SEQUENCE LISTING STATEMENT

The ASCII file, entitled 81245SequenceListing.txt, created on Feb. 27, 2020 comprising 12,510 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. The sequence listing submitted herewith is identical to the sequence listing forming part of the international application.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No. PCT/IL2018/050964 having International filing date of Aug. 30, 2018, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 62/552,477 filed on Aug. 31, 2017. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods for treating fibrosis and, more particularly, but not exclusively, to liver fibrosis.

Fibrosis is the excess accumulation of extracellular matrix (ECM), resulting from chronic, non-resolving inflammation. This inflammation triggers a wound-healing process that mitigates inflammatory tissue destruction but also leads to scar tissue formation. Multiple etiologies underlie development of liver fibrosis, such as chronic viral hepatitis B or C, autoimmune and biliary diseases, alcoholic steatohepatitis (ASH) and, nonalcoholic steatohepatitis (NASH). Fibrosis progression toward cirrhosis, i.e., replacement of functional parenchyma by scar tissue accompanied by severe architectural and vascular distortion, is the major cause of liver-related morbidity and mortality. Patient with cirrhosis are more prone to develop liver failure, portal hypertension or infection and are at higher risk of developing hepatocellular carcinoma (HCC). Liver fibrosis progression rates vary widely among patients and are thus not predictable.

In the normal liver, hepatic stellate cells (HSCs) constitute quiescent, vitamin A—storing cells. Upon activation by specific stimuli released by an injured liver, HSCs undergo “activation” or “transdifferentiation”, yielding a myofibroblast-like cell, with several new phenotypic characteristics, such as enhanced cell migration and adhesion, expression of αSMA, increased proliferation and production of chemotactic substances capable of recruiting inflammatory cells as well as other HSCs. Stellate cell activation is a tightly programmed response occurring in a reproducible sequence. The early events, known as “initiation”, are associated with transcriptional events and induction of immediate early genes as well as rapid changes in phenotype. These early changes are likely to result from paracrine stimulation by all neighboring cell types, including sinusoidal endothelium, Kupffer cells, hepatocytes, platelets, and leukocytes. “Perpetuation”, the second stage of stellate cell activation, involves several discrete changes in cell behavior. During this stage, pro-inflammatory, pro-fibrogenic and pro-mitogenic stimuli are released, and act in an autocrine and paracrine manner, maintaining the activated phenotype. Sustained activation of the cells involves at least seven discrete changes in cell behavior, i.e., proliferation, chemotaxis, fibrogenesis, contractility, matrix degradation, retinoid loss, and white blood cells chemoattractant/cytokine release, consequently leading to increased accumulation of extracellular matrix. Nevertheless, it has been demonstrated that fibrosis and even cirrhosis can be reversible if the underlying cause is successfully eliminated. Understanding the mechanistic pathways and the regulatory factors driving the development of fibrosis and its regression, may give rise to therapeutic approaches as well as novel diagnostic tools for this syndrome.

The ability of the fibrotic liver to revert to a less fibrotic stage or even to normal architecture has been convincingly demonstrated over the last two decades in both rodents and in patients following successful treatment of various underlying hepatic diseases. However not much is known about the mechanism of the “removal/clearance” of activated myofibroblasts from the liver or their inactivation during fibrosis resolution. Recently two studies have demonstrated that although a small number (˜2.6%) of activated HSCs are cleared during fibrosis resolution via apoptosis, most of them undergo a reversal mechanism back to their quiescent state [Troeger, J. S., et al., Gastroenterology, 2012. 143(4): p. 1073-83 e22; Kisseleva, T., et al., Proc Natl Acad Sci USA, 2012. 109(24): p. 9448-53].

Although there is a similarity between these inactivated HSCs, they are not identical to the quiescent non-activated cells, thus, they are more susceptible to a second round of fibrogenesis stimulation.

Background art includes Kitano and Bloomston, J Clin Med, 2016 March; 5(3):38; Hayes and Chayama, Int. J. Mol. Sci. 2016, 17, 280; doi:10.3390; Lee et al., Scientific Reports|7: 3710|DOI:10.1038/s41598-017-03389-2; Hyun et al., Nature Communications 7:10993|DOI: 10.1038/ncomms10993, Szabo and Bala., Nat Rev Gastroenterol Hepatol. 2013 September; 10(9): 542-552; and Shen et al., J Cell Mol Med. 2017 May; 21(5): 986-992.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one miRNA selected from the group consisting of hsa-miR-452-3p, hsa-miR-224-3p, hsa-miR-378i, hsa-miR-10b-5p, hsa-miR-99a-3p, hsa-miR-105-3p, hsa-miR-224-5p, hsa-miR-10b-3p, hsa-miR-338-5p, hsa-miR-767-5p, hsa-miR-196b-5p, hsa-miR-148a-3p, hsa-miR-452-5p, hsa-miR-873-5p, hsa-miR-873-3p, hsa-let-7a-3p, hsa-miR-208b-3p, hsa-miR-582-3p, hsa-miR-196a-5p, hsa-miR-99b-3p, hsa-miR-320d, hsa-let-7e-3p, hsa-miR-183-5p, hsa-miR-532-5p, hsa-miR-30c-2-3p, novel_mir18, hsa-miR-3529-3p, hsa-miR-3074-5p, hsa-miR-423-5p, hsa-let-7f-1-3p, hsa-let-7c-3p, hsa-miR-378c, hsa-miR-486-5p, hsa-miR-185-3p, hsa-miR-320e, hsa-miR-182-5p, novel_mir78, hsa-miR-340-3p, hsa-miR-24-2-5p, hsa-miR-330-3p, hsa-miR-192-3p, hsa-miR-26a-2-3p, hsa-miR-26b-3p, hsa-miR-99a-5p, hsa-miR-148b-3p, hsa-miR-374a-3p, hsa-miR-30a-3p, novel-mir32, hsa-miR-17-3p, hsa-miR-193b-5p, hsa-let-7i-5p and hsa-miR-19b-3p, thereby treating the disease associated with fibrosis.

According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least two miRNAs selected from the group consisting of hsa-miR-452-3p, hsa-miR-224-3p, hsa-miR-378i, hsa-miR-10b-5p, hsa-miR-99a-3p, hsa-miR-105-3p, hsa-miR-224-5p, hsa-miR-10b-3p, hsa-miR-338-5p, hsa-miR-767-5p, hsa-miR-196b-5p, hsa-miR-148a-3p, hsa-miR-452-5p, hsa-miR-873-5p, hsa-miR-873-3p, hsa-let-7a-3p, hsa-miR-208b-3p, hsa-miR-582-3p, hsa-miR-196a-5p, hsa-miR-99b-3p, hsa-miR-320d, hsa-let-7e-3p, hsa-miR-378a-3p, hsa-miR-183-5p, hsa-miR-532-5p, hsa-miR-30c-2-3p, novel_mir18, hsa-miR-3529-3p, hsa-miR-3074-5p, hsa-miR-423-5p, hsa-let-7f-1-3p, hsa-let-7c-3p, hsa-miR-378c, hsa-miR-486-5p, hsa-miR-185-3p, hsa-miR-320e, hsa-miR-182-5p, novel_mir78, hsa-miR-340-3p, hsa-miR-24-2-5p, hsa-miR-330-3p, hsa-miR-192-3p, hsa-miR-26a-2-3p, hsa-miR-26b-3p, hsa-miR-99a-5p, hsa-miR-148b-3p, hsa-miR-374a-3p, hsa-miR-30a-3p, novel-mir32, hsa-miR-17-3p, hsa-miR-193b-5p, hsa-let-7i-5p and hsa-miR-19b-3p, thereby treating the disease associated with fibrosis.

According to an aspect of some embodiments of the present invention there is provided a method of treating hepatic fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one miRNA selected from the group consisting of miRNA-30c-2-3p, miRNA-423-5p and miRNA-532-5p.

According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of exosomes isolated from a hepatic cell-conditioned medium, thereby treating the disease associated with fibrosis.

According to an aspect of some embodiments of the present invention there is provided a method of selecting an agent useful for treating a disease associated with fibrosis comprising:

(a) contacting the agent with primary hepatic stellate cells; and

(b) analyzing whether the primary hepatic stellate cells differentiate towards myofibroblasts, wherein if the agent prevents differentiation of the primary hepatic stellate cells towards the myofibroblasts, it is indicative that the agent is useful for treating the disease associated with fibrosis.

According to some embodiments of the invention, the miRNA is selected from the group consisting of hsa-miR-452-3p, hsa-miR-224-3p, hsa-miR-378i, hsa-miR-10b-5p, hsa-miR-99a-3p, hsa-miR-224-5p, hsa-miR-10b-3p, hsa-miR-148a-3p, has-let-7a-3p, hsa-miR-196a-5p, hsa-miR-99b-3p, hsa-miR-320d, hsa-let-7e-3p, hsa-miR-1835p, hsa-miR-532-5p, hsa-miR-30c-2-3p, hsa-miR-3529-3p, hsa-miR-3074-5p, hsa-let-7f-1-3p, hsa-let-7c-3p, hsa-miR-486-5p and hsa-miR-423-5p.

According to some embodiments of the invention, the miRNA is selected from the group consisting of miR-196a-5p and miR-378i.

According to some embodiments of the invention, the at least one miRNA is comprised in a particle.

According to some embodiments of the invention, the at least one miRNA comprises miRNA-423-5p and miRNA-532-5p.

According to some embodiments of the invention, the at least one miRNA comprises miRNA-30c-2-3p and miRNA-532-5p.

According to some embodiments of the invention, the particle is a liposome.

According to some embodiments of the invention, the particle is an exosome.

According to some embodiments of the invention, the exosome is secreted from hepatic cells.

According to some embodiments of the invention, the hepatic cells are non-diseased hepatic cells.

According to some embodiments of the invention, the exosome is an isolated exosome.

According to some embodiments of the invention, the miRNA is selected from the group consisting of hsa-miR-452-3p, hsa-miR-224-3p, hsa-miR-378i, hsa-miR-10b-5p, hsa-miR-99a-3p, hsa-miR-224-5p, hsa-miR-10b-3p, hsa-miR-148a-3p, has-let-7a-3p, hsa-miR-196a-5p, hsa-miR-99b-3p, hsa-miR-320d, hsa-let-7e-3p, hsa-miR-378a-3p, hsa-miR-1835p, hsa-miR-532-5p, hsa-miR-30c-2-3p, hsa-miR-3529-3p, hsa-miR-3074-5p, hsa-let-7f-1-3p, hsa-let-7c-3p, hsa-miR-486-5p and hsa-miR-423-5p.

According to some embodiments of the invention, the miRNA is selected from the group consisting of miR-196a-5p, miR378a-3p and miR-378i.

According to some embodiments of the invention, the hepatic cells comprise primary hepatic cells.

According to some embodiments of the invention, the hepatic cells comprise transformed hepatic cells.

According to some embodiments of the invention, the at least one miRNA is comprised in a hepatic cell-conditioned medium.

According to some embodiments of the invention, the at least one miRNA is an isolated miRNA.

According to some embodiments of the invention, the disease is a hepatic disease.

According to some embodiments of the invention, the hepatic disease is selected from the group consisting of hepatitis, an autoimmune hepatic disease, alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH).

According to some embodiments of the invention, the hepatitis is chronic viral hepatitis B or chronic viral hepatitis A.

According to some embodiments of the invention, the analyzing is effected by analyzing the expression of at least one of αSMA and/or Col1A.

According to some embodiments of the invention, the analyzing is effected by analyzing the expression of at least one marker selected from the group consisting of PDGF, TGFβ, TIMP1, TIMP2, αSMA and Col1A.

According to some embodiments of the invention, the analyzing is effected by visualizing the cells using a stain.

According to some embodiments of the invention, the agent is comprised in an exosome secreted from hepatic cells.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

