiologies, that cause interstitial lung damage, followed by fibrosis and eventually loss of lung elasticity. These conditions lead to symptoms such as persistent cough, chest pain, difficulty breathing and fatigue.

Pulmonary fibrosis that can be treated according to a method of the present invention may be caused by any of a variety of factors including, but not limited to, long-term exposure to certain toxins (e.g, silica dust, asbestos fibers, hard metal dusts, coal dusts, grain dusts, bird and animal droppings); certain medical conditions (e.g., dermatomyositis, polymyositis, mixed connective tissue disease, systemic lupus erythematosus, rheumatoid arthritis, sarcoidosis, scleroderma and pneumonia); radiation therapy (e.g., for lung or breast cancer); and some medications (e.g., chemotherapy drugs such as methotrexate and cyclophosphamide, heart medication such as amiodarone; some antibiotics such as nitrofurantoin and ethambutol; and anti-inflammatory drugs such as rituximab and sulfasalazine).

In some embodiments, pulmonary fibrosis that can be treated according to a method of the present invention may have no clear underlying cause The term idiopathic pulmonary fibrosis is then used.

In some embodiments, the pulmonary fibrosis to be treated using a method of treatment of the present invention is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonitis (NSIP), cryptogenic organizing pneumonia (COP), Hamman-Rich syndrome (also known as acute interstitial pneumonia), lymphocytic interstitial pneumonitis (LIP), respiratory bronchiolitis interstitial lung disease, desquamative interstitial pneumonitis or idiopathic lymphoid interstitial pneumonia, and idiopathic pleuroparenchymal fibroelastosis.

In certain preferred embodiments, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

In some embodiments, the pulmonary fibrosis is associated with chronic obstructive pulmonary disease. Chronic obstructive pulmonary disease (COPD) is a type of progressive respiratory disease characterized by airway obstruction, long-term breathing problems and poor airflow.

In certain embodiments, the pulmonary fibrosis is due to infection, such as C0VID19- associated fibrosis.

Administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to patients suffering from pulmonary fibrosis may slow, reduce, stop or alleviate the progression of the disease, in particular the development of complications such as pulmonary hypertension, right-sided heart failure, respiratory failure, lung cancer, or other lung complications such as blood clots in the lung, a collapsed lung or lung infections.

Alternatively, or additionally, administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to a patient suffering from pulmonary fibrosis may result in amelioration of at least one of the symptoms experienced by the individual including, but not limited to, dry cough, shortness of breath, fatigue, muscle pain, join pain, and weight loss. Alternatively, or additionally, administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to a patient suffering from pulmonary fibrosis may help avoiding or, at least delaying, lung transplantation.

In some embodiments, a method of the invention is applied to a subject with a risk of developing pulmonary fibrosis, for example someone who has been exposed to certain toxins known to be associated with lung fibrosis or someone who has received radiation therapy, or yet someone who has been treated with certain medications. Administration of an RNA interfering agent, or of a pharmaceutical composition thereof, may result in the prevention of the development of the pulmonary fibrotic disease or in the prevention of the progression of the pulmonary fibrotic disease beyond the very early stages.

The effects of a treatment according to the invention may be monitored using any of the assays and tests known in the art for the diagnosis of pulmonary fibrosis affecting the patient. Such assays and tests include, but are not limited to, imaging tests (such as chest X-ray, computerized tomography (CT) scan, and echocardiogram); lung function tests (such as pulmonary functions testing (e.g., spirometry), pulse oximetry, exercise stress test, and arterial blood gas test); or biopsy by bronchoscopy or surgical biopsy.

In certain embodiments of a method of prevention or treatment of pulmonary fibrosis according to the present invention, an RNA interfering agent, or a pharmaceutical composition thereof, is administered alone. In other embodiments, an RNA interfering agent, or a pharmaceutical composition thereof, is administered in combination with at least one additional therapeutic agent and/or therapeutic procedure. The RNA interfering agent, or pharmaceutical composition thereof, may be administered prior to administration of the therapeutic agent or therapeutic procedure, concurrently with the therapeutic agent or therapeutic procedure, and/or following administration of the therapeutic agent or therapeutic procedure.

Therapeutic agents that may be administered in combination with an RNA interfering agent, or a pharmaceutical composition thereof, may be selected among a large variety of biologically active compounds that are known in the art to have a beneficial effect in the treatment or management of pulmonary fibrosis. Examples of such therapeutic agents include, but are not limited to, immunosuppressive agents such as corticosteroids, anti-fibrotic agents such as ciclosporin or colchicine, new medications such as pirfenidone (ESBRIET®) and nintedanib (OFEV®), which have been approved by the Food and Drug Administration (FDA); and anti-acid medications to treat gastroesophageal reflux disease (GERD), a digestive condition that commonly occurs in people with idiopathic pulmonary fibrosis. Examples of therapeutic procedures include, but are not limited to oxygen therapy, which makes breathing and exercise easier, prevents or lessens complications from low blood oxygen levels, reduces blood pressure in the right side of the heart and improves sleep and sense of well-being; pulmonary rehabilitation, which helps manage the symptoms and improves daily functioning by improving physical endurance and lung efficiency; and lung transplant, which improves the quality of life and allows patients to live a longer life.

Thus, in certain embodiments, the method of treatment of pulmonary fibrosis according to the invention is administered in combination with a therapeutic agent selected from the group consisting of corticosteroids, ciclosporin, colchicine, pirfenidone, nintedanib and antiacid drugs to treat gastroesophageal reflux disease (GERD). Alternatively, or additionally, the method of treatment of pulmonary fibrosis according to the invention is administered in combination with a therapeutic procedure selected from the group consisting of lung transplantation, hyperbaric oxygen therapy and pulmonary rehabilitation.

It is also contemplated that RNA interfering agents as defined herein, be used in methods to prevent and/or treat mediastinal fibrosis. Mediastinal fibrosis (of fibrosing mediastinitis) is a condition characterized by calcified fibrosis that affects the area between the lungs (mediastinum), which contains the heart, large blood vessels, trachea, esophagus, and lymph nodes.

Kidney Fibrosis. In certain embodiments, the fibrotic disease to be prevented and/or treated using a method described herein is kidney fibrosis. The terms kidney fibrosis ' and “renal fibrosis’" are used herein interchangeably. Renal fibrosis is the hallmark of chronic kidney disease, regardless of underlying etiology. The pathological finding of renal fibrosis is characterized by progressive tissue scarring including glomerulosclerosis, tubulointerstitial fibrosis and loss of renal parenchyma (including tubular atrophy, loss of capillaries and podocytes). All the renal diseases are accompanied by kidney fibrosis, which is a progressive process that ultimately leads to end-stage renal failure (ESRD), a devastating disorder that requires dialysis or kidney transplant. Since chronic deterioration of renal function depends heavily on the extent of fibrosis of the kidney, it is thought that inhibiting the progress of fibrosis can result in suppression of the development of chronic renal failure. The term “chronic renal failure” refers to a state in which the renal functions gradually deteriorate irreversibly and homeostasis of a living body cannot be maintained. Renal fibrosis that can be treated according to a method of the present invention may be caused by any of a variety of factors including, but not limited to, certain medical conditions (nephropathies such as glomerular diseases (e.g., glomerulosclerosis, glomerulonephritis), chronic renal insufficiency, acute kidney injury, high blood pressure, polycystic kidney disease, vesicoureteral reflux, pyelonephritis (recurrent kidney infection), and autoimmune diseases (such as ANCA vasculitis or RPGN), metabolic disease such as diabetes mellitus; certain medical interventions (such as nephrectomy or kidney removal, a procedure which is sometimes performed on patients with kidney cancer and which may negatively impact kidney function of the remaining kidney; dialysis following kidney failure; and catheter placement); and some medications (chemotherapy and immunosuppressive therapy, that are a source of harmful effects to the kidney which result in most of the cases in renal fibrosis; long-time use of lithium and of non-steroidal anti-inflammatory drugs).

In some embodiments, renal fibrosis to be treated using a method of treatment of the present invention is selected from the group consisting of renal interstitial fibrosis and gl omerul oscl erosi s .

Administration of an RNA interfering agent described herein, or of a pharmaceutical composition thereof, to patients suffering from kidney fibrosis may slow, reduce, stop or alleviate the progression of the disease, in particular the development of complications such as fluid retention including pulmonary edema; hyperkalemia (sudden rise of potassium levels in the blood); cardiovascular disease; decreased immune response; pericarditis; and end-stage kidney disease.

Alternatively, or additionally, administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to a patient suffering from renal fibrosis may result in amelioration of at least one of the symptoms experienced by the individual including, but not limited to, nausea, vomiting, loss of appetite, fatigue and weakness, muscle cramps, fluid retention (puffiness or swelling), chest pain, shortness of breath, and hypertension.

Alternatively, or additionally, administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to a patient suffering from renal fibrosis may help avoiding, or at least delaying, dialysis or kidney transplant.

The effects of a treatment according to the invention may be monitored using any of the assays and tests known in the art for the diagnosis of renal fibrosis affecting the patient. Such assays and tests include, but are not limited to, imaging tests (such as ultrasound); blood tests (determination of creatinine and urea levels); urine tests, and biopsy.

In certain embodiments, an RNA interfering agent, or a pharmaceutical composition thereof, is administered alone. In other embodiments, an RNA interfering agent, or a pharmaceutical composition thereof, is administered in combination with at least one additional therapeutic agent and/or therapeutic procedure. The RNA interfering agent, or pharmaceutical composition thereof, may be administered prior to administration of the therapeutic agent or therapeutic procedure, concurrently with the therapeutic agent or therapeutic procedure, and/or following administration of the therapeutic agent or therapeutic procedure.

Therapeutic agents that may be administered in combination with an RNA interfering agent, or a pharmaceutical composition thereof, may be selected among a large variety of biologically active compounds that are known in the art to have a beneficial effect in the treatment or management of renal fibrosis. Examples of such therapeutic agents include, but are not limited to, anti-hypertensive drugs (in order to alleviate the burden on the glomerulus); supplementation in 1,25-dihydroxyvitamin D3 or erythropoietin, which are secreted by the kidney; angiotensin converting enzyme inhibitors (such as captoril, enalapril, delapril, imidapril, quinapril, temocapril, perindopril erbumine, and lisinopril) and angiotensin II receptor antagonists (such as losartan, valsartan, candesartan cilexetil, telmisartan, olmesartan medoxomil, and irbesartan), which are known to have a renal protective effect per se in addition to suppressing the progress of renal failure by decreasing glomerular blood pressure; diuretics, which relieve swelling; AST-120 (KREMEZIN®), an adsorptive carbon that adsorbs harmful substance in the intestine; and calcium polystyrene sulfonate, an ion-exchange resin that adorbs potassium in the intestine. Examples of therapeutic procedures include dialysis and kidney transplantation. The dialysis may be a hemodialysis or a peritoneal dialysis. In hemodialysis, a machine filters waste and excess fluids from the blood. In peritoneal dialysis, a catheter inserted in the abdomen fills the abdominal cavity with a dialysis solution that absorbs waste and excess fluids. After a period of time, the dialysis solution drains from the body, carrying the waste with it.

Thus, in certain embodiments, the method of treatment of kidney fibrosis according to the invention is administered in combination with a therapeutic agent selected from the group consisting of anti-hypertensive drugs 1,25-dihydroxyvitamin D3, erythropoietin, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists AST- 120 (KREMEZIN®), and calcium polystyrene sulfonate. Alternatively, or additionally, the method of treatment of kidney fibrosis according to the invention is administered in combination with a therapeutic procedure selected from the group consisting of dialysis and kidney transplantation.

It is also contemplated that RNA interfering agents, described herein, be used in methods to prevent and/or treat retroperitoneal fibrosis. Retroperitoneal fibrosis is a rare inflammatory disorder in which abnormal formation of fibrous tissue in the retroperitoneum, the compartment of the body containing the kidneys, aorta, renal tract, and various other structures.

Skin Fibrosis. In certain embodiments, the fibrotic disease to be prevented and/or treated using a method described herein is skin fibrosis. The terms “skin fibrosis”, “dermal fibrosis” and “cutaneous fibrosis” are used herein interchangeably. They refer to an excessive scarring of the skin which results from a pathologic wound healing response. Skin fibrosis is characterized by fibroblast proliferation and excessive synthesis as well as deposition of extracellular matrix (ECM) proteins, such as collagen, elastin, and fibrillin. Clinically, skin fibrosis manifests as thickened, tightened and hardened areas of skin. Ultimately, skin fibrosis may lead to dermal contractures that affect the ability to flex and extend the joints. Despite the morbidity and socioeconomic burdens associated with skin fibrosis, there are limited effective therapeutic options. Current therapies are associated with significant side effects and even with combination therapy, progression, and recurrence often occurs.

Skin fibrosis that can be treated according to a method of the present invention may be caused by any of a variety of factors including, but not limited to, certain medical conditions (scleroderma in both localized (morphea, linear scleroderma) and systemic forms, graft- versus-host disease (GVHD), nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleredema, scleromyxedema, eosinophilic fasciitis, chromoblastomycosis, hypertrophic scars and keloids); certain medical interventions (radiotherapy-induced skin fibroses); environmental or professional exposures to various chemicals (e.g., in eosinophiliamyalgia syndrome induced by L-tryptophan); and exposure to certain physical agents (e.g., skin fibroses induced by physical trauma, surgical injury, heat or ice skin burns).

Administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to patients suffering from skin fibrosis may slow, reduce, stop or alleviate the progression of the skin disease, for example the propagation of fibrosis to a non-affected skin area, and/or may slow, reduce, stop or alleviate the development of complications such as disfigurement, dermal contractures, diminished function of an affected limb and propagation to internal organs.

Alternatively, or additionally, administration of an RNA interfering agent, or of a pharmaceutical composition thereof, to a patient suffering from skin fibrosis may result in amelioration of at least one of the symptoms experienced by the individual including, but not limited to, thickened, tightened and hardened areas of skin.

The effects of a treatment according to the invention may be monitored using any of the assays and tests known in the art for the diagnosis of dermal fibrosis affecting the patient. Such assays and tests make use of, for example, durometers for measuring skin hardness and/or tautness, cutometers for quantifying skin elasticity, ultrasonographic devices for assessing local dermal and subcutaneous blood flow, and digital infrared thermal imaging of skin. Other non-invasive methods of skin fibrosis diagnosis include ultrasound scan, elastography, confocal microscopy, and optical coherence tomography.

In certain embodiments of a method of prevention or treatment of dermal fibrosis according to the present invention, an RNA interfering agent, or a pharmaceutical composition thereof, is administered alone. In other embodiments, an RNA interfering agent, or a pharmaceutical composition thereof, is administered in combination with at least one additional therapeutic agent and/or therapeutic procedure. The RNA interfering agent, or pharmaceutical composition thereof, may be administered prior to administration of the therapeutic agent and/or the therapeutic procedure, concurrently with the therapeutic agent and/or the therapeutic procedure, and/or following administration of the therapeutic agent and/or the therapeutic procedure.

Therapeutic agents that may be administered in combination with an RNA interfering agent, or a pharmaceutical composition thereof, may be selected among immunosuppressive drugs (such as methotrexate, mycophenolyate, mofetil, cyclophosphamide and cyclosporine), tocilizumab (an anti-IL-6 receptor antibody), rituximab (an anti-CD20 antibody), and fresolimumab (an anti-TGF-0 antibody, which show promising clinical outcomes.

Alternatively, or additionally, the RNA interfering agent, or a pharmaceutical composition thereof, may be administered in combination with a therapeutic procedure used in the treatment of skin fibrosis, such as ultraviolet phototherapy. Other Fibrotic Diseases Fibrosis. In certain embodiments, the fibrotic disease to be prevented and/or treated using a method described herein is pancreas fibrosis, cardiac fibrosis, ocular fibrosis, or bone marrow fibrosis.

Chronic pancreatitis is a progressive inflammatory disease of the pancreas, characterized by irreversible morphologic changes and gradual fibrotic replacement of the gland. Loss of exocrine and endocrine function results from parenchymal fibrosis. The primary symptoms of chronic pancreatitis are abdominal pain and maldigestion. Grossly, the pancreas may be enlarged or atrophic, with or without cysts or calcifications or tumors. The ducts may be dilated, irregular, or strictured. Essential pathologic features include irregular and patchy loss of acinar tissue, chronic inflammation, ductal changes, and fibrosis. The gross changes are end-manifestations of complex pathogenic mechanisms that are associated with gene mutations (including, but not limited to, cystic fibrosis, cationic trypsinogen gene, CFTR gene mutations in idiopathic acute and chronic pancreatitis, the pancreatic secretory trypsin inhibitor gene, the chymotrypinogen C gene and the calcium sensing receptor gene, alpha- 1 antitrypsine deficiency), metabolic (alcoholic, tobacco smoking, hypercalcemia, hyperlipidemia, chronic renal failure), environmental factors (nutritional factors such as micronutrient deficiencies (zinc, copper and selenium; also by radiation exposure), obstructive (tumors), ischemic (vascular diseases), and autoimmune or associated with primary sclerosing cholangitis, Sjogren’s syndrome, primary biliary disorder and type 1 diabetes mellitus.

Cardiac fibrosis, or heart fibrosis, a hallmark of heart disease, is thought to contribute to sudden cardiac death, ventricular tachyarrhythmia, left ventricular (LV) dysfunction, and heart failure. Cardiac fibrosis is characterized by a disproportionate accumulation of fibrillated collagen that occurs after myocyte death, inflammation, enhanced workload, hypertrophy, and stimulation by a number of hormones, cytokines, and growth factors. Cardiac fibrosis may also refer to an abnormal thickening of the heart valves due to inappropriate proliferation of cardiac fibroblasts but more commonly refers to the proliferation of fibroblasts in the cardiac muscle. Cardiac fibrosis can be the result of a wide array of chronic or acute injury such as coronary artery disease with myocardial infarction, myocarditis or arterial hypertension. Fibrocyte cells normally secrete collagen, and function to provide structural support for the heart. When over-activated this process causes thickening and fibrosis of the valve, with white tissue building up primarily on the tricuspid valve, but also occurring on the pulmonary valve. The thickening and loss of flexibility eventually may lead to valvular dysfunction and right-sided heart failure. Stopping the stimulatory drug or production of serotonin is sometimes used for cardiac valve fibrosis or fibrosis in other cardiac locations. Surgical tricuspid valve replacement for severe stenosis (blockage of blood flow) may be necessary in some patients. Also, a compound found in red wine, resveratrol, has been found to slow the development of cardiac fibrosis (Olson et al., Am. J. Physiol. Heart and Circulatory Physiology, 2005, 288 (3): Hl 131-1138; and Aubin et al., The Journal of Pharmacology and Experimental Therapeutics, 2008, 325 (3): 961-968). However, there is no medication on the market to prevent or treat cardiac fibrosis.

Ocular fibrosis is a complex biological process responsible for the pathogenesis or treatment failure of many blinding eye diseases, including corneal and conjunctival scarring, open-angle glaucoma and failure of glaucoma filtration surgery (GFS), fibrosis in the lens capsule post-cataract surgery, scarring in the tissue around the extraocular muscles in the strabismus surgery, subretinal fibrosis in neovascular age-related macular degeneration (nAMD), fibrovascular proliferative tissue in diabetic retinopathy, and failure of retinal detachment surgery due to proliferative vitreoretinopathy. In these conditions, the main components of the disease process include inflammation, fibroblast activation and extracellular matrix (ECM) accumulation, and resultant tissue contraction. Moreover, deficiency in the limbal stem cells is involved as it leads to the formation of vascularized scar tissue of conjunctival origin on the corneal surface. To date, the only available treatments are antimetabolite drugs that have significant potentially binding side effects, such as tissue damage and infection.

Bone marrow fibrosis, or myelofibrosis, is characterized by the increased deposition of reticulin fibers and in some cases of collagen fibers. The scarring in the bone marrow prevents the normal production of blood cells in the bone marrow. Myelofibrosis may lead to anemia, weakness, fatigue, and splenomegaly. There are a number of hematologic and non- hematologic disorders that are associated with increased bone marrow fibrosis, including but not limited to, primary and secondary myelofibrosis, chronic myeloid leukemia, acute myeloid leukemia, acute lymphocytic leukemia, Hodgskin lymphoma, non-Hodgskin lymphoma, multiple myeloma, systemic lupus erythematosus, systemic sclerosis, and Sjogren syndrome. There is not one treatment that is effective for all myelofibrosis patients, as patients have varying symptoms and circumstances that require different treatment options. There is no drug therapy that can cure bone marrow fibrosis. The only potential cure for myelofibrosis is allogeneic stem cell transplantation. But this procedure is risky for older patients and those with other health problems. For most patients with myelofibrosis, who are primarily older adults, stem cell transplantation is not an option, and treatment remains aimed at controlling disease symptoms and complications, enhancing quality of life and extending survival.

B. Administration

An RNA interfering agent described herein (optionally associated with a delivery system and/or after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients), in a desired dosage, can be administered to a subject in need thereof by any suitable route. Methods of administration include, but are not limited to via absorption, adsorption, aerosol, buccal, dermal, inhaling, intracentricular, intracranial, intradermal, intramuscular, intranasal, intraocular, intrapulmonary, intravenous, intraperitoneal, intrastemal, intrathecal, intraventricular, nasal, ocular, oral, optic, parenteral, patch, rectal, systemic, subcutaneous, sublingual, topical, or transdermal, or vaginal administration, for example. As will be appreciated by those of ordinary skill in the art, in embodiments where an inventive RNA interfering agent is administered in combination with an additional therapeutic agent, the RNA interfering agent and therapeutic agent may be administered by the same route e.g., intravenously) or by different routes (e.g, intravenously and orally).

C. Dosage

An RNA interfering agent described herein (optionally associated with a delivery system and/or after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients), will be administered in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation and dosage administered will depend on the therapeutic effect desired, the severity of the condition to be treated if already present, the presence of any infection, the age, sex, weight, and general health condition of the patient as well as upon the potency, bioavailability, and in vivo halflife of the RNA interfering agent or composition used, the use (or not) of concomitant therapies, and other clinical factors. These factors are readily determinable by the attending physician in the course of the therapy. Alternatively, or additionally, the dosage to be administered can be determined from studies using animal models (e.g., chimpanzee or mice). Adjusting the dose to achieve maximal efficacy based on these or other methods are well known in the art and are within the capabilities of trained physicians. As studies are conducted using RNA interfering agent according to the present invention, further information will emerge regarding the appropriate dosage levels and duration of treatment.

A treatment according to the present invention may consist of a single dose or multiple doses. Thus, administration of an RNA interfering agent described herein, or of a pharmaceutical composition thereof, may be constant for a certain period of time or periodic and at specific intervals, e.g., hourly, daily, weekly (or at some other multiple day interval), monthly, yearly (e.g., in a time release form). Alternatively, the delivery may occur at multiple times during a given time period, e.g., two or more times per week; two or more times per month, and the like. The delivery may be continuous delivery for a period of time, e.g., intravenous delivery.

In general, the RNA interfering agent, or pharmaceutical composition thereof, administered will preferably be in the range of about 1 ng/kg to about 100 mg/kg body weight of the subject, for example, between about 100 ng/kg and about 50 mg/kg body weight of the subject; or between about 1 pg/kg and about 10 mg/kg body weight of the subject, or between about 100 pg/kg and about 1 mg/kg body weight of the subject.

Ill - Pharmaceutical Compositions

As mentioned above, an RNA interfering agent according to the present invention may be administered per se or as a pharmaceutical composition. Accordingly, the present invention provides pharmaceutical compositions comprising an effective amount of an RNA interfering agent described herein (optionally associated with a delivery system) and at least one pharmaceutically acceptable carrier or excipient, for use in the prevention and/or treatment of fibrotic diseases, as defined above. In some embodiments, the composition further comprises one or more additional biologically active agents.

The RNA interfering agents or pharmaceutical compositions may be administered in any amount and using any route of administration effective for achieving the desired prophylactic and/or therapeutic effect. The optimal pharmaceutical formulation can be varied depending upon the route of administration and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered active ingredient.

The pharmaceutical compositions of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “unit dosage form”, as used herein, refers to a physically discrete unit of an RNA interfering agent described herein, for the patient to be treated. It will be understood, however, that the total daily dosage of the compositions will be decided by the attending physician within the scope of sound medical judgement.

A. Formulation

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 2,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid may also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.

Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be administered by, for example, intravenous, intramuscular, intraperitoneal or subcutaneous injection. Injection may be via single push or by gradual infusion. Where necessary or desired, the composition may include a local anesthetic to ease pain at the site of injection.

In order to prolong the effect of an active ingredient (here an RNA interfering agent), it is often desirable to slow the absorption of the ingredient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingredient may be accomplished by dissolving or suspending the ingredient in an oil vehicle. Injectable depot forms are made by forming micro-encapsulated matrices of the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active ingredient to polymer and the nature of the particular polymer employed, the rate of ingredient release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions which are compatible with body tissues. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and pressurized compositions. In addition to the RNA interfering agent, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilising agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, suspending agents, preservatives, sweetening, flavouring, and perfuming agents, thickening agents, colors, viscosity regulators, stabilizes or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potentially containing additives as above, e.g., cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

In certain embodiments, it may be desirable to administer an inventive composition locally to an area in need of treatment (e.g., the liver, the lung, the kidney or the skin). This may be achieved, for example, and not by way of limitation, by local infusion during surgery (e.g., transplantation), topical application, by injection, by means of a catheter, by means of a stent or other implant or yet by means of an inhaler.

For topical administration, the composition may preferably be formulated as a gel, an ointment, a lotion, or a cream which can include carriers such as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oil. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurat (5%) in water, or sodium lauryl sulphate (5%) in water. Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

Materials and methods for producing various formulations of therapeutic RNA interfering agents are known in the art and may be adapted for practicing the subject invention. B. Additional Biologically Active Agents

In certain embodiments, an RNA interfering agent described herein is the only active ingredient in a pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more biologically active agents. Examples of suitable biologically active agents include, but are not limited to, therapeutic agents such as anti-viral agents, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, kinase inhibitors, signalling inhibitors, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof. Examples of other suitable biologically active agents include the therapeutic agents suitable for the treatment of a fibrotic disease, such as those listed above.

In such pharmaceutical compositions, the RNA interfering agent and additional therapeutic agent(s) may be combined in one or more preparations for simultaneous, separate or sequential administration of the RNA agent interfering and therapeutic agent(s). More specifically, an inventive composition may be formulated in such a way that the RNA interfering agent and therapeutic agent(s) can be administered together or independently from each other. For example, an RNA interfering agent and a therapeutic agent can be formulated together in a single composition. Alternatively, they may be maintained (e.g., in different compositions and/or containers) and administered separately.

C. Pharmaceutical Packs of Kits

In another aspect, the present invention provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of an RNA interfering agent described herein.

Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Pharmaceutical packs or kits may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale.

In certain embodiments, a pharmaceutical pack or kit includes one or more additional therapeutic agent(s) as described above. Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of treatment disclosed herein.

An identifier, e.g., a bar code, radio frequency, ID tags, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.

Examples

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text is not intended to suggest that experiments were actually performed, or data were actually obtained.

Example 1: Targeting CLDN1 by GalNac SiRNA to Treat Liver Fibrosis in a Human Liver Chimeric Mouse Model

Materials and Methods

Human Subjects and Patient Cohorts. For transcriptomic analyses and ex vivo perturbation studies, human liver tissue samples were obtained from patients who had undergone liver resections for HCC, colorectal cancer metastasis, or cholangiocellular carcinoma between 2014 and 2022 at Strasbourg University Hospitals, France (DC-2016- 2616 and RIPH2 LivMod IDRCB 2019-A00738-49, Clinical Trial NCT04690972). All patients provided written informed consent and the protocol followed the ethical principles of the declaration of Helsinki and was approved by the local independent ethics committees. Datasets of clinical cohorts with chronic liver disease (GSE34798, GSE83148, GSE49541), chronic kidney disease (GSE11585 and GSE60685), IPF (GSE2052, GSE53845, GSE24206) were selected following comprehensive database analysis, where CLDN1 was identified as part of the microarray data. GSE34798, GSE83148, GSE49541, GSE34798, GSE115857, GSE53845 and GSE24206 were analyzed using shinyGEO (Dumas et al., Bioinformatics, 2016, 32(23): 3679-3681) with expression values shown as log2 expression. All other microarray data were analyzed using signal intensity values. Liver scRNA-seq data (GSE124395 and GSE136103) and snRNA-seq data (GSE185477) were obtained as Seurat objects and investigated. CLDN1 knockdown using GalNAc technology. The following siRNA sequences were designed to target human CLDN1 : siRNA CLDN1 : 5 ’-UAACAUUAGGACCUUAGAAUU-3 ’ (SEQ ID NO: 3) or used as non-targeting siRNA (CTRL): siRNA CTRL. 5 ’ -UAAGGCUAUGAAGAGAUAC-3 ’ (SEQ ID NO: 4).

Each siRNA was then coupled to GalNac group (Creative Biogene Inc.) for in vivo delivery. To validate the efficacy of GalNac siRNA, Huh7 cells were transfected with 12 pmol of regular siRNA or GalNac siRNA and the corresponding controls (CTRLs) by using lipofectamine RNAi Max (Invitrogen, Cat#13778-150) following the manufacturer’s instructions. Knock-down was validated by detecting CLDN1 at the cell surface by flow cytometry using CLDN1 -specific antibodies.

Humanized NASH liver fibrosis mouse models and GalNAc siRNA in vivo knockdown. All experiments were performed at the animal facility of Inserm Ul i 10 according to local laws and ethics committee approval (institutional protocol approval number APAFiS #3559, #7216 and #32429). The mice were housed in individually ventilated cages with 12h/12h light/dark cycles and ad libitum access to food and water. Humanized liver breeding mice were kept at the Inserm Unit 1110 SPF animal facility and maintained with 16 mg/L of 2-(2-nitro-4-trifluoro-methyl-benzoyl)-l,3 cyclohexanedione (NTBC; Swedish Orphan Biovitrum) in drinking water. Six-week-old mice were intravenously injected with 1.5 x 10⁹ plaque forming units (pfu) of an adenoviral vector encoding the secreted form of the human urokinase-like plasminogen activator (Ad-uP A) (Azuma et al., Nature Biotechnol., 2007, 25(8): 903-910). Forty-eight hours later, 10⁶ PHH were injected intrasplenically via a 27-gauge needle. For the procedure, the mice were kept under gaseous isoflurane anesthesia and received a subcutaneous injection of buprenorphine at the dose of 0.1 mg/kg. After transplantation, NTBC administration was gradually decreased and completely withdrawn in 7 d. Transplant success was evaluated 2 months after the procedure by dosing human albumin in mouse serum as previously described (Mailly et al., Nature Biotechnol., 2015, 33(5): 549-554). For the GalNAc siRNA in vivo knockdown study, mice successfully transplanted with PHH were fed with CDA-HFD for 12 weeks followed by subsequent subcutaneous injections of in vitro validated siRNA targeting the human CLDN1 or siCTRL (3 mg/kg/week) for 8 weeks. Mouse Liver Tissue Staining Analysis

Histological Analysis. All organs were immediately fixed in a 10% formalin solution after harvesting and subsequently included in paraffin. Liver slices stained with hematoxylin & eosin (H&E) and Sirius Red were obtained for all mice. For immunohistochemistry staining, the following antibodies were used: Fibronectin-1 (RRID: AB_732380, #ab45688, Abeam) or FAH (RRID: AB_2678806, #HPA044093, Sigma). Staining quantification was performed on entire histological slides or on 5 to 10 consecutive images at lOx or 20x magnification per staining. Images were analyzed using Imaged software vl .51 j 8 (Rasband W, National Institutes of Health, USA) or QuPath version 0.3.2 (Bankhead et al., Sci. Rep., 2017, 7(1): 16878).

For the collagen proportional area quantification in humanized areas, two consecutive liver cuts were stained with FAH and Sirius Red. The corresponding FAH-positive area in the Sirius Red histological slide was selected as region of interest and then the collagen proportional area quantified using Imaged software (Schneider et al., Nature Methods, 2012, 9(7): 671-675). For quantification of other immunostaining in representative humanized area, the humanized areas were selected from the hematoxylin staining based on the phenotype of human hepatocytes showing a brighter cytoplasm and different nucleus and cytoplasm size compared to mouse ones (Azuma etal., Nature Biotechnol., 2007, 25(8): 903-910).

CLDN1 Co-immunoprecipitation. For co-immunoprecipitation (co-IP), 5.10⁸ Huh7 cells (RRID: CVCL-0336) were harvested by scraping in cold PBS ^/_ and plasma membranes were extracted by using Plasma Membrane Protein Extraction Kit (Abeam ab65400) according to manufacturers’ instructions. Approximatively 100 pg of plasma membranes were resuspended in 250 pL of co-IP buffer (10 mM Tris HC1 pH 7.4, .15 M NaCl, 1 mM EDTA, 1 mM EGTA pH 8, 0.1% NP40, glycerol 10% in H2O milliQ water supplemented with protease inhibitors). For WB analysis, 50 pL of the total plasma membrane proteins were conserved and used as “Input” control. In parallel, 50 pL of magnetic Dynabeads (ThermoFisher) were coupled to 2 pg of CLDN1 antibody targeting the C-terminus part of the protein to capture membrane interactants (rabbit anti -human CLDN1 antibody, ab211737, Abeam) or CTRL antibody (Recombinant Rabbit IgG, monoclonal Isotype Control, RRID: AB_2687931, abl72730, Abeam) 10 minutes at room temperature. Dynabeads were then mixed with 150 pL of co-IP buffer and 100 pL of plasma membranes and incubated at 4°C on an orbital shaker. Last, Dynabeads were harvested using a magnet (the “Flow-through” was conserved for WB analysis) and washed 3 times with cold PBS ^A. CLDN1 interactants were eluted in IX Laemmli buffer by heating the beads 5 minutes at 95°C. CLDN1 IP was validated by WB analysis before analyzing the interactants by mass-spectrometry (IGBMC proteomic platform, Illkirch-Graffenstaden).

Single-Cell and Single Nucleus RNAseq Analyses.

Single-cell (sc) RNAseq data'. Based on the clustering and data normalization of the whole human liver cell atlas (GSE124395) (Aizarani et al., Nature, 2019, 572(7768): 199- 204), CLDN1 expression was analyzed in different cell types. Thereby, RacelD methods were used to draw expression t-SNE maps and ggplot2 (Wickham, ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York (2016)) to draw corresponding boxplots of normalized expression values. Based on the clustering and provided data of the fibrotic liver cell atlas (GSE136103), CLDN1 expression was analyzed in different cell types. Seurat (Hao et al., Cell, 2021, 184(13): 3573-3587) was used to generate expression UMAPs and violin plots, and ggplot2 to generate correlation figures.

Single nucleus (sn) RNAseq data. Based on the clustering of the whole snRNA-seq data set as previously published (Azuma et al., Nature Biotechnol., 2007, 25(8): 903-910), CLDN1 expression was analyzed using Seurat (Hao et al, Cell, 2021, 184(13): 3573-3587). All epithelial cell clusters (hepatocytes, cholangiocytes and bipotent progenitor cells) were extracted and a pseudotime analysis was performed applying slingshot (Street et al., BMC Genomics, 2018, 19(1): 477). Cell types were annotated based on specific marker gene expression including ALB (hepatocytes), EPCAM (bipotent progenitor cells), and CK19 (cholangiocytes). An expression plot overlay ed with the resulting pseudotime trajectories was generated using ggplot2 (Wickham, ggplot2: Elegant Graphics for Data Analysis. Springer- Verlag New York (2016)).

RNAi Treatment Studies in A549 Lung Cells and Renal Epithelial Cells (REpiC) Cells Modeling Fibrotic Pathways and Disease.

A549 cells purchased from ATCC and were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 supplemented with 10% heat-decomplemented fetal bovine serum and gentamycin (0.05 mg/mL) at 37°C with 5% CO2.

REpiC primary cells were purchased from ScienCell Research Laboratories and cultured on poly-L-lysine coated plates in Epithelial Cell Medium supplemented with epithelial cell growth supplement, 2% of FBS and 1% penicillin/streptomycin (ScienCell Research Laboratories ) at 37°C with 5% CO2 For RNAi treatment studies, cells were transfected with siRNA targeting CLDN1 expression or non-targeting siRNA CTRL using Lipofectamine RNAi Max according to the manufacturer instructions. After 48 hours, cells were lysed and RNA extracted using ReliaPrep(TM) RNA Cell Miniprep System (Promega) for qRT-PCR assay.

Real-Time qRT-PCR. cDNAs were synthetized by reverse transcription using SuperScript III First-Strand Synthesis SuperMix (Life Technologies). Expression of ACTA2, COLIAI, TGFB1 and TNFA was analyzed by quantitative real-time PCR using iTaq™ Universal SYBR® Green Supermix (Bio-Rad). Expression of CLDN1, CDH 1, TWIST, VIM, CD44, FN1 and SNAI was analyzed by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific). qRT-PCR assays were performed on Applied Biosystems™ Kit QuantStudio 3 PCR system (Thermo Fisher Scientific). The 2-ACT method was applied for relative quantification of mRNA with normalization to either 18S (TaqMan) or GAPDH (SYBR) mRNA.

Results

CLDN1 Expression is Associated with Liver Fibrosis and Disease Progression. CLDN1 gene expression was first analyzed in liver tissues of patients with chronic liver disease (HBV, HCV, or NASH) in several independent cohorts from Gene Expression Omnibus (GEO) and in a NASH cohort from the University of Strasbourg. CLDN1 is upregulated in liver tissue of patients with liver disease of all major etiologies (Figure 1(A)). CLDN1 expression is associated with fibrotic disease progression in patients with NASH and HCV-infected individuals post transplantation (Rasmussen et al., Hepatology, 2012, 56(1): 17-27) (Figure 1(B)). Corroborating a pathogenetic role of CLDN1 for liver fibrosis, CLDN1 was found to be the most highly expressed CTDN family member in fibrotic liver and the only family member significantly upregulated in fibrosis (data not shown).

CLDN1 expression was then investigated in the liver at the single-cell level. Comprehensive expression studies including analysis of single cell (sc) and single nucleus (sn) RNA-seq data (Figure l(C-E), fig. S2A-D) showed that CLDN1 was highly expressed by hepatocytes and cholangiocytes (p<0.0001, U-test, respectively) with increasing expression towards liver bipotent progenitor cells (Andrews et al., Hepatol. Commun. 2022, 6(4): 821- 840) (Figure 1(D)). Within the liver microenvironment, activated hepatic stellate cells (aHSCs)Zhuman liver myofibroblasts (HLMFs) robustly expressed CLDN1 (Figure 1(C)). In contrast, CDLN1 abundance in liver endothelial cells, monocytes/macrophages and lymphocytes appeared low or absent (Figure 1(C), Figure 1(E)). CLDN1 expression in hepatocytes, cholangiocytes, progenitor cells and fibroblasts was confirmed at the protein level by in situ hybridization (ISH), immunohistochemistry (IHC) and double color immunofluorescence (data not shown).

In the fibrotic liver, scRNA analyses revealed that CLDN1 was differentially expressed in a subpopulation of diseased hepatocytes at the epithelial-stromal interface (Figure 1(E)) and correlated with grade of de-differentiation towards a progenitor or bile-duct like cell type (Ramachandran et al., Nature, 2019, 575(7783): 512-518) (p=3.75 x 10’⁸). Within the mesenchymal cell compartment in fibrotic liver, CLDN1 was highly expressed by mesothelial cells (p<0.0001, U-test, GSE136103, Figure 1(E)) that have previously been described as fibrosis-associated fibroblast progenitor cells (Li et al., Proc. Natl. Acad. Sci. USA, 2013, 110(6): 2324-2329; Buechler etal., Nature, 2021, 593(7860): 575-579).

Taken together, these studies show that CLDN1 is robustly expressed in hepatocytes, liver progenitor cells, cholangiocytes, mesenchymal cells including aHSCs/HLMFs and mesothelial cells. The up-regulation of CLDNl expression in fibrotic liver and its association with disease progression among different etiologies suggests a functional role in liver fibrosis.

In vivo Knockdown of CLDNl using GlaNac siRNA Technology Reduces Liver Fibrosis. To assess CLDNl as a therapeutic target using RNAi -based therapies, the effect of in vivo CLDNl knockdown was next assessed in a patient-derived human liver chimeric mouse model that expresses human CLDNl and closely recapitulates key features of clinical liver fibrosis based on Fah ^/ /Rag2 ^/ /Il2rg^ (FRG)-NOD mice robustly repopulated with primary human hepatocytes (Azuma et al., Nature Biotechnol., 2007, 25(8): 903-910). To model NASH-induced advanced fibrosis, a well-established long-term choline-deficient, L- amino acid-defined, high fat diet (CDA-HFD) (Kishida et al., BMC Gastroenterol., 2016, 16(1): 61) was applied. For hepatocyte-specific in vivo knockdown of human CLDNl, CLDNl specific siRNAs covalently linked to a ligand containing three A'-acetylgalactosamine (GalNAc) residues (Debacker et al., Mol. Then, 2020, 28(8): 1759-1771) (Figure 2).

After 12 weeks of diet (Figure 3(A)), mice were randomized to 2 groups receiving either siRNA targeting CLDNl or control siRNA at 3mg/kg/week for 8 weeks. GalNAc siRNAs were found to reduce CLDNl protein expression in Huh7 liver cells and in vivo (data not shown). Mice treated with CLDN1 -targeting GalNAc siRNA showed significantly reduced fibrosis compared to mice treated with GalNac control siRNA (Figure 3(B-C)). Moreover, mice in the CLDN1 knockdown group developed significantly fewer liver nodules (Figure 3(B-C), p=0.044).

Taken together, these data suggest that CLDN1 plays a functional role in liver fibrosis.

Mechanistic Studies. To study the molecular events mediating the fibrotic effects of CLDN1, the molecular partners of CLDN1 were identified at the membrane of epithelial cells using Huh7 cells with high CLDN1 expression as a tractable model for immature liver epithelial cells. Co-immunoprecipitation of CLDN1 identified more than 300 proteins as potential CLDN1 interactants. String analysis with clustering revealed 3 clusters of proteins interacting with CLDN1 (Figure 4(A)): cell adhesion proteins, integrins and ECM proteins as well as proteins related to cell proliferation and fate. Epithelial growth factor receptor (EGFR), EPCAM, the ECM receptor integrin alpha 5 (ITGA5), and ECM component laminin 5 (LAMA5) were confirmed as molecular partners by Western blot analyses (Figure 4(B)).

Collectively, the data obtained indicate that CLDN1 interacts with EPCAM, EGFR, ITGA5, laminin, and other proteins at the cell membrane of liver epithelial cells which have been shown to play a pathogenic role in fibrosis and cancer. Reduction of CLDN1 expression may inhibit downstream profibrogenic and pro-carcinogenic signaling mediating cell plasticity, fibrogenesis, and carcinogenesis.

CLDN1 is a Candidate Target for RNAi-based Treatment of Lung and Kidney Fibrosis. The mechanistic role of CLDN1 during fibrosis is not necessarily limited to the liver. Several studies have suggested a role of CLDN1 in the pathogenesis of chronic kidney disease (Hasegawa et al., Nature Med., 2013, 19(11): 1496-1504). Upregulation of CLDN1 expression in patients with glomerulonephritis as well as murine fibrotic kidneys (Lovisa et al., Nature Med., 2015, 21(19): 998-1009) (Figure 5(A)) suggests the involvement of CLDN1 in the pathogenesis of renal fibrotic disease. Furthermore, CLDN1 was significantly overexpressed in patients with idiopathic pulmonary fibrosis (IPF) with increasing expression along disease progression (p=0.03, Figure 5(B)).

Discussion

In this study, the present Inventors uncovered CLDN1 as a mediator and therapeutic target for tissue fibrosis. Using the liver as a model of chronic inflammation-associated fibrogenesis and carcinogenesis they showed that targeting CLDN1 by GalNac siRNA robustly reduced fibrosis and tumor development in a mouse model for MASH (also known as NASH).

Their comprehensive analyses provide the following model for the molecular mechanism of CLDN1 mediated anti-fibrotic effects: hepatocytes and their progenitors are the primary target cells of CLDN1 -targeting RNAi antifibrotic effect. Reduction of CLDN1 by siRNA may interfere with the interaction of CLDN1 with EPCAM, EGFR, ITGA5 as well as the ECM, potentially resulting in inhibition of downstream signaling of well-established pro- fibrotic and pro-carcinogenic signaling pathways across organs, such as SRC and MAPK signaling.

The large majority of liver disease therapeutics target metabolism, inflammation or cell death, which are relevant in the early stage of disease (Mullar, Nature Rev. Drug Discov., 2020, 19(8): 501; Newsome et al., New Engl. J. Med., 2021, 384(12): 1113-1124). A key differentiator of CLDN1 siRNA is the combination of robust anti-fibrotic and tumor preventive effect, which addresses the key unmet medical need in advanced liver fibrosis.

Beyond the liver, the expression data (Figure 5) suggest that CLDN1 is also a candidate target for kidney and lung fibrosis (Richeldi et al., Eur. Respir. Rev., 2018, 27(150): 180074; Ruiz-Ortega et al., Nature Rev. Nephrol., 2020, 16(5): 269-288). Collectively, the development of RNAi-based provides an opportunity for the clinical development of a first- in-class compound for the treatment of fibrotic diseases, a major and rapidly growing unmet medical need world-wide.

Example 2: Liposome-mediated Delivery of CLDN1 RNAi into Lung and Kidney Epithelial Cells to Treat Lung and Renal Fibrosis

Results

Treatment with CLDN1 RNAi Reduces Expression of Drivers and Mediators of Lung Fibrosis Including Collagen and TGF0 Expression as well as Epithelial- Mesenchymal Transition (EMT). Idiopathic pulmonary fibrosis (IPF) is a chronic and inflammatory fibrotic lung disease leading to respiratory failure and ultimately to death (Richeldi et al., Lancet, 2017, 389: 1941-1952). Previously, the present Inventors have observed that CLDN1 is significantly overexpressed in patients with IPF with increasing expression along disease progression demonstrating that CLDN1 is a therapeutic target for IPF (Roehlen et al., Sci. Transl. Med., 2022, 14: eabj4221) (Figure 5). Although the pathogenic mechanisms of lung fibrosis remain to be fully elucidated, functional studies have suggested that fibroblasts, an important contributor and mediator of lung fibrosis, may be derived from alveolar epithelial cells by epithelial-mesenchymal transition (EMT) (Rout-Pitt et al., Respir. Res., 2018, 19: 136).

To assess CLDN1 as a therapeutic target using RNAi-based therapies for lung fibrosis, the Inventors performed perturbation studies using liposome-mediated delivery of CLDN1- specific siRNA in A549 cells derived from human adenocarcinoma alveolar basal epithelial cells (Lee et al., Clin. Cancer Res., 2013, 19: 5879-5889), which model key pathways of lung fibrosis. Treatment with CLDN1 RNAi decreases expression of the key pro-fibrogenic mediators collagen 1 (COL1A1) and TGFB1. Collagen-1 is a key component of the fibrosis extracellular matrix and TGF0 is recognized as a key mediator and driver across organs (Frangogiannis et al., J. Exp. Med., 202, 217: e20190103). Moreover, treatment with CLDN1 RNAi was found to significantly reduced expression of ACTA2, coding for alpha smooth muscle actin (ocSMA) - a marker significantly associated with EMT-induced fibrosis in IPF (Figure 6) (Rout-Pitt et al., Respir. Res., 2018, 19(1): 136; Alipio et al., Differentiation, 2011, 82: 89-98. Moreover, the upregulation of ocSMA is also a marker for activation of myofibroblasts, which are key mediators of fibrosis across organs.

EMT of epithelial cells has been shown to be an important mediator of fibrosis across organs (Liu et al., Burns Trauma, 2022, 10:tkac011). Indeed, alveolar epithelial cells undergoing mesenchymal transition express several other secreted profibrotic factors and are capable of activating lung fibrosis (Yang et al., Am. J. Pathol., 2013, 183: 1559-1770). Therefore, the Inventors have studied the effect of CLDN1 RNAi treatment on EMT markers. Treatment with CLDN1 RNAi was found to decrease the expression of EMT markers and transcription factor TWIST and vimentin VIM and to increase the expression of E-Cadherin (CDH1), an epithelial marker (Figure 6) demonstrating that treatment with CLDN1 RNAi inhibits EMT implicated in fibrosis.

Together, these functional data obtained in a human lung epithelial cell-based model confirm that targeting CLDN1 expression using RNAI provides a previously undiscovered therapeutic approach for lung fibrosis, including IPF or cystic fibrosis by inhibiting the expression of key profibrogenic pathways such as collagen production, TGF0, ocSMA and EMT.

CLDN1 RNAi-based Treatment of Glomerular Kidney Disease and Kidney Fibrosis. Glomerular diseases are the leading causes of end-stage kidney disease. Glomerular cells and in particular parietal epithelial cells (PECs) activation, migration and proliferation are involved in glomerulosclerosis by either producing excessive extracellular matrix (ECM) proteins or by accumulating and leading to crescent formation (Kuppe et al., Kidney Int., 2019, 96; 80-93). CLDN1 is a well-described marker of activated PECs (Yu, J. Am. Soc. Nephrol., 2015, 26: 11-19). Previously, the present Inventors observed that CLDN1 is significantly overexpressed in patient with kidney disease (Roehlen et al., Sci. Transl. Med., 2022, eabj4221) (Figure 5). Therefore, they investigated CLDN1 as therapeutic target for glomerular disease using RNAi technology (Figure 7). As a cell-based model for PECs, they used the Human Renal Epithelial Cells (HREpic), which are primary cells isolated from human kidney recapitulating PECs functions. Treatment with CLDN1 RNAis delivered by liposomes was found to lead to a decrease in CD44 expression, a marker associated with disease progression. Moreover, CLDN1 KD reduced ECM component expression (fibronectin, FN1, and COL1A1) and inflammation markers (TNFA), as well as expression of SNAI and TGFB1, two actors involved in EMT, fibrosis, and crescent formation (Kuppe et al., Kidney Int., 2019; 96(1): 80-93) (Figure 7).

Together, these functional data in a human renal cell-based model confirm that targeting CLDN1 expression using RNAi is a new therapeutic approach for glomerular disease and kidney fibrosis.

Claims

Claims What is claimed is:

1. An RNA interfering agent targeting CLDN1 for use in the prevention and/or treatment of a fibrotic disease in a subject.

2. An RNA interfering agent targeting CLDN1 for the use according to claim 1, wherein the fibrotic disease affects the liver, lung, kidney, heart, skin, pancreas, intestine, eye, nerve system, mediastinum, retroperitoneum, joint or tendon, in particular wherein the fibrotic disease is hepatic fibrosis, pulmonary fibrosis or renal fibrosis.

3. An RNA interfering agent targeting CLDN1 for the use according to claim 2, wherein the fibrotic disease is hepatic fibrosis.

4. An RNA interfering agent targeting CLDN1 for the use according to claim 3, wherein the hepatic fibrosis is associated with hepatitis B, C, D or E infection, alcoholic liver disease, primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver disease, obesity, nonalcoholic steatohepatitis, cystic fibrosis, hemochromatosis, or autoimmune hepatitis.

5. An RNA interfering agent targeting CLDN1 for the use according to claim 2, wherein the fibrotic disease is pulmonary fibrosis, and the pulmonary fibrosis is one of: idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonitis (NSIP), cryptogenic organizing pneumonia (COP), Hamman-Rich syndrome, lymphocytic interstitial pneumonitis (LIP), respiratory bronchiolitis interstitial lung disease, desquamative interstitial pneumonitis or idiopathic lymphoid interstitial pneumonia, and idiopathic pleuroparenchymal fibroelastosis.

6. An RNA interfering agent targeting CLDN1 for the use according to claim 2, wherein the fibrotic disease is renal fibrosis, and the renal fibrosis is renal interstitial fibrosis or glomerular disease such as glomerulosclerosis or ANCA vasculitis or RPGN or diabetic kidney disease.

7. An RNA interfering agent targeting CLDN1 for the use according to any one of claims 1 to 6, wherein the RNA interfering agent is a single-stranded RNA interfering molecule or a double- stranded RNA interfering molecule. An RNA interfering agent targeting CLDN1 for the use according to claim 7, wherein the RNA interfering agent is a siRNA, a shRNA, a micro-RNA or an aiRNA An RNA interfering agent targeting CLDN1 for the use according to claim 8, wherein the RNA interfering agent is a double-stranded short interfering nucleic acid molecule comprising a first strand comprising a sense region and a second strand comprising an antisense region, wherein the sense region targets a portion of an exon of a CLND1 mRNA sequence, and the antisense region comprises a nucleotide sequence that is complementary to the sense region, wherein the exon of the CLDN1 mRNA sequence is Exon 2 or Exon 4. An RNA interfering agent targeting CLDN1 for the use according to claim 9, wherein the sense region of the RNA interfering agent: targets a portion of Exon 2 of the CLND1 mRNA sequence, wherein the portion consists of the sequence set forth in

SEQ ID NO : 1 : 5 ’ -UGAAGUGUAUGAAGUGCUU-3 ’ ; or targets a portion of Exon 4 of the CLDN1 mRNA sequence, wherein the portion consists of the sequence set forth in

SEQ ID NO: 2: 5’-CACCAAGGCCCUAUCCAAA-3’; or targets a portion of Exon 4 of the CLND1 mRNA sequence, wherein the portion consists of the sequence set forth in

SEQ ID NO : 3 : 5 ’ -UAAC AUUAGGACCUUAGAAUU-3 ’ . An RNA interfering agent targeting CLDN1 for the use according to claim 9, wherein the RNA interfering agent targeting CLDN1 is a double-stranded short interfering nucleic acid molecule comprising a first strand comprising a sense region and a second strand comprising an antisense region, wherein the sense region consists of, or comprises, the sequence set forth in SEQ ID NO: 3 (5’- UAAC AUUAGGACCUUAGAAUU-3’), or a sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity with SEQ ID NO: 3, and the antisense region comprises a nucleotide sequence that is complementary to the sense region. An RNA interfering agent targeting CLDN1 for the use according to claim 10, wherein the sense region consists of the sequence set forth in SEQ ID NO: 3, and the antisense region consists of a nucleotide sequence that is perfectly complementary to the sense region. An RNA interfering agent targeting CLDN1 for the use according to any one of claims 1 to 12, wherein the RNA interfering agent is associated with a delivery system, in particular wherein the RNA interfering agent is conjugated to a N- acetylgalactosamine (GalNAc) moiety or to a molecule comprising at least one GalNAc moiety, or wherein the RNA interfering agent is encapsulated into a lipid nanoparticle (LNP). A pharmaceutical composition comprising an effective amount of an RNA interfering agent targeting CLDN1 and at least one pharmaceutically acceptable carrier or excipient, for the use in the prevention or treatment of a fibrotic disease according to any one of claims 1 to 13. A pharmaceutical composition for the use according to claim 14, wherein the pharmaceutical composition further comprises at least one additional therapeutic agent, wherein the at least one additional therapeutic agent is selected from anti-viral agents, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, kinase inhibitors, signalling inhibitors, antibacterial agents, antibiotics, antioxidants, antiseptic agents, therapeutic agents suitable for the treatment of a fibrotic disease, and combinations thereof. A pharmaceutical composition for the use according to claim 14 or claim 15, wherein the prevention or treatment of the fibrotic disease is associated with a modulation of expression or function of epithelial growth factor receptor (EGFR), EPCAM, ECM receptor integrin alpha 5 (ITGA5), and/or ECM component laminin 5 (LAMA5). An RNA interfering agent targeting CLDN1, wherein the RNA interfering agent is as defined in any one of claims 9 to 12. A pharmaceutical composition comprising an effective amount of an RNA interfering agent targeting CLDN1 according to claim 17 and at least one pharmaceutically acceptable carrier or excipient, and optionally at least one additional therapeutic agent as defined in claim 15. A pharmaceutical composition according to claim 18, wherein said pharmaceutical composition results in modulation of expression or function of epithelial growth factor receptor (EGFR), EPCAM, ECM receptor integrin alpha 5 (ITGA5), and/or ECM component laminin 5 (LAMA5). An antisense oligonucleotide (ASO) targeting CLDN1 for use in the prevention and/or treatment of a fibrotic disease in a subject.