Patent Grant
10684272
This patent application is a U.S. National Phase Application under 35 U.S.C. § 371 of International Application No. PCT/SG2016/050338, filed on 15 Jul. 2016, entitled MODULATION OF HEPATITIS B VIRUS REPLICATION, which claims the benefit of priority of Singapore application No. 10201505551U, filed 15 Jul. 2015, the contents of which were incorporated by reference in the entirety for all purposes.
This patent application incorporates by reference the material (i.e., Sequence Listing) in the ASCII text file named sequence_listing_ST25_2153326_1.txt, created on Jan. 11, 2018, having a file size of 57,344 bytes, and the written sequence listing identified as sequence_listing_ST25 2153326 2, which is a PDF of an ASCII text file in computer readable form (CRF) named sequence_listing_ST25 2153326 2.txt, created Feb. 8, 2019, having a file size of 90,112 bytes.
The present invention relates to the field of virology, and more specifically the study of the underlying mechanism and factors involved in HBV replication. More specifically, the present invention relates to the identification of factors that modulate HBV replication for the subsequent development of agents that target these factors for use in in vitro methods or for treating HBV infection and associated diseases or conditions.
Hepatitis B virus (HBV) affects 240 million chronic carriers globally and is linked to ˜800,000 deaths annually from complications including liver cancer and cirrhosis.
Current HBV antiviral therapeutics target the late stage replicative phase by inhibiting viral polymerase/reverse transcriptase (Pol/RT) when viral load has risen significantly, limiting their effectiveness for viral clearance. Targeting early stage viral replication has not been feasible due to a lack of understanding of the precise host factors that drive viral replication.
Accordingly, there is a need to elucidate the fundamental molecular mechanism and associated factors underlying HBV replication. The identification of such factors will allow for the development of novel HBV intervention approaches as well as therapeutics for treating HBV infections and associated conditions or diseases.
According to one aspect, there is provided a method of modulating HBV replication in a cell, comprising: contacting the cell with at least one agent that modulates the expression of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
In another aspect, there is provided a use of at least one agent in the manufacture of a medicament for treating a HBV infection in a subject, wherein the at least one agent modulates the activity of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
In another aspect, there is provided a use of at least one agent in the manufacture of a medicament for treating a disease or condition associated with a HBV infection in a subject, wherein the at least one agent modulates the activity of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
In another aspect, there is provided a method of treating a HBV infection in a subject, comprising administering to the subject of at least one agent that modulates the activity of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
In another aspect, there is provided a method for treating a disease or condition associated with a HBV infection in a subject, comprising administration to the subject of at least one agent that modulates the activity of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
In another aspect, there is provided a use of at least one peptide derived from SOX7 or SNAI2 in the manufacture of a medicament for inhibiting HBV replication or treating a HBV infection in a subject.
In another aspect, there is provided a method of screening at least one agent for modulating HBV replication, comprising; a) contacting a cell expressing the HBV virus with the at least one agent, wherein the at least one agent modulates the expression of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A; b) obtaining a HBV expression profile of the cell contacted with the at least one agent; and c) comparing the HBV expression profile of the cell in b) with an HBV expression profile of a control cell that has not been contacted with said at least one agent, wherein a decrease or increase in the expression of the HBV virus in the cell, relative to the control cell, indicates a modulation of HBV replication by said at least one agent.
In another aspect, there is provided a method of identifying at least one factor that modulates replication of a virus, comprising: a) transfecting at least two cell lines of different origins with an expression construct comprising a selection marker operably linked to a viral promoter of the virus; b) detecting the expression of the selection marker to classify the at least two cell lines into permissive and non-permissive cell lines; c) transfecting the permissive and non-permissive cell lines with an expression construct comprising a viral replicon of the virus; d) screening for expression of at least one factor in said permissive and non-permissive cell lines of c) and comparing the expression of the at least one factor in the permissive cell line to the non-permissive cell line, to identify at least one candidate factor, wherein the differential expression of the at least one factor between the permissive and non-permissive cell line is indicative of identifying at least one candidate factor; e) contacting said permissive cell line expressing the viral replicon with an agent to knock out expression of said at least one candidate factor; and f) comparing the pre-genomic RNA level of said viral replicon in said permissive cell line of e) relative to a control cell line that has not been contacted with said agent, wherein a decrease or increase in the expression level of the pre-genomic RNA level in the permissive cell line, relative to the control cell line, indicates the identification of at least one factor that modulates replication of the virus.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
d) Luciferase assay for effect of Sox7 motif deletion on transcription at the HBVCP when Sox7 is overexpressed.
Hepatitis B virus (HBV) is known mainly as a liver specific pathogen that causes chronic hepatitis infection and liver cancer. However, the present disclosure is based upon the discovery that HBV may replicate efficiently in a number of non-liver cells. Accordingly, this suggests that host cellular factors that are required for HBV propagation in liver cells may also be present in non-liver cells.
In particular, HBV is dependent on host transcription factors for pre-genomic RNA (pgRNA) synthesis at the HBV core promoter (HBVCP) (nt1600-1860) during its early replicative phase. These host factors may include but are not limited to Sp 1, PARP1 and HNF4α which are widely expressed in a variety of tissues, and hence cannot account for liver-specific replication of HBV.
Furthermore, since viral transmission may occur through exosomes, and clathrin-dependent endocytosis, it may be possible that HBV replication initiation is prohibited by mechanisms intrinsic to the infected non-liver cell. In particular, mechanisms blocking pgRNA synthesis may silence HBV replication as its loss would prevent the generation of the HBV DNA genome, viral polymerase/reverse transcriptase (Pol/RT) and capsid protein hepatitis B core antigen (HBcAg).
Accordingly, the present disclosure is based upon the further elucidation of the mechanism of HBV replication and transcription in identifying associated host factors required for HBV transcription and replication in various cells and/or tissues. The identification of such host factors will allow for the generation of new intervention approaches as well as the development of cell based screening methods which are critical for HBV therapeutic discovery.
In this regard, an advantage of the present disclosure is related to the identification of a number of factors that may influence or modulate the efficiency of HBV replication. In one embodiment, these factors may include but are not limited to transcriptional factors, signal pathway factors, protein binding factors and protein maturation factors.
In particular, the genes encoding these factors may be targeted to influence or modulate the efficiency of HBV replication. Exemplary, non-limiting embodiments of factors and associated gene targets that may influence the efficiency of HBV replication are listed in Table 1. All gene IDs listed in Table 1 are publicly disclosed in well-known gene or protein databases that may be referred to in obtaining further information on each gene ID, such as their sequence, location, family and function. These gene or protein databases may include but are not limited to the HUGO Gene Nomenclature Committee (HGNC), NCBI GeneBank or Uniprot.
In one embodiment there is provided a method of modulating HBV replication in a cell. In particular, the method comprises contacting the cell with at least one agent that modulates the expression of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
In an alternative embodiment the cell may be contacted with at least one agent that modulates the expression of at least one factor selected from the group consisting of Clorf131, ETS1, FSTL1, HNF4A, INHBA, KCTD12, MAK16, NOSTRIN, PNPT1, PTCD3, SEMA4G, SNAI2, SNRPD1, SUSD1, WDR43, ZFHX4, ALDH2, ALDH5A1, B3GNT2, CHUK, DDX18, EPT1, ERMP1, GCNT3, GSR, HMGCS1, NAE1, PDSS1, PPID, SMURF2, TANC2, BCAR3, C16orf70, C9orf100, EPB41L5, HSPA14, HSPA9, LRP12, NSMCE4A, NUP35, PSMD11, RNF43, EXOC6, FUZ, STOML2, WASF2, ARHGAP12, MYO9B, TBC1D14, FLVCR1, SLC39A14, GPX2, IDH1 and FAM35A.
In one embodiment, the HBV replication in the cell may be inhibited. In some embodiments, the at least one agent may be selected from the group consisting of a chemical compound, a small molecule, an oligonucleotide, a protein, a peptide, a stapled peptide, a peptidomimetic, an antibody and an antigen binding molecule. The oligonucleotide may be a siRNA or a shRNA.
The at least one factor may be selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8 and HNF4α9. In one embodiment, the at least one factor may be SNAI2 or SOX7.
In one embodiment, the at least one agent may be a siRNA. In one embodiment, the siRNA decreases the expression of SNAI2 or SOX7 in the cell such that HBV replication in the cell is increased relative to the HBV replication in the cell in the absence of the siRNA. In another embodiment, the siRNA decreases the expression of HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8 or HNF4α9 such that HBV replication in the cell is decreased relative to the HBV replication in the cell in the absence of the siRNA.
In some embodiments, the cell may be selected from the group consisting of a liver cell, colon cell, stomach cell, blood cell and lung cell.
In one embodiment, the method may be performed in vitro.
In some embodiments, the cell may be derived from a cell line selected from the group consisting of HepG2, HuH6, HuH7, HuH4, PLC/PRF/5, Kato III, AGS, HCT116, Caco-2, HL-60, HEK293 and A549.
In the method as described herein the contacting step may comprise culturing the cell in the presence of the at least one agent.
The present disclosure also provides the use of at least one agent in the manufacture of a medicament for treating a HBV infection in a subject, wherein the at least one agent modulates the activity of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
The present disclosure also provides the use of at least one agent in the manufacture of a medicament for treating a disease or condition associated with a HBV infection in a subject, wherein the at least one agent modulates the activity of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
In some embodiments, the disease or condition may be liver disease. In another embodiment, the disease, disorder or condition is jaundice, liver inflammation, liver fibrosis, inflammation, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, liver steatosis, hepatocellular carcinoma, liver disease-related transplantation, glomerulonephritis, dyslipidemia, hematopoietic malignancies or pancreatitis.
In one embodiment, the at least one agent inhibits HBV replication. In some embodiments, the at least one agent may be selected from the group consisting of a chemical compound, a small molecule, an oligonucleotide, a protein, a peptide, a stapled peptide, a peptidomimetic, an antibody and an antigen binding molecule. The oligonucleotide may be a siRNA or shRNA.
In some embodiments, the at least one factor may be selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8 and HNF4α9. In one embodiment, the at least one factor may be SNAI2 or SOX7.
The present disclosure also provides the use of at least one peptide derived from SOX7 or SNAI2 in the manufacture of a medicament for inhibiting HBV replication or treating a HBV infection in a subject. In another embodiment, the at least one peptide may be derived from any one of the factors disclosed herein.
In one embodiment, the at least one peptide described herein may include a peptide analogue, modified peptide or peptide derivative of the native protein encoded by SOX7 or SNAI2. Analogues or functional equivalents of the native protein may be a peptidic molecule that retains the activity and function of the native protein, and may, for instance be a peptidomimetic. A peptide derivative or variant may be a peptide identical to the reference protein or peptide sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%, such as at least 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identical. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion. Modified peptides may be molecules derived from a peptide or protein by the introduction of substituents or functional groups which are, generally, not present in naturally occurring amino acids. A peptidomimetic or peptide mimic, may include synthetic compounds with a functional structure more or less similar to a reference peptide or protein, but which may also contain non-peptidic bonds in the backbone, or D-amino acids. In general, peptidomimetics may serve as substitutes for native proteins in being designed to mimic the function and activity of the protein. An example of a peptide mimic may include but is not limited to a stapled peptide that is a peptide comprising a synthetic brace (“staple”) such as a hydrocarbon staple.
In one embodiment, the at least one peptide may be derived from DNA binding domains of the native protein encoded by SOX7 or SNAI2. Accordingly, in one embodiment, the at least one peptide may be derived from a peptide selected from the group consisting of Slug-ZF1s (SEQ ID NO: 215); Slug-ZF2s (SEQ ID NO: 216); Slug-ZF3s (SEQ ID NO: 217); Slug-ZF4s (SEQ ID NO: 218); Slug-ZF5s (SEQ ID NO: 219) Sox7-H1s (SEQ ID NO: 220); and Sox7-H2s (SEQ ID NO: 221).
In one embodiment, the at least one peptide may be derived from a peptide selected from the group consisting of Slug-ZF4s (SEQ ID NO: 218); Slug-ZF5s (SEQ ID NO: 219), Sox7-H1s (SEQ ID NO: 220); and Sox7-H2s (SEQ ID NO: 221).
In one embodiment, the at least one peptide may be a stapled peptide. In another embodiment, the stapled peptide may comprise a hydrocarbon staple.
In one embodiment, the at least one peptide may be formulated as a composition suitable for a convenient mode of administration to the subject. In one embodiment, the at least one peptide may be formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
The present disclosure also provides a method for inhibiting HBV replication or treating a HBV infection in a subject, comprising administering to the subject at least one peptide derived from SOX7 or SNAI2.
In one embodiment, the at least one peptide may be derived from a peptide selected from the group consisting of Slug-ZF1s (SEQ ID NO: 215); Slug-ZF2s (SEQ ID NO: 216); Slug-ZF3s (SEQ ID NO: 217); Slug-ZF4s (SEQ ID NO: 218); Slug-ZF5s (SEQ ID NO: 219) Sox7-H1s (SEQ ID NO: 220); and Sox7-H2s (SEQ ID NO: 221).
In one embodiment, the at least one peptide may be derived from a peptide selected from the group consisting of Slug-ZF4s (SEQ ID NO: 218); Slug-ZF5s (SEQ ID NO: 219), Sox7-H1s (SEQ ID NO: 220); and Sox7-H2s (SEQ ID NO: 221).
In one embodiment, the at least one peptide may be a stapled peptide. In another embodiment, the stapled peptide may comprise a hydrocarbon staple.
The sequence, structure and location of the hydrocarbon staple may be varied for functionality and synthesized using well-known techniques in the art or obtained from commercial companies such as GenScript. As may be appreciated by those skilled in the art, the sequence, structure and location of the hydrocarbon staple may vary as long as it does not disrupt the interaction of the peptide comprising the hydrocarbon staple with a nucleic acid, for example DNA.
In particular, the hydrocarbon staple may attach 2 amino acid residues within each peptide at specific residue positions. The residue positions as described herein may correspond to the residue positions within the amino acid sequence of the full-length native protein. For example, the residue position for the first hydrocarbon attachment may be denoted as “i”, and the subsequent second hydrocarbon attachment may be positioned at least 2 or more amino acid residues from “i”. In one embodiment, the hydrocarbon staple may be attached at residue positions “i” and “i+4” or “i” and “i+7”, wherein the +4 or +7 denotes the number of amino acid positions from “i” that the second hydrocarbon attachment is positioned.
As readily known in the technical field, once a peptide is stapled with a hydrocarbon staple, the original amino acid at the attachment positions may be replaced by a non-natural amino acid. The non-natural amino acid may include but is not limited to S-pentenylalanine, S-octenylalanine, R-octenylalanine or R-pentenylalanine. In one embodiment, the hydrocarbon staple may be attached at residue positions “i” and “i+4”, wherein the amino acids at both “i” and “i+4” positions may be S-pentenylalanine. In another embodiment, the hydrocarbon staple may be attached at residue positions “i” and “i+7”, wherein the amino acids at both “i” and “i+7” positions may be selected from R-octenylalanine, S-pentenylalanine, S-octenylalanine or R-pentenylalanine. In particular, the amino acid at position “i” may be R-octenylalanine or S-octenylalanine and the amino acid at position “i+7” may be S-pentenylalanine or R-pentenylalanine.
In one embodiment, the stapled peptide may comprise a hydrocarbon staple that is attached in Slug zinc fingers (ZF) at the following locations of ZF1s (Zinc Finger 1 stapled) at F130 and A134, ZF2s (Zinc Finger 2 stapled) at L171 and 1178, ZF3s (Zinc Finger 3 stapled) at P197 and Q201, ZF4s (Zinc Finger 4 stapled) at R229 and Q233 or ZF5s (Zinc Finger 5 stapled) at M253 and H257 (
In one embodiment, the stapled peptide may comprise a hydrocarbon staple that is attached in Sox7 helices (H) at the following locations of H1 s (Helix 1 stapled) at V54 and D58 or H2s (Helix 2 stapled) at L75 and S82 (
In one embodiment, the at least one peptide may be formulated as a composition suitable for a convenient mode of administration to the subject. In one embodiment, the at least one peptide may be formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In one embodiment, the at least one peptide may be formulated as a composition suitable for administration by injection. In another embodiment, the at least one peptide may be incorporated into a nanoparticle complex for a suitable mode of administration.
The present disclosure also provides a method of treating a HBV infection in a subject, comprising administering to the subject at least one agent that modulates the activity of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
The present disclosure also provides a method for treating a disease or condition associated with a HBV infection in a subject, comprising administration to the subject of at least one agent that modulates the activity of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A.
In some embodiments, the disease or condition may be liver disease. In another embodiment, the disease, disorder or condition is jaundice, liver inflammation, liver fibrosis, inflammation, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, liver steatosis, hepatocellular carcinoma, liver disease-related transplantation, glomerulonephritis, dyslipidemia, hematopoietic malignancies or pancreatitis.
In one embodiment, the at least one agent inhibits HBV replication. In some embodiments, the at least one agent may be selected from the group consisting of a chemical compound, a small molecule, an oligonucleotide, a protein, a peptide, a stapled peptide, a peptidomimetic, an antibody and an antigen binding molecule.
The oligonucleotide may be a siRNA or shRNA. In some embodiments, the at least one factor may be selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8 and HNF4α9. In one embodiment, the at least one factor may be SNAI2 or SOX7.
In the context of this disclosure, the term “administering” and variations of that term including “administer” and “administration”, includes contacting, applying, delivering or providing a an agent, peptide, compound or composition as described herein to an organism, or a surface by any appropriate means. Convenient modes of administration may include injection (subcutaneous, intravenous, and the like), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration. Depending on the route of administration, the agent may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound. The agent may also be administered parenterally or intraperitoneally.
Dispersions of the agent, peptide, compound or composition as described herein may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi.
In one embodiment, an agent, peptide, compound or composition as described herein may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The agent, peptide, compound or composition may be incorporated into a nanoparticle complex. Alternatively, the agent, peptide, compound or composition as described herein and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet. For oral therapeutic administration, the agent, peptide, compound or composition as described herein may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Suitably, such compositions and preparations may contain at least 1% by weight of the agent. The percentage of the compound(s) of formula (I) and/or (II) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit. The amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained.
The language “pharmaceutically acceptable carrier” is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, use thereof in the therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may also be incorporated into the compositions according to the present disclosure. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of an agent, peptide, compound or composition as described herein is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The agent, peptide, compound or composition as described herein may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. In one embodiment, the carrier may be an orally administrable carrier.
Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration. Also included in the scope of this invention are delayed release formulations.
An agent, peptide, compound or composition as described herein may also be administered in the form of a “prodrug”. A prodrug is an inactive form of a compound which is transformed in vivo to the active form.
In one embodiment, the agent, peptide, compound or composition as described herein may be administered by injection. In the case of injectable solutions, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or anti-fungal agents. Suitable anti-bacterial and/or anti-fungal agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the agent, peptide, compound or composition as described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the agent, peptide, compound or composition as described herein into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the analogue can be incorporated into sustained-release preparations and formulations.
Preferably, the pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.
Single or multiple administrations of the agent, peptide, compound or composition as described herein may be carried out. One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the agent according to the disclosure and an administration pattern which would be suitable for treating the diseases and/or infections to which the agent, peptide, compound or composition as described herein is applicable.
Further, it will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the agent, peptide, compound or composition as described herein given per day for a defined number of days, can be ascertained using convention course of treatment determination tests.
Generally, an effective dosage per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; suitably, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight. More suitably, an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight.
Alternatively, an effective dosage may be up to about 500 mg/m2. For example, generally, an effective dosage is expected to be in the range of about 25 to about 500 mg/m2, about 25 to about 350 mg/m2, about 25 to about 300 mg/m2, about 25 to about 250 mg/m2, about 50 to about 250 mg/m2, and about 75 to about 150 mg/m2.
There is also provided a method of screening at least one agent for modulating HBV replication. The method comprises a) contacting a cell expressing the HBV virus with the at least one agent, wherein the at least one agent modulates the expression of at least one factor selected from the group consisting of SNAI2, SOX7, HNF4α1, HNF4α2, HNF4α3, HNF4α7, HNF4α8, HNF4α9, ARID3A, ATF2, ATF3, ATF4, CALCOCO1, CHD3, CPD, CSNK2A2, CNOT11, DCP1A, DDX39B, DYRK1B, E2F6, E2F7, EPAS1, FOXN2, HIVEP2, HERPUD1, KPNA3, KANK2, LIN54, LSD1, NCL, PAK1IP1, PNPT1, POLR3E, PRDX3, PTP4A1, RNASEH2A, RHOB, RNF4, RNF43, SERBP1, SKA1, SMAD3, SRPK1, STAM, STRADB, SSB, STT3B, TFAP2A, TFAP2C, TFB2M, TRIM24, TRIM68, TRIM27, WDR54 and ZNF518A; b) obtaining a HBV expression profile of the cell contacted with the at least one agent; and c) comparing the HBV expression profile of the cell in b) with an HBV expression profile of a control cell that has not been contacted with said at least one agent, wherein a decrease or increase in the expression of the HBV virus in the cell, relative to the control cell, indicates a modulation of HBV replication by said at least one agent.
In one embodiment, the at least one agent may be selected from the group consisting of a chemical compound, a small molecule, a oligonucleotide, a protein, a peptide, a stapled peptide, a peptidomimetic, an antibody and an antigen binding molecule. In one embodiment, the oligonucleotide may be a siRNA or shRNA.
In one embodiment, the contacting step comprises transfecting the cell with the siRNA or shRNA.
In some embodiments, the cell may be permissive or non-permissive of HBV replication.
A “permissive” cell as disclosed herein relates to a cell that allows the virus to circumvent its defences and replicate. Usually this occurs when the virus has modulated one or several of the cells intrinsic defences. There are conventional methods and techniques known to the skilled person in determining or validating if a cell or cell line is permissive to a certain virus. In contrast, a “non-permissive” cell as disclosed herein relates to a cell that does not allow the virus to circumvent its defences and replicate.
The cell may be selected from the group consisting of a liver cell, colon cell, stomach cell, blood cell, kidney cell and lung cell. In one embodiment, the cell may be a liver cell or a non-liver cell. In another embodiment, the cell may be derived from a HBV liver permissive cell line, such as HepG2, HuH7, PLC/PRF/5 or Hep3B. In another embodiment, the cell may be derived from a non-liver HBV permissive cell line such as Caco-2, Kato III, AGS or A549. In yet another embodiment, the cell may be derived from a non-liver HBV non-permissive cell line, such as T24, 5637, PC-3, Saos-2, FS-4 or COLO316.
In some embodiments, the cell may be comprised in a biological sample. A biological sample may be a sample of tissue or cells from a patient that has been obtained from, removed or isolated from the patient. Non-limiting examples of biological samples include whole blood or a component thereof (e.g. plasma, serum), urine, saliva lymph, bile fluid, sputum, tears, cerebrospinal fluid, bronchioalvcolar lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast milk and pus. In one embodiment, the sample of nucleic acid is obtained from blood, amniotic fluid or a buccal smear. [SF: Please confirm if this is correct]
A biological sample, as contemplated herein, may also include cultured biological materials, including a sample derived from cultured cells, such as culture medium collected from cultured cells or a cell pellet. Accordingly, a biological sample may refer to a lysate, homogenate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. A biological sample may also be modified prior to use, for example, by purification of one or more components, dilution, and/or centrifugation. In some embodiments the biological sample may be obtained from an HBV infected subject.
In some embodiments, the cell may be derived from a cell line selected from the group consisting of HepG2, HuH6, HuH7, HuH4, PLC/PRF/5, Kato III, AGS, HCT116, Caco-2, HL-60, HEK293 and A549.
In one embodiment, the contacting step may comprise culturing the cell in a suitable culture medium that promotes HBV replication. In another embodiment the contacting step may comprise transfecting the cell with a HBV replicon.
In one embodiment, the step of obtaining the HBV expression profile may comprise measuring one or more markers of HBV replication. The one or more markers of HBV replication may be selected from the group consisting of pre-genomic RNA levels of the HBV replicon, hepatitis B surface antigen levels, and hepatitis B core antigen levels.
In some embodiments, the step of obtaining the HBV expression profile may comprise western blot analysis of the at least one factor in the cell and measuring a band intensity of the at least one factor. The band intensity of the at least one factor may be normalized to a control. In some embodiments, a relative difference of 1 to 35% or greater between the band intensity of the at least one factor and the control may indicate a modulation of HBV replication by said at least one agent.
Also provided is a method of identifying at least one factor that modulates replication of a virus. The method comprises a) transfecting at least two cell lines of different origins with an expression construct comprising a selection marker operably linked to a viral promoter of the virus; b) detecting the expression of the selection marker to classify the at least two cell lines into permissive and non-permissive cell lines; c) transfecting the permissive and non-permissive cell lines with an expression construct comprising a viral replicon of the virus; d) screening for expression of at least one factor in said permissive and non-permissive cell lines of c), and comparing the expression of the at least one factor in the permissive cell line to the non-permissive cell line, to identify at least one candidate factor, wherein the differential expression of the at least one factor between the permissive and non-permissive cell line is indicative of identifying at least one candidate factor; e) contacting said permissive cell line expressing the viral replicon with an agent to knock out expression of said at least one candidate factor; and f) comparing the pre-genomic RNA level of said viral replicon in said permissive cell line of e) relative to a control cell line that has not been contacted with said agent, wherein a decrease or increase in the expression level of the pre-genomic RNA level in the permissive cell line, relative to the control cell line, indicates the identification of at least one factor that modulates replication of the virus. (
In one embodiment, the at least one candidate factor may be selected from the group consisting of a transcription factor, regulation factor, RNA processing factor and signaling molecule.
In another embodiment, the selection marker may be green fluorescent protein (GFP) or luciferase.
In one embodiment the agent may be a siRNA or a shRNA.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
Non-limiting examples of the invention, including the best mode will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention and are merely illustrative of the general concept of the present invention.
Materials and Methods
Cells and Reagents
Cell lines HepG2, HuH7, PLC/PRF/5, Hep3B, AGS, A549, PC-3, Saos-2 and FS-4 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Caco-2 was grown in Eagle's minimum essential medium (EMEM) supplemented with 20% FBS, while T24, 5637, and COL0316 were grown in RPMI-1640 supplemented with 10% FBS. Kato III was maintained in DMEM supplemented with 20% FBS.
All cells were grown at 37° C. in a humidified incubator containing 5% CO2, and cultured according to the manufacturing instructions. Primary human hepatocytes were purchased from Triangle Research Labs and cultured as recommended. Total RNA from primary human tissues were purchased from Zyagen.
Primary antibodies used are as follows: HBcAg rabbit polyclonal antibody (Dako), HNF4α7/8/9 mouse monoclonal antibody clone H6939 (R & D Systems), HNF4α1/2/3 mouse monoclonal antibody clone K9218 (R & D Systems), Slug mouse monoclonal antibody clone A-7 (Santa Cruz Biotechnology), Sox7 goat polyclonal antibody (R & D Systems), lamin A/C goat polyclonal antibody (Santa Cruz Biotechnology), β-actin mouse monoclonal antibody (Santa Cruz Biotechnology), albumin mouse monoclonal antibody clone AL-01 (Santa Cruz Biotechnology), transferrin mouse monoclonal antibody clone D-9 (Santa Cruz Biotechnology). HRP-conjugated secondary antibodies were purchased from Dako. Fluorophore conjugated antibodies (Alexa Fluor® 488 and Alexa Fluor® 546) for immunofluorescence staining was obtained from Life Technologies.
Primers for full-length Slug detection were Slug-F (5′ CGATGCTGTAGGGACCGC 3′ (SEQ ID NO: 222)) and Slug-R (5′TGGTCAGCACAGGAGAAAATGC 3′(SEQ ID NO: 223). The primer pair to detect for full-length Sox7 were Sox7-F (5′ TATGCTAGCATGGCTTCGCTGCTGG 3′(SEQ ID NO: 224) and Sox7-R (5′ TAATCTAGACTATGACACACTGTAGCTGTTGTAG 3′(SEQ ID NO: 225). RNA was extracted using the Nucleospin RNA kit (Machery Nagel), and 100 ng RNA was used for first strand cDNA synthesis using the AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Stratagene) according to manufacturers' instructions.
Plasmids
The HBV replicon was cloned from 1.1× of HBV genotype A (nt 1535-1937) and inserted into pcDNA3.1+ vector upstream of the CMV promoter (
Thus, pgRNA synthesis is dependent only on active transcription at the HBVCP (nt 1600-1860), and full-length pgRNA generated terminates at the HBV poly-adenylation signal after initial read-through transcription. HBVCP-Luc was generated by insertion of HBVCP into the PGL3 Basic (Promega) plasmid through KpnI and HindIII restriction sites. HBVCP-GFP was generated by swapping the coding sequence of luciferase in HBVCP-Luc with the coding sequence of green fluorescent protein (GFP) from pTurboGFP-C plasmid (Evrogen).
HNF4α overexpression constructs for each isoform were generated by insertion of amplification products from 2-step PCR (polymerase chain reaction) outlined in Tables 2, 3 and 4 into pIVEX2.5d vector through NotI and XmaI sites and sub-cloned into pcDNA3.1+ using NotI and XbaI restriction sites. cDNA from HepG2 was used as template to generate the HNF4α amplicons.
Overexpression constructs for human Slug and Sox7 were obtained from Origene. Plasmids carrying mutations in HBVCP-Luc were generated by site-directed mutagenesis using the QuickChange II Site-Directed Mutagenesis kit (Agilent Technologies).
Human Transcriptome Array (HTA 2.0)
Total RNA was extracted from cells 30 hours after seeding in 10 cm dishes till 50% confluent. Only good quality RNA with RIN (RNA integrity number) value >9.80 was used. Processed cRNA from the cell lines was hybridized to the Affymetrix Human Transcriptome Array 2.0 according to the manufacturer's protocols. The Affymetrix® Expression Console™ Software (version 1.3.1.187) was used to normalize the data for both gene level and exon level analysis in log2 scale. Only protein coding genes were considered for evaluation.
The global gene expression profiles were assessed using principal component analysis (PCA). The resultant principle components were used as input to determine sample clustering topology by k-means. Differential expression analysis was performed using the Limma package and the p-values were adjusted for multiple testing with Benjamini and Hochberg. Only genes with adjusted p-value<0.05 and |log2(fold-change)|>0.5 were identified as differentially expressed. All the above statistical analysis was implemented using R version 3.1.1.
Differentially expressed genes were subjected to gene ontology annotation analysis using PANTHER classification system (version 10.0). The microarray data is available at Gene Expression Omnibus under the accession number GSE72779.
Validation Assay—(siRNA with HBV pgRNA Readout)
In order to determine if HBV replication is modulated by the listed genes in Tables 5 and 8, over-expression and siRNA knock-down studies were performed to determine if HBVCP activity and pgRNA synthesis would be affected by altered gene expression.
The validation assays were performed in HBV permissive cells. In HBVCP activity assays, 50 ng over-expression constructs or 10 nM siRNA were co-transfected with HBVCP-Luc (luciferase reporter). The relative luminescence was compared to a negative siRNA control or an empty vector control and determined 48 hours post-transfection. In pgRNA assays, 50 ng over-expression constructs or 10 nM siRNA were co-transfected with 800 ng full-length HBV replicon and the relative amount of pgRNA 72 hours post-transfected normalized against GAPDH loading control was compared with that of empty vector or negative siRNA controls.
HBV Replication Assays
For pgRNA assay and immunofluorescence staining for HBcAg, HepG2 and Caco-2 cells were seeded in 24-well plates at 1.0-1.5×105 cells per well and transfected with 800 ng HBV replicon, 1.1 μl Lipofectamine® 2000 (ThermoFisher Scientific) in 100 μl OPTI-MEM. Overexpression constructs were added when indicated at 50 ng per well per construct. Primary human hepatocytes were transfected with 5.6 μl Lipofectamine® 2000.
Silencer® Select Validated siRNA specific for Slug, Sox7 and negative control siRNA were purchased from ThermoFisher Scientific and co-transfected at 10 nM concentration unless stated otherwise. 10 μM peptides in 0.2% DMSO were added 24 hours post-transfection.
Transfected cells were analyzed 48-72 hours post-transfection. Full-length pgRNA was amplified using pgRNA-F (5′ ACACCGCCTCAGCTCTGTATCGAG 3′ (SEQ ID NO: 9)) and pgRNA-R (5′ TTCTTTATAAGGGTCAATGTCCATGCCCC 3′ (SEQ ID NO: 10)) primers with reagents from Expand™ Long Template PCR System (Sigma Aldrich). HBcAg immunofluorescence staining was performed after 20 minute fixation using 4% paraformaldehyde, antigen retrieval with 0.1% TritonX-100 in PBS for 10 minutes, blocking at 4° C. overnight with blocking buffer (1% bovine serum albumin in PBS). Primary antibodies were added at 1:100 dilution in blocking buffer for 2 hours at room temperature, washed thrice with PBS and incubated with 1:1000 secondary antibodies for 1 hour. DAPI was used to stain nuclei.
HBVCP Activity Assays
Luciferase reporter assay for HBVCP transcription activity was performed as recommended (Promega). Cells at a density of 3×104 cell per well were transfected with 160 ng wild type or mutant HBVCP-Luc constructs into 96-well black clear bottom plates with 0.22 μl Lipofectamine® 2000. Additional overexpression constructs were co-transfected at 10 ng per well per construct, and siRNA at 10 nM unless indicated otherwise.
Cells were lysed and luminescence determined 30 hours post-transfection for experiments with HBVCP-Luc alone, and at 48 hours after transfection for experiments with overexpression or knockdown.
In experiments involving transcription factor mimetics, 10 μM peptides in 0.2% DMSO were added 24 hours post-transfection unless stated otherwise.
HBVCP transcription activity was also assessed using the HBVCP-GFP construct and analyzed by flow cytometry using 1.6 μg HBVCP-GFP alone or together with 100 ng overexpression construct, 2.2 μl Lipofectamine® 2000 in 12-well plates, and harvested 48 hours post-transfection. Cells were washed twice in PBS, re-suspended in staining buffer (2% FBS, 10 mM EDTA in PBS), passed through 40 μm cell strainer prior to flow cytometry. Data was acquired using BD FACSCanto II (BD Biosciences) and analyzed using Flowjo v10.
Electrophoretic Mobility Shift Assay (EMSA).
Nuclear lysates were obtained using NEPER (Nuclear and Cytoplasmic Extraction Reagents; ThermoFisher Scientific) as instructed from 4.5×106 cells transfected with empty vector or overexpression constructs using 30 μg plasmids, 33 μl Lipofectamine® 2000 in 10 cm dishes 48 hours after transfection.
EMSA was performed in 20 μl reactions as recommended using the LightShift™ Chemiluminescent EMSA kit (ThermoFisher Scientific), with 0.5 ng biotinylated probes, 2 μg nuclear lysates in the presence of 1-2 μg poly-dIdC. DNA-protein complexes were allowed to form at 37° C. for 45 minutes, and electrophoresis performed on 5% gels with TBE buffer system.
siRNA's
Stapled Peptides
Multiple sequence alignments were performed using Clustal Omega to identify conserved DNA-binding domains and residues between Slug, its orthologues and human Snail and Smuc, as well as between Sox7, its orthologues and human Sox17 and Sox18.
Relative positions of α-helices within the C2H2 zinc finger domains of Slug were predicted using CFSSP (Chou & Fasman Secondary Structure Prediction Server) and JPred4 to identify DNA binding residues at positions −1, +2, +3 and +6 of each α-helix.
Helical wheel diagrams were drawn with HeliQuest to determine DNA-binding surfaces so that hydrocarbon staples may be positioned with low probability of disrupting peptide-DNA interaction. Peptides (GeneScript) were synthesized at >95% purity, dissolved in DMSO and incubated with cells at final concentration of 10 μM in 0.2% DMSO unless stated otherwise. WST-1 assay (Sigma-Aldrich) for cell proliferation was performed as recommended in the manufacturer's instructions.
In Vivo Animal Models
As readily understood and appreciated by those skilled in the art, there are no conventional small animal models for HBV replication as HBV does not naturally infect non-primates. In this regard, the most relevant mouse model for HBV involves injection of primary human hepatocytes into immuno-compromised mice. Since data is already provided herein using primary human hepatocytes, it was determined that injecting the hepatocytes into small animals would not provide significant added advantage to the conclusions drawn and described herein.
Statistical Analysis
Data is expressed as means±s.e.m. and unpaired Student's t-test was performed. P<0.05 was considered significant.
Using a panel of liver and non-liver cell lines, a number of host factors were identified to influence the efficiency of HBV replication.
Through the use of siRNAs to knock down the genes associated with the factors, it was shown that transcriptional activity controlled by a HBV core promoter, as well as the replication of the entire HBV genome, may be modulated. The siRNAs applied for each gene target were in an equimolar mixture for down-regulating the gene (gene ID). This supporting the theory these genes may function as targets to develop interventions that can be used to modulate and control HBV replication.
Development of a HBV Transcription Screen
A HBV core promoter (HBVCP)-green fluorescence protein (GFP) construct was developed for use in transfecting different human cell lines. Subsequently, cell lines that possess host factors that bind to the HBVCP will lead to transcription as evidenced by the increased expression of GFP. Using this screening method, it was shown that cells from liver (n=5), colon (n=2), stomach (n=2), lymphoid (n=1), lung (n=1), were positive for GFP after transfection with the HBVCP-GFP construct.
In comparison, a control CMV-GFP vector was able to produce GFP in all cell lines assayed. This demonstrates that the transcription of HBVCP in non-liver permissive cell lines i.e. KATOIII, AGS, HCT116, Caco-2, HL-60 and A549 is authentic (
Accordingly, the discovery of this unusual shared property among a panel of non-liver cell lines is a powerful tool that can be harnessed for the identification of host cellular factors that drive transcription and replication of HBV.
Screening for Host Factors
The transcriptomic profiles of the various cell lines were assayed using Affymetrix GeneChip Human Trancriptome Array 2.0, and differentially expressed genes between permissive (liver, colon, stomach, blood and lung) and non-permissive cell lines (bladder, breast, prostate, cervix, lung) were identified.
Table 5 lists the 50 genes that have the most significant expression level difference between HBV replication permissive (n=11) and non-permissive (n=6). Table 5 list of transcription factors, RNA processing and signalling molecules that were shown to have the most significant correlation values with the efficiency of HBV replication.
Thus, the gene list in Table 5 refers to the set of genes differentially expressed between HBV permissive and HBV non-permissive cells, which when downregulated using siRNAs resulted in an altered HBVCP activity. This suggests that the encoded transcriptional factors or signalling pathway modulators that regulate HBVCP activity of the identified genes or their products may be targeted for controlling HBV replication.
Validation Assay (siRNA with Luciferase Readout)
HepG2 cells were treated with siRNA and transfected with the HBVCP-luciferase construct to assess the functional role of these factors in the activity of the HBVCP. Table 6 outlines the results and the effect of the siRNA treatment on HBVCP activity as reflected by the luciferase expression level detected (TBC=to be confirmed).
Validation Assay—(siRNA with HBV pgRNA Readout)
Various cell lines were transfected with a HBV full length (1.1×) replicon and the efficacy of the transfection was evaluated.
The results showed that non-liver cell lines such as Kato III (stomach), HCT116, Caco-2 (colon) were able to support the generation of the full length HBV genome (
HepG2 cells were treated with siRNA and transfected with the HBV replicon. Consequently, genes that were shown to be down-regulated by the siRNA treatment and associated with reduced pgRNA levels were compared to controls. The results suggested that the replication of the complete HBV viral genome is affected by these genes (
Table 7 shows that each target (shown as gene code) is tested by both overexpression and inhibition of expression. In order to satisfy these stringent criteria, the overexpressed target should enhance HBV replication, and the downregulation of the target will result in the reduction of HBV replication. Among our earlier, 50 gene list, some 80-90% of targets do not meet this stringent criteria.
Accordingly, genes that are promising targets for modulating HBV replication are recognised as either modulating HBVCP activity and/or modulating pgRNA expression when down-regulated or over-expressed. In this regard, the genes in Tables 6 and 7 were shown to meet such screening criteria and hence have the potential for developing agents/therapeutics for modulation of HBV replication.
Development of a HBV Transcription Screen
To identify tissue restrictive host transcription factors, as in Example 1 described herein, a GFP reporter construct driven by HBVCP was transfected into a panel of liver and non-liver cell lines (n=14,
Screening for Host Factors
Transcriptomic profiles for all 14 cell lines (Affymetrix HTA 2.0, (
Thus, the gene list in Table 8 refers to the genes that are differentially regulated between HBV permissive and HBV non-permissive cells, but their expression correlates significantly with the property of HBV permissiveness in the cells.
SNAI2 (Snail)
SNAI2 codes for Slug, a member of snail protein family of zinc finger transcription factors. Its homologs SNAI1 (Snail) and SNAI3 (Smuc/ZNF293) showed no correlation with HBV permissiveness (
Slug binds to the E-box recognition motif which overlaps the pgRNA initiator within the basal core promoter (BCP) of HBVCP (
The whole virus replication was effectively inhibited as Slug overexpression in HBV permissive cells shut down HBcAg expression (
HNF4α and Isoforms
HNF4α displayed mutually exclusive pattern of mRNA expression with Slug (
As HNF4α isoforms are differentially expressed between permissive and non-permissive cells (
Therefore expression of specific HNF4α isoforms in different tissues was shown to regulate the efficiency of HBV transcription.
Sox7 and interaction with HNF4α
Situated at the 3′ end of the HNF4α binding motif is another motif in the opposite orientation that appears to bind Sox7, a SOX (SRY-related HMG-box) family transcription factor (
Since the Sox7 and HNF4α binding motifs overlap by one nucleotide, Sox7 may affect HNF4α binding. Using an EMSA probe bearing Sox7 motif “TTTGTA”, a prominent Sox7 band was pulled down which greatly diminished when it was mutated to “TCCATA” (
Discussion
Thus, it was shown that Sox7 binding interfered with HNF4α function as Sox7 overexpression in permissive cells reduced transcription at the HBVCP in a dose-dependent manner (
While Slug and Sox7 are independent repressors, co-expression of both factors result in more potent and enhanced repression of HBV replication together (
In primary human hepatocytes, co-expression of Slug and Sox7 significantly diminished whole virus replication as pgRNA was barely detected (
Similarly in 5637 cells, simultaneous mutation of Slug and Sox7 binding motifs lifted the barrier to transcription, activating the HBVCP in the presence of HNF4α3 (
Validation Assay (siRNA)
As further confirmation_of their cooperative effects, HNF4α3 was overexpressed in PC-3 (Sox7+) and Saos-2 (Sox7−) cells treated with Slug siRNA. Only the Saos-2 cell line responded positively (
Taken together, these results show that HNF4α alone cannot overcome the Slug repression signal in the presence of Sox7, and that Slug and Sox7 determine HBV non-permissiveness to silence HBV replication (
Validation of SNAI2(Slug) and Sox7 on HBV Transcription
To validate the functionality of host Slug and Sox7 short stapled peptides were generated from their respective DNA-binding domains to test for their ability to silence HBV transcription.
Slug binds DNA through conserved residues −1, +2, +3 and +6 relative to the α-helices within its five C2H2 zinc fingers (ZFs) that span amino acid residues 128-264 of slug (
A stapled peptide was designed for each α-helix (
The underlining in these Slug sequences indicates the amino acid residues the staples are attached and cross-link. Specifically, the staple in Slug-ZF1s cross-links residues 130 and 134; the staple in Slug-ZF2s cross-links residues 171 and 178; the staple in Slug-ZF3s cross-links residues 197 and 201; the staple in Slug-ZF4s cross-links residues 229 and 233; and the staple in Slug-ZF5s cross-links residues 253 and 257.
All peptides recapitulated Slug function by inhibiting transcription at the HBVCP (
Sox7 binds DNA through its HMG-Box spanning amino acid residues 45-116 and contains 3 α-helices (H1-H3), wherein H1=residues 51-64, H2=residues71-85 and H3=residues 88-107.
Based on Sox17-DNA crystallographic data and sequence conservation between orthologues and human Sox17 and Sox18, only α-helices H1 and H2 have high affinity for DNA (
The underlining in these Sox7 sequences indicates the amino acid residues the staples are attached and cross-link. Specifically, the staple in Sox7-H1s cross-links residues 54 and 58; and the staple in Sox7-H2s cross-links residues 75 and 82.
The stapled peptides inhibited transcription to a similar extent (
Peptide stapling was necessary to preserve peptide function as non-stapled versions could not suppress HBVCP transcription as well (
Discussion
As Slug and Sox7 together prohibit HBV replication, the mimetics Slug-ZF4s, Slug-ZF5s, Sox7-H1s and Sox7-H2s may achieve the greatest inhibition when used together. Sequential addition of mimetics confirmed this, as Slug mimetics outperformed either Slug-ZF4s or Slug-ZF5s alone, adding Sox7-H2s reduced HBVCP activity further with maximal loss when Sox7-H1s was also added (
Accordingly, the stapled peptides and mimics described herein are capable of inhibiting HBV replication in cell lines (
In this regard, the stapled peptides or mimics only require minor modification in terms of length and staple positions to be functionally optimized. Moreover, the stapled peptides or mimics are not cytotoxic to HepG2 cells when used at concentrations sufficient to inhibit HBV replication (
While nucleoside/nucleotide analogues and interferons are available for the treatment of HBV infection, they require long-term use because they cannot suppress HBV replication to sufficiently low levels for immune clearance. Thus, stapled peptides or mimics developed on the findings and data disclosed herein may bring HBV replication to a minimum for current therapeutics to be more effective, hence resulting the advantage of reducing drug dependency of patients for viral clearance. In this regard, the representative stapled peptides or mimics described herein were shown to be significantly advantageous as they were almost as potent as the native whole proteins (for example, Slug and Sox7). In that regard, the specific peptide sequences of a length of about 13-14 amino acids appear to be sufficiently specific as they possess the required potency to inhibit HBVCP.
In summary, the present invention has elucidated that Slug and Sox7 are independent transcription repressors that decide the ability of a cell to support HBV replication. While HNF4α is an activator that enhances viral replication efficiency, it can only do so when Slug and Sox7 are absent. Since the potent HBVCP activator HNF4α1 is preferentially enriched in liver cells, the data disclosed herein provides fundamental insights on HBV replication primarily in the liver.
Using stapled peptides and mimics derived from DNA-binding domains of Slug and Sox7, HBV replication has been shown to be successfully silenced by inhibiting HBVCP activity and pgRNA transcription, demonstrating that novel HBV therapeutics can be developed by targeting the identified host factors described herein. Moreover, as viruses are dependent on host factors for replication, this approach of host repressor molecular mimicry may also be useful for inhibiting replication of other viruses.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201505551 U | Jul 2015 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2016/050338 | 7/15/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/010950 | 1/19/2017 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7939115 | Huang | May 2011 | B2 |
| 20020045161 | Allaway et al. | Apr 2002 | A1 |
| 20020045191 | Schneider et al. | Apr 2002 | A1 |
| 20130131094 | Nakache | May 2013 | A1 |
| 20140256742 | Baker | Sep 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 02094796 | Nov 2002 | WO |
| WO 2010127246 | Nov 2010 | WO |
| WO 2011106106 | Sep 2011 | WO |
| WO 2013106548 | Jul 2013 | WO |
| WO 2013139895 | Sep 2015 | WO |
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