# Patent

## Title
Modulation of NLGn4 expression, NK cell activity in non-alcoholic fatty liver disease (NAFLD)

## Abstract

The present invention provides a method of treating, attenuating or preventing a liver disorder by inhibiting NGn4 expression and thereby modulating the activity of NK cells. The present invention further relates to diagnosing a liver disorder by evaluating NLGn4 expression in NK cells.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Provisional application Ser. No. 61/884153 filed on Sep. 30, 2013.


FIELD OF THE INVENTION
The present invention relates to the involvement of NK cells in Nonalcoholic-Fatty-Liver-Disease (NAFLD), mediated by a novel Neuroligin-4 (NLGn4) synaptic pathway. The present invention provides compositions and methods for modulating the action of NLGn4 to attenuate Nonalcoholic-Fatty-Liver-Disease (NAFLD).
BACKGROUND OF THE INVENTION
Nonalcoholic fatty-liver disease (NAFLD) is one of the most prevalent liver diseases in western countries. The full pathophysiology of NAFLD is still unknown. Both obesity and insulin resistance are considered to play a strong role in the disease process. Indeed, the rising rates of obesity and diabetes mellitus correlate with the increasing incidence of NAFLD, which is the hepatic and early manifestation of metabolic syndrome. Estimates suggest that about 20% to 30% of adults in developed countries have excess fat accumulation in the liver, 50% among people with diabetes, and about 80% in the obese and morbidly obese individuals.
Non-alcoholic steatohepatitis (NASH) is the most severe form of NAFLD, and can progress to more severe forms of liver disease, including fibrosis progression, cirrhosis, and even hepatocellular carcinoma.
The disease begins with the aberrant accumulation of triglycerides in the liver, resulting in simple steatosis; most patients who develop steatosis are stable and further disease does not develop. However, some individuals progress to NASH, the severe form of NAFLD. In NASH, up to 20% of patients' progress into cirrhosis.
The normal liver is composed of hepatocytes and non-parenchymal cells, which include kupffer cells, sinusoidal endothelial cells, and myofibroblasts known as Hepatic Stellate Cells (HSCs). HSCs are considered to be involved in the pathogenesis of liver fibrosis from any etiology, including NASH-related hepatic fibrosis. In normal liver, HSCs are described as being in a quiescent state and serve to store retinoids (vitamin A). Quiescent stellate cells represent 5-8% of the total number of liver cells. When the liver is damaged, HSCs can change into an activated state characterized by contractions, loss of lipid droplets and enhanced of proliferation, cell migration as well as cellular adhesion. HSCs are also unequivocally the main cells involved in the production of excessive ECM seen in liver fibrosis. Since activated HSCs themselves secrete inflammatory chemokines, a vicious cycle is formed, whereby fibrogenic and inflammatory cells stimulate each other and perpetuate a process of liver damage and repair.
Natural killer (NK) cells are a key component of the innate immune system, and play a critical role in the early stages of the immune response against tumor cells, as well as those infected by viral and microbial pathogens.
In humans, two NK-cell subsets have been characterized according to the cell-surface density of CD56 and expression of CD16. CD56dimCD16bright NK cells (hereinafter CD56dim) compose approximately 90% of circulating NK cells; CD56brightCD16dim NK cells (hereinafter CD56bright) constitute approximately 10%. CD56bright NK cells proliferate and produce interferon in response to stimulation with interleukin-12 (IL-12), whereas CD56dim NK cells are more cytolytic and produce significant amounts of cytokine when their activating receptors are engaged.
In a paper published by some of the inventors it was found that, as opposed to CD8 immune cells, NK cells have anti-fibrotic activity through stimulation of HSC killing. (Melhhem et al., J.Hepatology; 2006; 45: 60-71). It has also been reported that the function of NK cells decreases when the liver disease progresses into cirrhosis, suggesting that attenuating NK function is a prerequisite for the progression of the disease (Seki et al.; Clin Dev Immunol.; 2011; Article ID 868345).
Human neuroligin-4 (NLG4, NLGn4, NLGn4X) encodes a member of a family of neuronal cell surface proteins called the Neuroligins. FIG. 1 illustrates the neuroligins and their interactions. Members of this family may act as splice site-specific ligands for beta-neurexins and may be involved in the formation and remodeling of central nervous system synapses. The encoded protein interacts with discs, large (Drosophila) homolog 4 (DLG4). Mutations in this gene have been associated with autism and Asperger syndrome. NLGn4 is also detected with high levels of expression in heart and lower in liver, skeletal muscle and pancreas.
The clinical implications of NAFLD are derived mostly from its potential to progress to cirrhosis and liver failure. There is an unmet medical for compositions and methods for treating NAFLD and preventing the progression to cirrhosis. Nowhere in the art has it been suggested that disease progression of NAFLD can be modulated by attenuating NLGn4 expression and thereby NK cell activity.
SUMMARY OF THE INVENTION
The present invention relates to preventing, treating and attenuating liver disease by inhibiting NLGn4 expression and thereby modulating the activity of NK cells. The invention, according to some embodiments relates to attenuation of the progression of NAFLD into cirrhosis and liver failure by modulating the expression of human neuroligin-4 (NLGn4, NLGn4, NLGn4X) and thereby activating cytotoxic NK cells.
There is provided herein according to some embodiments, a method of treating, attenuating and/or preventing progression of a liver disorder in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of an agent capable of inhibiting expression of a NLGn4 gene product, thereby treating, attenuating and/or preventing progression of the liver disorder.
According to some embodiments, the human NLGn4 gene product is encoded by a nucleic acid sequence comprising SEQ ID NO: 1. According to some embodiments, the NLGn4 gene product is encoded by a nucleic acid sequence with the accession number NM_020742. According to some embodiments, the NLGn4 gene product is encoded by a nucleic acid sequence with the accession number NM_181332. According to some embodiments, the NLGn4 gene product is encoded by a nucleic acid sequence with the accession number NM_001282145. According to some embodiments, the NLGn4 gene product is encoded by a nucleic acid sequence with the accession number NM_001282146.
According to some embodiments, the NLGn4 gene product comprises an mRNA sequence set forth in SEQ ID NO: 2. According to some embodiments, the accession number of the NLGn4 mRNA is AY358562. According to some embodiments, the accession number of the NLGn4 mRNA is BC032567. According to some embodiments, the accession number of the NLGn4 mRNA is BC034018.
According to some embodiments, the NLGn4 gene product comprises a peptide sequence set forth in SEQ ID NO: 4. According to some embodiments, the accession number of the NLGn4 polypeptide is NP_001269075.1. According to some embodiments, the accession number of the NLGn4 polypeptide is NP_001269074.1. According to some embodiments, the accession number of the NLGn4 polypeptide is NP_851849.1. According to some embodiments, the accession number of the NLGn4 polypeptide is NP_065793.1.
According to some embodiments, the agent comprises one or more inhibitory nucleic acids complementary to at least a portion of SEQ ID NO: 2.
According to some embodiments, the one or more inhibitory nucleic acids is selected from the group consisting of: an antisense molecule, an siRNA, and an shRNA. Each possibility is a separate embodiment of the invention.
According to some embodiments, the siRNA comprises a sequence set forth in SEQ ID NO: 3. According to some embodiments, the accession number of the siRNA sequence is SI03083395.
According to some embodiments, the liver disorder is selected from the group consisting of: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, liver adenoma, insulin hypersensitivity, liver cancer and any combination thereof. Each possibility is a separate embodiment of the invention.
According to some embodiments, the liver disorder is characterized by NLGn4 overexpression. According to some embodiments, NLGn4 overexpression comprises a 2,3,4,5-10 fold or more increase in NLGn4 expression relative to the expression level obtained in normal subjects. According to some embodiments, the overexpression attenuates NK cell activity, inhibits the expression of NLGn4 and modulates and/or activates the function of the NK cell.
According to some embodiments, administering to the subject the composition comprising a therapeutically effective amount of an agent capable of inhibiting expression of a NLGn4 gene product comprises administering the composition to an immune cell population of the subject. According to some embodiments, administering the composition to an immune cell population comprises infecting the immune cell population with a vector comprising the agent capable of inhibiting NLGn4 expression.
According to some embodiments, inhibiting the expression of the NLGn4 gene product reduces the activity of hepatic stellate cells. According to some embodiments, inhibiting the expression of the NLGn4 gene product increases apoptosis of the hepatic stellate cells.
According to some embodiments, the composition further comprises a GLUT4 antagonist. According to some embodiments, NLGn4 expression is regulated by a specific type of ionotropic glutamate receptor N-methyl-D-aspartate (NMDA or GLUT4 receptor; NMDAR). According to some embodiments, NLGn4 is linked to NMDR and both localize and bind PSD-95; a post synaptic density protein (PSD) According to some embodiments, the composition comprises an NMDAR antagonist selected from the group consisting of: Ketamin, Amantadine, Phencyclidine, Nitrous oxide, Dextromethorphan (and dextrorphan), Memantine, Ethanol, Riluzole, Xenon, HU-211, Lead (Pb2+), Conantokins, and Huperzine A.
According to an alternative embodiment, administering an N-methyl D aspartate receptor (NMDAR) agonist can increase NMDAR-mediated NLGn4 expression and as a result attenuate NK cell activity. Non-limiting examples of NMDAR agonists are Aminocyclopropanecarboxylic acid, D-Cycloserine, cis-2,3-Piperidinedicarboxylic acid, L-aspartate, L-alanine, Quinolinate, Homocysterate, D-serine, and ACPL.
There is provided herein according to some embodiments, a pharmaceutical composition for the use in treating, attenuating and/or preventing progression of a liver disorder in a subject, the composition comprising a therapeutically effective amount of an agent capable of inhibiting expression of a NLGn4 gene product, wherein the composition is capable of treating, attenuating and/or preventing progression of the liver disorder.
According to some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
There is provided herein according to some embodiments, a method of diagnosing and/or monitoring a liver disorder in a subject, the method comprising: isolating an immune cell population from a biological sample of the subject; detecting expression level of an NLGn4 gene product in the immune cell population and diagnosing and/or monitoring the liver disorder according to the NLGn4 gene product expression level.
According to some embodiments, the NLGn4 gene product is encoded by a nucleic acid sequence comprising SEQ ID NO: 1. According to some embodiments, the NLGn4 gene product comprises SEQ ID NO: 2.
According to some embodiments, the agent comprises one or more inhibitory nucleic acids complementary to at least a portion of SEQ ID NO: 2. According to some embodiments, the one or more inhibitory nucleic acids are selected from the group consisting of: an antisense molecule, an siRNA, and an shRNA. Each possibility is a separate embodiment of the invention.
According to some embodiments, the siRNA comprises a sequence set forth in SEQ ID NO: 3.
According to some embodiments, the liver disorder is selected from the group consisting of: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, liver adenoma, insulin hypersensitivity, liver cancer and any combination thereof. Each possibility is a separate embodiment of the invention.
According to some embodiments, the immune cell population is a natural killer (NK) cell population. Additionally or alternatively, the immune cell population is a subpopulation of NK cells According to some embodiments; the NK subpopulation is the CD56dim subpopulation. According to some embodiments; the NK subpopulation is the CD56bright subpopulation. According to some embodiments, the method comprises modulating the activity of the NK cells and/or a subpopulation of NK cells. According to some embodiments, modulating the activity of NK cells comprises enhancing the cytotoxicity of the NK cells. According to some embodiments, enhancing the cytotoxicity of NK cells comprises, but is not limited to, elevating CD107a expression in the NK cell and/or NK subpopulation.
According to some embodiments, the NK cell is a liver NK cell, and the activity of the NK cell is attenuated in patients with a liver disorder. According to yet another embodiment, NK cells from patients with a liver disorder, overexpresses NLGn4.
According to some embodiments, the biological sample comprises a blood sample, a tissue sample, a biological fluid, or any combination thereof.
According to some embodiments, the NLGn4 gene product expression level is detected by Polymerase Chain Reaction (PCR), Reverse-Transcriptase-PCR (RT-PCR), Northern Blot, Real-time PCR, hybridization to an oligonucleotide or any combination thereof. Each possibility is a separate embodiment of the invention.
According to some embodiments, the oligonucleotide comprises deoxyribonucleic acid (DNA), RNA, complementary deoxyribonucleic acid (cDNA), genomic DNA, synthetic oligonucleotide, or any combination thereof. Each possibility is a separate embodiment of the invention.
There is provided herein according to some embodiments, a kit for diagnosing a liver disorder, the kit comprising: means for isolating an immune cell population from a biological sample of a patient; and at least one reagent capable of detecting NLGn4 gene product expression level.
According to some embodiments, the reagent comprises NLGn4 specific primers.
According to some embodiments, the NLGN4 primers were designed to specifically amplify the NLGN4 copy on the X chromosome (Xp22.32-p22.31).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic representation of the Neuroligins and NLG interactions.
FIG. 2 shows NLGn4 expression upon NLGn4 siRNA expression in mouse NK cells.
FIG. 3A presents the percentage of viable NK cells expressing CD107a (control or infected with NLGn4 siRNA) co-cultured with HSCs isolated from a WT mouse.
FIG. 3B presents αSMA intensity of HSCs co-cultured with control or NLGn4 siRNA infected NK cells.
FIG. 3C shows apoptosis of HCS upon co-culturing with control or NLGn4 siRNA infected NK cells.
FIG. 4 presents NLGn4 expression in the CD56bright and CD56dim NK subpopulation.
FIG. 5 presents CD107a expression in NAFLD patients and healthy controls.
FIG. 6 presents on NK cell activity as assessed by CD107a expression.
FIG. 7A presents the effects of NLGn4 KD on NK cell viability, as estimated by PI incorporation.
FIG. 7B presents the effects of NLGn4 KD on NK cell viability, as estimated by annexin binding.
FIG. 8 shows the correlation between insulin and NLGn4 expression.



DETAILED DESCRIPTION
The present invention provides methods and compositions for treating and diagnosing liver disorders by activating attenuated natural killer (NK) cells and thereby reducing Hepatic stellate cell (HCSs) induced fibrosis.
In the following description, various aspects of the invention will be described. For the purpose of explanation, specific details are set forth in order to provide a thorough understanding of the invention. However, it will also be apparent to one skilled in the art that the invention may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the invention.
The following are terms which are used throughout the description and which should be understood in accordance with the various embodiments to mean as follows:
As referred to herein, the terms “liver disorder”, “liver disease” and “hepatic disease” are used interchangeably and refer to diseases and disorders that cause the liver to function improperly or stop functioning.
As referred to herein, the term “gene product” refers to a DNA sequence that is transcribed into mRNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide. Hence it is understood by the skilled in the art that the term gene product encompasses non-processed RNA, mRNA, splice variants thereof, corresponding cDNA sequences, polypeptides and proteins.
As used herein the terms “polynucleotide” “polynucleotide molecules”, “oligonucleotide”, “nucleic acid” and “nucleotide” may interchangeably be used. The terms are directed to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded, double stranded, triple stranded, or hybrids thereof. The term also encompasses RNA/DNA hybrids. The polynucleotides may include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA. The DNA or RNA molecules may be, for example, but not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, and the like. Accordingly, as used herein, the terms “polynucleotide molecules”, “oligonucleotide”, “polynucleotide”, “nucleic acid” and “nucleotide” sequences are meant to refer to both DNA and RNA molecules and refers to nucleic acid or ribonucleic acid sequence.
As used herein the term “complementary” is directed to base pairing between strands of nucleic acids. As known in the art, each strand of a nucleic acid may be complementary to another strand in that the base pairs between the strands are non-covalently connected via two or three hydrogen bonds. Two nucleotides on opposite complementary nucleic acid strands that are connected by hydrogen bonds are called a base pair. According to the Watson-Crick DNA base pairing, adenine (A) forms a base pair with thymine (T) and guanine (G) with cytosine (C). In RNA, thymine is replaced by uracil (U). The degree of complementarity between two strands of nucleic acid may vary, according to the number (or percentage) of nucleotides that form base pairs between the strands. For example, “100% complementarity” indicates that all the nucleotides in each strand form base pairs with the complement strand. For example, “95% complementarity” indicates that 95% of the nucleotides in each strand from base pair with the complement strand. The term sufficient complementarity may include any percentage of complementarity from about 30% to about 100%.
As used herein the term “short hairpin RNA” and “shRNA are used interchangeably and refer to, refer to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
As used herein the term “small interfering RNA” and “siRNA” are used interchangeably and refer to a nucleic acid molecule mediating RNA interference or gene silencing. The siRNA inhibits expression of a target gene and provides effective gene knock-down.
As used herein the term “antisense oligonucleotide” refer to nucleic acids, preferably, DNA, RNA or its derivatives, that are complementary to the nucleotide sequences of a target mRNA, characterized in that they binds to the target mRNA and interfere its translation to protein.
As used herein the term “vector” refers to expression constructs engineered to express shRNAs such as, but not limited to, retroviral and lentiviral vectors. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or H1 RNA polymerase III promoters, or other promoters known in the art.
According to an aspect of the invention, provided is a method of treating, attenuating or preventing a liver disorders such as Non-alcoholic fatty liver disease (NAFLD), and Non-alcoholic steatohepatitis (NASH) in a patient in need thereof. Alternatively other disorders such as cirrhosis, hepatitis, liver adenoma, insulin resistance, and liver cancer, or any NK related inflammatory or neoplastic disorder, can be the subject of treatment as well. The clinical implications of NAFLD are derived mostly from its potential to progress to Non-alcoholic steatohepatitis, cirrhosis and liver failure. In accordance, the invention, addresses the long felt need to attenuate the progression of NAFLD into cirrhosis and liver failure by inhibiting NLGn4 expression and thereby modulating the cytotoxic activity of NK cells. According to some embodiments, the invention provides a method for modulating the activity of a natural killer (NK) cell.
According to some embodiments, the method comprises administering to the patient in need thereof, a composition comprising a therapeutically effective amount of an agent capable of inhibiting the expression of the ribonucleic acid (RNA) encoded by NLGn4 nucleic acid molecule. The agent can for example be one or more polynucleotides, capable of hybridizing with the NLGn4 nucleic acid, such an inhibitory nucleic acid that is complementary and specific to at least a portion of NLGn4. The inhibitory nucleic acid can for example be an antisense molecule, an siRNA, or an shRNA. According to some embodiments, the siRNA comprises the sequence set forth in SEQ ID NO: 3. CGGCTGCAACTTCTCGCGCAA.
The NLGn4 mRNA sequence is set forth in the following sequence SEQ ID NO:2:








agaaggggaaggctcctgggctttcaatacatcctcctgaatcatacctcgtttcgggttccctagaaaaatctggacgtgtaaa



 


aagaactcttaacggccgatgcagctcttccaaagctaaggctgccttggagttttcataagaaattgtccctggaggtgttgga


 


tgatcacagcttccttggagcattgcagttgctggaatccagtttcaggattaagggagggctgcctccttgcaatgggctgcc


 


aagaaaacggctgtgcttgttcttaacctcaggctctgtctgtgatcagtctgagagtctctcccaggtctactgctccctggaaa


 


gccctatctctctgcaggctcgcctctgggctttgtctccttggagccacatcactgggacagctgtggatgtggatgcagattt


 


gaaccatgtcacggccccagggactgctatggcttcctttgttgttcaccccggtctgcgtcatgttaaactccaatgtcctcctg


 


tggttaactgctcttgccatcaagttcaccctcattgacagccaagcacagtatccagttgtcaacacaaattatggcaaaatcc


 


ggggcctaagaacaccgttacccaatgagatcttgggtccagtggagcagtacttaggggtcccctatgcctcaccccccact


 


ggagagaggcggtttcagcccccagaacccccgtcctcctggactggcatccgaaatactactcagtttgctgctgtgtgccc


 


ccagcacctggatgagagatccttactgcatgacatgctgcccatctggtttaccgccaatttggatactttgatgacctatgttc


 


aagatcaaaatgaagactgcctttacttaaacatctacgtgcccacggaagatgatattcatgatcagaacagtaagaagcccg


 


tcatggtctatatccatgggggatcttacatggagggcaccggcaacatgattgacggcagcattttggcaagctacggaaac


 


gtcatcgtgatcaccattaactaccgtctgggaatactagggtttttaagtaccggtgaccaggcagcaaaaggcaactatggg


 


ctcctggatcagattcaagcactgcggtggattgaggagaatgtgggagcctttggcggggaccccaagagagtgaccatct


 


ttggctcgggggctggggcctcctgtgtcagcctgttgaccctgtcccactactcagaaggtctcttccagaaggccatcattc


 


agagcggcaccgccctgtccagctgggcagtgaactaccagccggccaagtacactcggatattggcagacaaggtcggc


 


tgcaacatgctggacaccacggacatggtagaatgcctgcggaacaagaactacaaggagctcatccagcagaccatcacc


 


ccggccacctaccacatagccttcgggccggtgatcgacggcgacgtcatcccagacgacccccagatcctgatggagca


 


aggcgagttcctcaactacgacatcatgctgggcgtcaaccaaggggaaggcctgaagttcgtggacggcatcgtggataa


 


cgaggacggtgtgacgcccaacgactttgacttctccgtgtccaacttcgtggacaacctttacggctaccctgaagggaaag


 


acactttgcgggagactatcaagttcatgtacacagactgggccgataaggaaaacccggagacgcggcggaaaaccctg


 


gtggctctctttactgaccaccagtgggtggcccccgccgtggccaccgccgacctgcacgcgcagtacggctcccccacc


 


tacttctatgccttctatcatcactgccaaagcgaaatgaagcccagctgggcagattcggcccatggtgatgaggtcccctat


 


gtcttcggcatccccatgatcggtcccaccgagctcttcagttgtaacttttccaagaacgacgtcatgctcagcgccgtggtca


 


tgacctactggacgaacttcgccaaaactggtgatccaaatcaaccagttcctcaggataccaagttcattcacacaaaaccca


 


accgctttgaagaagtggcctggtccaagtataatcccaaagaccagctctatctgcatattggcttgaaacccagagtgaga


 


gatcactaccgggcaacgaaagtggctttctggttggaactcgttcctcatttgcacaacttgaacgagatattccagtatgtttc


 


aacaaccacaaaggttcctccaccagacatgacatcatttccctatggcacccggcgatctcccgccaagatatggccaacc


 


accaaacgcccagcaatcactcctgccaacaatcccaaacactctaaggaccctcacaaaacagggcctgaggacacaact


 


gtcctcattgaaaccaaacgagattattccaccgaattaagtgtcaccattgccgtcggggcgtcgctcctcttcctcaacatctt


 


agcttttgcggcgctgtactacaaaaaggacaagaggcgccatgagactcacaggcgccccagtccccagagaaacacca


 


caaatgatatcgctcacatccagaacgaagagatcatgtctctgcagatgaagcagctggaacacgatcacgagtgtgagtc


 


gctgcaggcacacgacacactgaggctcacctgcccgccagactacaccctcacgctgcgccggtcgccagatgacatcc


 


cacttatgacgccaaacaccatcaccatgattccaaacacactgacggggatgcagcctttgcacacttttaacaccttcagtg


 


gaggacaaaacagtacaaatttaccccacggacattccaccactagagtatagctttgccctatttcccttcctatccctctgccc


 


tacccgctcagcaacatagaagagggaaggaaagagagaaggaaagagagagagaaagaaagtctccagaccaggaat


 


gtttttgtcccactgacttaagacaaaaatgcaaaaaggcagtcatcccatcccggcagacccttatcgttggtgttttccagtatt


 


acaagatcaacttctgaccctgtgaaatgtgagaagtacacatttctgttaaaataactgctttaagatctctaccactccaatcga


 


tgtttagtgtgataggacatcaccatttcaaggccccgggtgtttccaacgtcatggaagcagctgacacttctgaaactcagcc


 


aaggacacttgatattttttaattacaatggaagtttaaacatttctttctgtgccacacaatggatggctctccttaagtgaagaaa


 


gagtcaatgagattttgcccagcacatggagctgtaatccagagagaaggaaacgtagaaatttattattaaaagaatggactg


 


tgcagcgaaatctgtacggttctgtgcaaagaggtgttttgccagcctgaactatatttaagagactttgtaaaaaagaaaaatgt


 


atatagctgtgagtttaaacaaaaaccacaaacagacaaacaagaaaaaaagcttttattggtgttttcactttgaaagagctttta


 


gcaaggttgtgcttttcattgtgctctgtacgtatataaatatatatatatatacacacacacacacacattagtcatatcacctctgt


 


ttcctccccaacaaaagaggcttttcttcttaattacttgtggtaaacaaagacatgggattttcttacatgagattctcatttgtagg


 


aggatgtgatgtcccacagaagacccagacggtctgtgtggcctatttcccccgtcaggttgcacaggtgcatgcaagagcat


 


tcttaggagaccactgttttgaaaaacttttgacttgtacgtgttagccttcatgaaattgcagtacagagatgggtccccaaagt


 


ggagtgtatttacagcttgttaaattagagacatgcacacacaaagaatcagtagggagaaacaaaaatacaagtcccgttctg


 


tagctctggccctttgaatatgtttaggaagagttgcttcccatttcagggccctgccaaaaaaagaagaaagcttgcctttggtg


 


gggctatgccccttggagtaaatacggctctgtgttccctagcagctgcgggagggtttggccgatgaagtacctgctcagctt


 


agctaatcagattgaaggaagacatgtgtctttcctttttgtttaagcactcggtcccttatttatcagtaagcaggtttttaaaaatct


 


tttatatcatttatgggatcaaacatatgattgtctgaaaacatcactttttgtggatttgtgtatccggtcaccaaacggtgaatatta


 


tagaagaatgggggaagaaaggatagaatattaaaactgctttgcatgggttttctgggaaattaggataacttcactgagaag


 


acattgaatggaaattattcacccattttaaattggtgacctagggatcagagatttgtctttccaacagcttgtcattttttcatttctc


 


ttctcatttttcaggaaagttttgagtgttataaggtggaaggaaacatagtagcaatggatacttttttgaaaaattattgcattacc


 


aagaaacagtagccaaagatatttgaagatcatgttcctcggctccattgtgggttattctagaaatccagtcttaaatctctccgc


 


taaagtggacattccccataaaaattgtccagctgcctggctcttttgcaataacaacctttgattactgaatccctacactcaaac


 


tatagtgatatatcagtgtttgagagtgacctctagaaaaaagaaaagtgtttttagaaatgcgtacaagtcacccccaaatccta


 


ttgcttatcttgggttaaatttgagagtgattctctgtatataaatatgtgaaatattattatctcaacttagcacacgtgaagcaacat


 


ttctttcctacagagaggtgtcatggtaagatttcattccgaattcattgtttcatagagctatgatcaggccatttctgcaagcaat


 


gtatgaccccacctgagcaaccacaaataggctctctgtgaaactacaaaggaagttatgtgtggcatccatgttggtttcgtct


 


gtctgtaatgtgaattccagtatttgtttagtatttccagttgtctcctgctagcaatatgtacagtaacgcgtcaggcttgtgacattt


 


gaataaggaaaaacagagttcctgttaagtgaataactttagcttttacaggggattatgatcaaaagtgattttagtacatcttaa


 


atgatatcttatttctacatggaaagaagttatagaatcttcatagagttctatgagaaaaaatatacttgctatctataaaaaagag


 


aaaaaagaaaaaaaatgagaaaaaagtaagaaaaaaaaaaatcctgtcctaggcttttactcttgatcttcaaaggcacgcag


 


ggtttaatggttccttgggttattattttgcagttttgttttttattttgccttaagtaatgatagaagatatatatggccggacacatatg


 


tataaacttttcagcagcatttttaataataaaatatcacagtattttctaaaaaaaaaaaaaaaaaa






Additionally or alternatively, the method comprises administering to the patient in need thereof, a composition comprising a therapeutically effective amount of an agent (such as for example an antibody) capable of inhibiting the expression and/or function of NLGn4 protein.
The NLGn4 polypeptide sequence is set forth in the following sequence SEQ ID NO: 4:








MSRPQGLLWLPLLFTPVCVMLNSNVLLWLTALAIKFTLIDSQAQYPVVNTNYGKIRGLRTPLPNEILGPV



 


EQYLGVPYASPPTGERRFQPPEPPSSWTGIRNTTQFAAVCPQHLDERSLLHDMLPIWFTANLDTLMTYVQ


 


DQNEDCLYLNIYVPTEDDIHDQNSKKPVMVYIHGGSYMEGTGNMIDGSILASYGNVIVITINYRLGILGF


 


LSTGDQAAKGNYGLLDQIQALRWIEENVGAFGGDPKRVTIFGSGAGASCVSLLTLSHYSEGLFQKAIIQS


 


GTALSSWAVNYQPAKYTRILADKVGCNMLDTTDMVECLRNKNYKELIQQTITPATYHIAFGPVIDGDVIP


 


DDPQILMEQGEFLNYDIMLGVNQGEGLKFVDGIVDNEDGVTPNDFDFSVSNFVDNLYGYPEGKDTLRETI


 


KFMYTDWADKENPETRRKTLVALFTDHQWVAPAVATADLHAQYGSPTYFYAFYHHCQSEMKPSWADSAHG


 


DEVPYVFGIPMIGPTELFSCNFSKNDVMLSAVVMTYWTNFAKTGDPNQPVPQDTKFIHTKPNRFEEVAWS


 


KYNPKDQLYLHIGLKPRVRDHYRATKVAFWLELVPHLHNLNEIFQYVSTTTKVPPPDMTSFPYGTRRSPA


 


KIWPTTKRPAITPANNPKHSKDPHKTGPEDTTVLIETKRDYSTELSVTIAVGASLLFLNILAFAALYYKK


 


DKRRHETHRRPSPQRNTTNDIAHIQNEEIMSLQMKQLEHDHECESLQAHDTLRLTCPPDYTLTLRRSPDD


 


IPLMTPNTITMIPNTLTGMQPLHTFNTFSGGQNSTNLPHGHSTTRV






According to some embodiments, the NK cells are liver NK cells which are attenuated in patients having a liver disorder. According to yet another embodiment, the liver disorder is characterized by overexpression of NLGn4 RNA. Such overexpression can attenuate NK cell activity.
According to some embodiments inhibiting the expression of NLGn4 modulates the function of the NK cell for example by activating the NK cell and/or the CD56dim NK cell subset. As a result of NK activation, the activity of hepatic stellate cells (HSCs) and hence fibrosis is reduced. In addition, and according to yet another embodiment, modulating and/or activating the NK cells increases the apoptosis of the HSCs.
According to yet another embodiment there is provided a method for modulating the activity of a natural killer (NK) cell and/or treating, preventing and/or attenuating a liver disorder by administering to a patient a composition comprising a GLUT4 antagonist. Such antagonist can according to the present invention inhibit GLUT4 mediated NLGn4 expression. The antagonist can be selected from the group comprising Ketamine, Amantadine, Phencyclidine, Nitrous oxide, Dextromethorphan (and dextrorphan), Memantine, Ethanol, Riluzole (used in ALS), Xenon, HU-211 (also a cannabinoid), Lead (Pb2+), Conantokins, and Huperzine A. According to an alternative embodiment administering a NMDAR (also known as GLUT4) agonist can increase GLUT4 mediated NLGn4 expression and as a result attenuate NK cell activity. Examples of a GLUT4 agonists are Aminocyclopropanecarboxylic acid, D-Cycloserine, cis-2,3-Piperidinedicarboxylic acid, L-aspartate, L-alanine, Quinolinate, Homocysterate, D-serine, and ACPL.
According to another aspect of the invention, there is provided a method of modulating the expression of the ribonucleic acid (RNA) encoded by NLGn4 nucleic acid. According to one embodiment, modulating the expression NLGn4 can serve to treat, attenuate or prevent a liver disorder, such as Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, liver adenoma, insulin resistance, a liver cancer, any NK related inflammatory or neoplastic disorder, or any combination thereof.
According to another embodiment, modulating the expression of NLGn4 comprises contacting the immune cell, such as an NK cell and/or a CD56dim NK cell subset, with a composition comprising an effective amount of an agent that inhibits NLGn4 expression. Such agent can for example be an inhibitory nucleic acid that is complementary and specific to at least a portion of the NLGn4 nucleic acid molecule.
According to yet another embodiment, the inhibitory nucleic acid can for example be an antisense molecule, an siRNA, or an shRNA.
Inhibiting NLGn4 can according to the present invention enhance the cytotoxicity of the NK cells and or specific NK cell subpopulations. According to certain embodiments enhancing the cytotoxicity comprises enhancing the expression of CD107a on said NK cell.
In certain liver disorders NK cell function can be attenuated. According to the present invention such attenuation can be a result of NLGn4 overexpression. In accordance, inhibiting the expression of NLGn4 modulates and/or activates the function of attenuated NK cell. In turn, activating the NK cell may reduce HSC activity and/or increase their apoptosis.
According to yet another aspect of the invention there is provided a method of diagnosing or monitoring a liver disorder and/or the severity of a liver disorder in a patient such as Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, a liver adenoma, insulin resistance, a liver cancer, any NK related inflammatory or neoplastic disorder, or any combination thereof. The method comprises, according to one embodiment, detecting the expression level of a ribonucleic acid (RNA) encoded by NLGn4 nucleic acid molecule in a biological sample, such as a blood sample, a tissue sample and/or a biological fluid, of a patient.
According to some embodiments, the method further comprises isolating the RNA from the biological sample prior to detecting the NLGn4 RNA expression level. The detection of NLGn4 expression comprises Polymerase Chain Reaction (PCR), Reverse-Transcriptase-PCR (RT-PCR), Northern Blot, Real-time PCR, Flow Cytometry (FACS) or any combination thereof.
Alternatively, the expression level of NLGn4 is detected by hybridization to an oligonucleotide such as a deoxyribonucleic acid (DNA), an RNA, complementary deoxyribonucleic acid (cDNA), a genomic DNA, a synthetic oligonucleotide, or any combination thereof.
According to yet another aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of an agent that inhibits the expression or function of NLGn4.
The agent can be one or more polynucleotides, capable of hybridizing with said nucleic acid. For example the agent can be an inhibitory nucleic acid, such as an antisense molecule, an siRNA, or an shRNA that is complementary and specific to at least a portion of said NLGn4 nucleic acid molecule
According to some embodiments, the pharmaceutical composition further comprises a vector capable of expressing the inhibitory nucleic acid molecule. Non-limiting examples of vectors comprise lentiviral vectors, retroviral vectors, plasmids as well as other suitable vectors.
According to another embodiment, the composition comprises or additionally comprises a GLUT4 antagonist. Such antagonist can according to the present invention inhibit GLUT4 mediated NLGn4 expression. The antagonist can be selected from the group consisting of Ketamin, Amantadine, Phencyclidine, Nitrous oxide, Dextromethorphan (and dextrorphan), Memantine, Ethanol, Riluzole (used in ALS), Xenon, HU-211 (also a cannabinoid), Lead (Pb2+), Conantokins, and Huperzine A. According to an alternative embodiment administering a GLUT4 agonist can increase GLUT4 mediated NLGn4 expression and as a result attenuate NK cell activity. Examples of a GLUT4 agonists are alanine, Aminocyclopropanecarboxylic acid, D-Cycloserine, cis-2,3-Piperidinedicarboxylic acid, L-aspartate, L-alanine, Quinolinate, Homocysterate, D-serine, and ACPL.
According to yet another aspect of the invention, there is provided a kit for prevention, treatment or attenuation of a liver disorder such as, but not limited to, Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, a liver adenoma, insulin resistance, a liver cancer, any NK related inflammatory or neoplastic disorder, or any combination thereof. The kit comprises the pharmaceutical composition as essentially described above and a pharmaceutically acceptable carrier.
According to yet another aspect of the invention, there is provided a kit for diagnosing a liver disorder such as but not limited to Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, a liver adenoma, insulin hypersensitivity, a liver cancer or any combination thereof. The kit comprises at least one reagent capable of detecting the expression of a nucleic acid in a biological sample such as a blood sample, a tissue sample, and/or a biological fluid.
According to some embodiments, the reagent comprises NLGn4 specific primers. According to some embodiments, the NLGn4 specific primers are selected from the group set forth in table 1 below.







TABLE 1







NLGn4 specific primers









Exon
Forward
Reverse





2.1
AAAGCCCTATCTCTCTGCAGG 
TGAGTAGTATTTCGGATGCCAG



(SEQ ID NO: 5)
(SEQ ID NO: 6)


 


2.2
AAGAACACCGTTACCCAATGAG 
GAGACATTATAAAACCCTCCTAG



(SEQ ID NO: 7)
(SEQ ID NO: 8)


 


3
TTAGCATTGGTGAGTCAGTGTG 
CCGTCAAAACGAGAAGTGGACT



(SEQ ID NO: 9)
(SEQ ID NO: 10)


 


4
CTTTTTCTATTTGGCCACCA
TTCTTGGTTCAGGGTATTTGC



(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


5.1
AGCTGCATTTCTGTCCTGTG
TCTCCCGCAAAGTGTCTTTC



(SEQ ID NO: 13)
(SEQ ID NO: 14)


 


5.2
CCAACTTCGTGGACAACCTT
ACCCCAACACGAAGATGAAC



(SEQ ID NO: 15)
(SEQ ID NO: 16)


 


6.1
CACGTCACATGTGGAAGAGT
GACGGCAATGGTGACACTTA



(SEQ ID NO: 17)
(SEQ ID NO: 18)


 


6.2
TCCTCATTGAAACCAAACGA
AACATTCCTGGTCTGGAGAC



(SEQ ID NO: 19)
(SEQ ID NO: 20)









The following examples are presented to provide a more complete understanding of the invention. The specific techniques, conditions, materials, proportions and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention.
EXAMPLES
Example 1
Methods used for evaluating the role of NLGn4 in NK activity


    a) Knockdown of NLGn4: Lentivirus expressing NLGn4 siRNA were used to infect NK cells of either mouse or human origin and thereby inhibiting NLGn4 expression.
    b) NLGn4 expression: NLGn4 expression level was evaluated by real-time PCR. In short, RNA was extracted from the cells using Tri Reagent. The extracted RNA was converted to cDNA using random hexamers and reverse transcriptase. NLGn4 expression level was assessed by real time PCR using NLGn4 specific primers. The results were normalized to the expression levels of α-actin using α-actin specific primers.
    c) Isolation of NK cells from human blood samples: Blood samples obtained from patients were centrifuged at 4000 rpm for 5 min. After centrifugation, the buffy coat fraction of the blood containing most of the leukocytes was collected and NK cells were isolated using the RosetteSep NK isolation kit according to manufactures instruction.
    d) Flow cytometry using FACS analysis of CD107a, NLGn4, α-SMA, annexin.


Example 2
NLGn4 is Overexpressed in Patients with Cirrhosis and in a Non-alcoholic Fatty Liver Disease (NAFLD) Mouse Model
Human peripheral blood cells (PBLs) were isolated in accordance with Example 1c from cirrhotic patients and healthy controls, as well as from NAFLD/control mice. RNA was extracted and converted into cDNA and a gene array analysis was performed using an Affymetrix expression array. The results were collated in order to identify the genes having an at least two-fold change in the expression profile. It was found that NLGn4 showed the most significant change in that an approximately 4-fold up-regulation was observed among the cirrhotic patients.
Example 3
NLGn4 Expression Can Be Reduced Using siRNA
Mouse liver NK cells were infected with a lentiviral vector expressing an siRNA against NLGn4 or a scrambled control. 48 hours post infection, the cells were harvested, RNA extracted and converted into cDNA in accordance with example 1b. NLGn4 Expression levels in cells infected with the NLGn4 siRNA or the scrambled control were evaluated using real-time PCR using primes specific for NLGn4. The expression levels obtained were normalized to those obtained for α-actin. As seen in FIG. 2, a significant reduction in NLGn4 expression is observed in cells infected with the siRNA expressing vector, as compared to the control.
Example 4
NLGn4 Knockdown (KD) Increases NK Activation and Hepatic Stellate Cell (HSC) Apoptosis and Reduces HSC Activity
NK cells obtained from mice livers were pre-incubated with IL2 in order to obtain a mature NK cell population. Following infection with the NLGn4 siRNA or with the scrambled control, the cells were co-cultured with freshly isolated HSC from a NAFLD mouse model. The activity of the NK cells was evaluated by the expression of CD107a, a marker of active NK cells. The percentage of viable NK cells expressing CD107a was evaluated by FACS using an anti-CD107a antibody and gating annexin negative cells. As seen in FIG. 3A, as a result of the KD of NLGn4 a significant increase in CD107a positive cells amongst the viable NK cell population was observed.
The impact of NK cell activation by NLGn4 KD on HCSs was evaluated by co-culturing the HSCs with the control or the NLGn4 KD NK cells and assessing αSMA intensity (marker of HSC activation). α-SMA intensity was significantly decreased upon co-culture with NLGn4 KD NK cells (FIG. 3B) and amongst the α-SMA expressing cells an increase in apoptosis was observed (FIG. 3C).
Example 5
NLGn4 is Expression is High in the CD56bright NK Subpopulation and Low in the CD56dimNK Subpopulation
Human peripheral blood cells (PBLs) were isolated in accordance with Example 1c. The isolated NK cells were then co-stained with an anti-CD56 antibody and with an anti-NLGn4 antibody. FACS analysis of the cells showed that NLGn4 is significantly more abundant in the CD56bright cell population as compared to the CD56dim cell population (FIG. 4).
Example 6
NK activity as Assessed by CD107a Expression is Attenuated in NAFLD Patients
Human peripheral blood cells (PBLs) from NAFLD patients (n=9) and healthy controls (n=3) were isolated in accordance with Example 1c. The isolated NK cells were then co-stained with an anti-CD56 antibody and with an anti-CD107a antibody. FACS analysis showed that CD107a expression was reduced, corresponding to an attenuated NK activity in NAFLD patients (FIG. 5).
Example 7
NLGn4 KD Increases CD56dim NK Cell Activity as Assessed by CD107a Expression
Human peripheral blood cells (PBLs) from NAFLD patients (n=9) and healthy controls (n=3) were isolated in accordance with Example 1c. The isolated NK cells were then infected with a lentiviral vector expressing an siRNA against human NLGn4 or a scrambled control. NK activity in response to NLGn4 KD was assessed by CD107a expression. That is, the isolated NK cells were co-stained with an anti-CD56 antibody and with an anti-CD 107a antibody. As seen from FIG. 6, CD107a expression was significantly elevated in the CD56dim subpopulation. This might suggest that reducing the expression of NLGn4 can effectively enhance NK cytotoxicity. Since NLGn4 is primarily expressed in CD 56bright cells it may be suggested that overexpression of NLGn4 by CD56bright cells inhibits the cytotoxicity of CD56dim cells.
Example 8
NLGn4 KD Does Not Alter NK Viability
Human peripheral blood cells (PBLs) from NAFLD patients (n=9) and healthy controls (n=3) were isolated in accordance with Example 1c. The isolated NK cells were then infected with a lentiviral vector expressing an siRNA against human NLGn4 or a scrambled control. The viability of the NK cells was assessed by FACS analysis estimating annexin binding and PI incorporation. As seen in FIGS. 7A and B, NLGn4 knockdown did not alter cellular viability neither of CD56bright nor of CD56dim NK cells in either NAFLD patients or healthy controls. This indicates that CD56dim cytotoxicity toward foreign cells is elevated without compromising self-recognition.
Example 9
NLGn4 Overexpression Correlates with High Insulin Levels
Human peripheral blood cells (PBLs) from patients with low insulin levels (n=3) and patients with high insulin levels controls (n=3) were isolated in accordance with Example 1c. The isolated NK cells were stained with an anti-NLGn4 antibody. FACS analysis of the cells showed that NLGn4 was significantly higher in NK cells from patients with high insulin levels as compared to those with low insulin levels (FIG. 8). This may suggest that the increased prevalence of NAFLD among insulin resistant subjects may be due to insulin mediated NLGn4 overexpression.
Example 10
Treatment of Mice With a GLUT4 Agonist Elevates NLGn4 Expression
NK cells from livers of mice treated with the GLUT4 agonist alanine or control mice are isolated. The isolated NK cells are then co-stained with an anti-CD56 antibody and with an anti-NLGn4 antibody.
Example 11
Treatment of Mice With a GLUT4 Antagonist Reduces NLGn4 Expression
NK cells from livers of mice treated with the GLUT4 agonist or control mice are isolated. The isolated NK cells are then co-stained with an anti-CD56 antibody and with an anti-NLGn4 antibody.

## Claims

1. A method of treating and/or attenuating hepatocellular carcinoma in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of an agent capable of specifically inhibiting expression of a human NLGn4 gene product, said human NLGn4 gene product comprising SEQ ID NO: 2, and said agent comprising one or more siRNAs having a length of 20-25 base pairs and a sequence with at least 95% complementarity to at least a portion of SEQ ID NO: 2, thereby treating and/or attenuating the hepatocellular carcinoma.

2. The method of claim 1, wherein the siRNA has a sequence set forth in SEQ ID NO: 3.

3. The method of claim 1, wherein the NLGn4 gene product is encoded by a nucleic acid sequence comprising SEQ ID NO: 1.

4. The method of claim 1, wherein inhibiting the expression of the NLGn4 gene product reduces activity of hepatic stellate cells.

5. The method of claim 1, wherein inhibiting the expression of the NLGn4 gene product increases apoptosis of the hepatic stellate cells.

6. The method of claim 1, wherein said composition further comprises a GLUT4 antagonist.