Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses

Definitions

• the present invention relates to methods and compositions for treating and preventing human hepatitis B virus (HBV) and/or human hepatitis D virus (HDV) infection.
• HBV human hepatitis B virus
• HDV human hepatitis D virus
• HBV human hepatitis B virus
• HDV satellite hepatitis D virus
• HBV is an enveloped virus containing a small genome of 3.2 Kb partially double-stranded DNA encoding four overlapping reading frames.
• HBV envelope consists of the small (S), middle (M) and large (L) envelope proteins that are multiple transmembrane spanners and share the same C-terminal domain corresponding to the S protein but differ at their N-terminal domains (Fig. 1 A) (Seeger et al., in Field's Virology, D. M. Knipe, P. M. Howley, Eds. (Lippincott, Williams, and Wilkins, Philadelphia, 2007), vol. 2, pp. 2977; Heermann et al, J Virol 52, 396 (1984)).
• HDV is a small satellite RNA virus of HBV carrying all three HBV envelope proteins, and can only propagate when it coexists with HBV.
• L protein and the integrity of S protein are critical for HBV and HDV infections.
• the pre-Sl domain of the L protein is a key determinant for both HBV and HDV entry and is believed to mediate viral binding to a specific cellular receptor(s) on hepatocytes (Seyec et al, / Virol 73, 2052 (1999); Blanchet & Sureau, / Virol 81, 5841 (2007); Duff et al, / Virol 83, 12443 (2009); Chouteau et al, J Virol 75, 11565 (2001)).
• a number of receptor candidates that are pre-Sl binding proteins have been reported in the past, however none of them has been confirmed to be essential for viral infection (Glebe & Urban, World J Gastroenterol 13, 22 (2007)).
• NTCP Sodium taurocholate cotransporting polypeptide
• the present invention relates to compositions and methods of treating or preventing HBV and/or HDV infection or a disease associated with said infection in a mammal through modulation of the production and/or function of the NTCP protein and polynucleotide, or its interaction with the viruses.
• the present invention provides a method for treating and/or preventing HBV and/or HDV infection or a disease associated with said infection in a mammal, which method comprises administering, to a mammal in need of such treatment and/or prevention, an effective amount of an agent that prevents or reduces production and/or function of NTCP in said mammal, and/or an agent that prevents or reduces interaction between NTCP in said mammal and HBV and/or HDV.
• the mammal being treated may be a human, a chimpanzee, or a tupaia, or other mammals, e.g., mouse or rat, expressing the NTCP from human, chimpanzee, or tupaia.
• the mammal being treated may be a mammal transplanted with human, chimpanzee or tupaia hepatocytes.
• the method may be used for treating HBV and/or HDV infection.
• the method may be used for preventing HBV and/or HDV infection.
• the method may be used for treating and/or preventing HBV infection.
• the method may be used for treating and/or preventing HDV infection.
• the agent is not a polypeptide corresponding to the N-terminal 2-48 residues of the pre-S 1 domain of the HBV L envelope protein or an antibody that binds to said polypeptide.
• the agent being administered may prevent or reduce production and/or function of NTCP.
• the agent comprises an siRNA targeting the gene encoding NTCP, wherein the siRNA comprises a nucleotide sequence set forth in siRNA-1 to siRNA-4 targeting tupaia SLC10A1 gene, or siRNA- 11, siRNA-405, siRNA- 406, siRNA-pool(4), siRNA-pool(5), siRNA-pool(6) or siRNA-pool(7) targeting human SLC10A1 gene.
• the agent comprises an antisense RNA targeting the gene encoding NTCP.
• the agent inhibits or modifies a nuclear factor that controls NTCP transcription.
• the agent may be TTNPB, a pan- RAR agonist, all-trans retinoic acid (ATRA), 9-cis retinoic acid (9CRA), etc.
• the agent inhibits or modifies a histone or genomic DNA modification that controls NTCP transcription.
• DNA methylation inhibitors including nucleoside analogs of cytosine, such as 5-azacytidine, 5-aza-2'-deoxycytidine (5-azadC); MG98 (an antisense oligodeoxynucleotide directed against the 3 ' untranslated region of the DNA methyltransferase-1 enzyme mRNA); histone deacetylase inhibitors (HDAC inhibitors, HDI), such as vorinostat (suberoylanilide hydroxamic acid; SAHA), and mocetinostat (MGCD0103), are all contemplated by the invention.
• the agent inhibits or modifies the phosphorylation and/or glycosylation of NTCP, such as
• Tunicamysin which is a mixture of homologous nucleoside antibiotics, which blocks all N-glycosylation of proteins.
• the agent is a PI3K inhibitor, e.g., LY294002, wortmannin, etc.
• the agent being administered may prevent or reduce interaction between NTCP and HBV and/or HDV in the mammal.
• the agent comprises an antibody that specifically binds to NTCP, wherein the antibody specifically binds to a portion of NTCP that interacts with HBV and/or HDV, e.g., an epitope in the extracellular domains.
• the extracellular domain comprises amino acids 17-27, 73-89, 142-152, 207-217 or 275-278 of NTCP (Fig. 2).
• the agent comprises a variant form of NTCP, wherein the variant form of NTCP is selected from the group consisting of a mutant NTCP, a fragment of NTCP and a soluble NTCP polypeptide.
• the fragment comprises an extracellular domain of NTCP.
• the variant form of NTCP does not support HBV and/or HDV infection while functions as the transporter, for example, the mouse or rat NTCP.
• the agent comprises a NTCP substrate or an NTCP substrate derivative or analogue.
• the NTCP substrate comprises a bile acid, wherein the bile acid is selected from the group consisting of taurolithocholate, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid and hyodeoxycholic acid.
• the bile acid is selected from the group consisting of taurolithocholate, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid and hyodeoxycholic acid.
• the method may further comprise administering a pharmaceutically acceptable carrier or excipient with the agent.
• the agent is administered via an oral, nasal, inhalational, parental, intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, topical, or rectal route.
• the agent is administered to the liver of the mammal.
• the agent is administered with a drug for the treatment and/or prevention of HBV and/or HDV infection, wherein the drug is selected from the group consisting of an interferon, a nucleoside analogue, a non-nucleoside antiviral and a non-interferon immune enhancer.
• the drug is selected from the group consisting of Intron A
• Telbivudine Tyzeka (Telbivudine), Hepsera (Adefovir), Baraclude (Entecavir), Viread (Tenofovir), Clevudine (L-FMAU), Emtricitabine (FTC), MIV-210, Amdoxovir (DAPD), NOV-205 (BAM 205), LB80380 (ANA380), Myrcludex B, HAP Compound Bay 41- 4109, REP 9AC, Alinia (Nitazoxanide), and Zadaxin (Thymosin alpha- 1).
• the drug is Myrcludex B.
• a pharmaceutical composition for treating and/or preventing HBV and/or HDV infection or a disease associated with said infection in a mammal which pharmaceutical composition comprises an effective amount of an agent that prevents or reduces production and/or function of NTCP in said mammal, and/or an agent that prevents or reduces interaction between NTCP in said mammal and HBV and/or HDV, and a pharmaceutically acceptable carrier or excipient.
• mutant human NTCP protein comprising SN105/106AA and/or E257A or an isolated form thereof.
• the mutant human NTCP protein may further comprise Q68A and/or S226A.
• an isolated antibody that specifically binds to said mutant human NTCP protein but not the wild-type human NTCP protein.
• an isolated polynucleotide encoding said mutant human NTCP protein, and a vector comprising said polynucleotide.
• a cell and a non-human animal comprising said vector.
• dsRNA ribonucleic acid
• a first strand of the dsRNA is substantially identical to at least 19 consecutive nucleotides of the human SLCIOAI gene, and a second strand of the dsRNA is substantially complementary to the first strand, wherein the first strand of the dsRNA targets nucleotides 87-105, 652- 670, or 1 136-1 154 of the human SLCIOAI cDNA.
• the dsRNA is encoded by a polynucleotide, wherein the first strand and the second strand of the dsRNA are transcribed from said polynucleotide and form a hairpin loop.
• an isolated single stranded oligonucleotide that is complementary to a portion of the human SLCIOAI mPvNA of at least 10 consecutive nucleotides, wherein the oligonucleotide targets nucleotides 87-105, 652-670, or 1 136-1 154 of the human SLCIOAI cDNA.
• an isolated polypeptide comprising a tupaia NTCP protein or a variant thereof. Also provided is an isolated antibody or antigen-binding fragment thereof that specifically binds to a tupaia NTCP protein or a variant thereof. Further provided is an isolated polynucleotide encoding a tupaia NTCP protein or a variant thereof. Additionally provided is a vector comprising an isolated polynucleotide encoding a tupaia NTCP protein or a variant thereof, and a cell or a non- human transgenic animal comprising said vector.
• dsRNA ribonucleic acid
• a first strand of the dsRNA is substantially identical to at least 19 consecutive nucleotides of the tupaia SLCIOAI gene, and a second strand of the dsRNA is substantially complementary to the first strand.
• the first strand of the dsRNA targets nucleotides 83- 101 , 342-360, 465-483, or 630-648 of the coding region of the tupaia SLCIOAI gene.
• the dsRNA is encoded by a polynucleotide, wherein the first strand and the second strand of the dsR A are transcribed from said polynucleotide and form a hairpin loop.
• an isolated single stranded oligonucleotide that is complementary to a portion of the tupaia SLC10A1 m NA of at least 10 consecutive nucleotides.
• the oligonucleotide targets nucleotides 83-101, 342- 360, 465-483, or 630-648 of the coding region of the tupaia SLC10A1 gene.
• a vector for the production of NTCP comprising a polynucleotide sequence encoding an NTCP protein from human, tupaia, chimpanzee, mouse, rat, or another mammal, wherein the NTCP protein comprises an EGFP or C9 tag at the C-terminus.
• a cell or a non-human animal comprising said vector.
• the drug candidates are small molecules, a polypeptide library comprising mutants and/or fragments of NTCP, antibodies that specifically bind to NTCP, siRNAs or antisense RNAs or NTCP substrates or NTCP substrate derivatives or analogues.
• the antibodies specifically bind to the extracellular domain of NTCP.
• the antibodies are monoclonal antibodies. Further provided is a drug candidate identified by said use.
• a no-longer-susceptible primary hepatocyte comprising a polynucleotide encoding an exogenous NTCP.
• the hepatocyte may be susceptible to HBV and/or HDV infection, or binds to HBV and/or HDV.
• the no-longer-susceptible primary hepatocyte is a primary human or tupaia hepatocyte.
• the drug candidates are small molecules, a polypeptide library comprising mutants and/or fragments of NTCP, antibodies that specifically bind to NTCP, siRNAs or antisense RNAs or NTCP substrates or NTCP substrate derivatives or analogues.
• the antibodies specifically bind to the extracellular domain of NTCP.
• the antibodies are monoclonal antibodies. Further provided is a drug candidate identified by said use.
• a vector system for constructing a non- human NTCP knockout/knockin animal model comprising a vector capable of homologous recombination at the SLC10A1 locus and a vector encoding an exogenous NTCP protein.
• a method of making an animal model for HBV and/or HDV infection using said vector system using, e.g., the Zinc-finger and TALEN nuclease technology.
• a non-human animal e.g., mouse or rat, model for HBV and/or HDV infection made using said method.
• a non-human, e.g., mouse or rat, animal model for HBV and/or HDV infection or a disease associated with said infection which comprises a vector comprising a polynucleotide encoding an exogenous NTCP or a variant thereof.
• the non-human animal model may be susceptible to HBV and/or HDV infection, or binds to HBV and/or HDV.
• the exogenous NTCP is from human, chimpanzee or tupaia.
• the drug candidates are small molecules, a polypeptide library comprising mutants and/or fragments of NTCP, antibodies that specifically bind to NTCP, siRNAs or antisense RNAs or NTCP substrates or NTCP substrate derivatives or analogues.
• the antibodies specifically bind to the extracellular domain of NTCP.
• the antibodies are monoclonal antibodies. Further provided is a drug candidate identified by said use.
• a method for treating HBV and/or HDV infection or a disease associated with said infection in a mammal comprises administering an effective amount of an agent that increases the production and/or function of NTCP in said mammal.
• a pharmaceutical composition for treating HBV and/or HDV infection or a disease associated with said infection in a mammal which pharmaceutical composition comprises an effective amount of an agent that increases the level and/or function of NTCP in said mammal, and a pharmaceutically acceptable carrier or excipient.
• the disease associated with HBV and/or HDV infection may be cirrhosis and/or liver cancer.
• the agent being administered may be a polypeptide.
• the polypeptide is a mouse or rat NTCP protein.
• the polypeptide is a variant form of NTCP.
• the variant form of NTCP does not support HBV and/or HDV infection while functions as the transporter.
• the variant form of NTCP comprises a mutation in the extracellular domains.
• the variant form of NTCP comprises Q68A.
• the agent being administered may be a polynucleotide.
• a method of diagnosis, prognosis or treatment monitoring of HBV and/or HDV infection or a disease associated with said infection comprising assessing the level and/or function of NTCP in a subject suspected of or being treated for HBV and/or HDV infection or said disease.
• polypeptide may be assessed. Also provided herein is a method of companion
• a screening system for drug candidates for treating and/or preventing HBV and/or HDV infection comprising: a) a surrogate of HBV L protein; b) a hepatic cell line stably expressing a mammalian NTCP that interacts with HBV and/or HDV; c) cell culture ingredients; and d) a detecting agent.
• the surrogate of HBV L protein is HDV pseudotyped with L envelope protein from HBV or a Woolly monkey hepatitis virus (WMHV).
• the detecting agent is an antibody to the HDV or an antibody to the WMHV.
• the antibody to the HDV is the HDV delta antigen monoclonal antibody 4G5.
• screening system for screening drug candidates for treating and/or preventing HBV and/or HDV infection or a disease associated with said infection. Further provided herein is a drug candidate identified by the screening system.
• the agent being administered may prevent or reduce production and/or function of NTCP.
• the agent comprises an siRNA targeting the gene encoding NTCP, wherein the siRNA comprises a nucleotide sequence set forth in siRNA-1 to siRNA-4 targeting tupaia SLC10A1 gene, or siRNA-11, siRNA-405, siRNA- 406, siRNA-pool(4), siRNA-pool(5), siRNA-pool(6) or siRNA-pool(7) targeting human SLC10A1 gene.
• the agent comprises an antisense RNA targeting the gene encoding NTCP.
• the agent inhibits or modifies a nuclear factor that controls NTCP transcription.
• the agent being administered may prevent or reduce interaction between NTCP and HBV and/or HDV in the mammal.
• the agent comprises an antibody that specifically binds to NTCP, wherein the antibody specifically binds to a portion of NTCP that interacts with HBV and/or HDV, e.g., an epitope in the extracellular domains.
• the extracellular domain comprises amino acids 17-27, 73-89, 142-152, 207-217 or 275-278 of NTCP (Fig. 2).
• the agent comprises a variant form of NTCP, wherein the variant form of NTCP is selected from the group consisting of a mutant NTCP, a fragment of NTCP and a soluble NTCP polypeptide.
• the fragment comprises an extracellular domain of NTCP.
• the variant form of NTCP does not support HBV and/or HDV infection while functions as the transporter, for example, the mouse or rat NTCP.
• the agent comprises a NTCP substrate or an NTCP substrate derivative or analogue.
• the NTCP substrate comprises a bile acid, wherein the bile acid is selected from the group consisting of taurolithocholate, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid and hyodeoxycholic acid.
• the genotype calls may be made using an imputation algorithm, wherein the HapMap may be used for the imputation algorithm.
• the use may further comprise administering a pharmaceutically acceptable carrier or excipient with the agent.
• the agent is administered via an oral, nasal, inhalational, parental, intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, topical, or rectal route.
• the agent is administered to the liver of the mammal.
• the agent is
• Telbivudine Tyzeka (Telbivudine), Hepsera (Adefovir), Baraclude (Entecavir), Viread (Tenofovir), Clevudine (L-FMAU), Emtricitabine (FTC), MIV-210, Amdoxovir (DAPD), NOV-205 (BAM 205), LB80380 (ANA380), Myrcludex B, HAP Compound Bay 41- 4109, REP 9AC, Alinia (Nitazoxanide), and Zadaxin (Thymosin alpha- 1).
• the drug is Myrcludex B.
• Figure 1 shows the identification of pre-Sl binding molecule(s) on primary tupaia hepatocytes (PTHs) with photo-reactive peptide Myr-47/WTt,.
• PTHs primary tupaia hepatocytes
• FIG. 1 shows the identification of pre-Sl binding molecule(s) on primary tupaia hepatocytes (PTHs) with photo-reactive peptide Myr-47/WTt,.
• A Schematic diagram of HBV envelope proteins and N-terminal peptides of pre-Sl domain.
• Pre-Sl (2- 47) 2 -47 th residues of the pre-Sl domain of the L protein of HBV (S472 strain, genotype C). Residue numbering is based on genotype D.
• Star indicates highly conserved residues among genotypes.
• B Myr-47/WTb dose dependently inhibited HDV virion binding.
• FIG. 4 shows activities of photo-reactive bait peptides for inhibiting HDV binding and HBV infection.
• HBV envelope-bearing HDV viruses were prepared and verified for specific binding and infection on primary tupaia hepatocytes (PTHs).
• PTHs primary tupaia hepatocytes
• Left panel HDV binding with PTH is L protein dependent.
• HDV virions enveloped with HBV envelope protein(s) of LS, LMS, MS and S only, respectively, were incubated with PTH for 4 hours at 16°C followed by extensive wash with cold PBS for 5 times.
• Cell- bound virions were quantified by real time RT-PCR with GAPDH as an internal control and normalized against total input virus genome copy numbers. Means ⁇ s.d. of representative result from three independent experiments are shown.
• FIG. 5 shows that mouse monoclonal antibody 2D3 targets residues 19-33 on pre-Sl .
• Pre-Sl(-11-47) the first 58 amino acids of the pre-Sl domain of HBV (strain S472), amino acids were numbered according to HBV L protein of genotype D; NC36 conjugated with keyhole limpet hemocyanin (KLH) was the immunogen peptide for generating 2D3 in mice; SP15, SD15, LAI 5, LD15 and FG15 peptides were used for epitope mapping of 2D3.
• B 2D3 epitope mapped by peptide competition assay. 2D3 was purified with protein-G Sepharose beads.
• FIG. 6 shows Western blot analysis of WTb cross-linked molecule(s).
• WT Non-photo-reactive Myr-47/WT peptide
• B The abundance of the target protein(s) decreased over time of PTH cells in culture.
• PTHs plated on collagen- coated 3.5 cm dishes were analyzed for crosslinking with 200 nM WTb on days 1-6 after cell plating.
• C WTb crosslinked with primary hepatocytes from human (PHHs). PHH cells were plated on collagen coated 3.5 cm dishes one day before crosslinking.
• Figure 7 shows mass spectrometry analysis of target protein(s).
• PTH cells photo-crosslinked with 200 nM of WT b or N9K b bait peptide were lysed and purified in tandem with Streptavidin Dynal Tl beads, 2D3 conjugated beads, and Streptavidin Dynal Tl beads in 1XRIPA buffer. Extensive wash was applied to the beads for each purification step. The samples were treated with or without PNGase F as indicated prior to the last step of Streptavidin beads precipitation. The final purified samples were subjected to SDS-PAGE and followed by silver staining. Boxed areas indicate the bands cut for Mass spectrometry analysis.
• M protein molecular weight marker.
• Figure 9 shows binding of NTCP with N-terminal peptide of pre-Sl and HDV virions.
• A 293T cells transfected with hNTCP, tsNTCP, tsNTCP-EGFP (tsNTCP fused with C-terminal EGFP) plasmids or a vector control were crosslinked with 200nM Myr- 47/WTb similarly as in Fig. 1D-E 24hrs post transfection. PTHs crosslinked samples were run as positive controls.
• FIG 10 shows HBV and HDV infection of hepatocytes requires NTCP.
• A- B Freshly isolated PTH cells were transfected with siRNAs against tsNTCP or a control siRNA and re-seeded onto supporting fibroblast cells one day after transfection. Three days later, lxl 0 5 PTHs were inoculated with 1x10 genome equivalent copies of purified HBV.
• A infected cells were visualized on 8 days post infection (dpi) by intracellular staining of HBV S antigen (HBsAg) with mAb 17B9 (Red). Hepatocytes were visualized by antibody against CYP3A4 (Green), cell nuclei were stained with DAPI (Blue).
• Figure 11 shows recombinant viruses AAV8 and Lenti-VSV-G infected primary tupaia hepatocytes.
• PTHs were isolated and plated on collagen-coated plates, three days after cell plating, recombinant adenovirus-associated -virus 8 (AAV8) carrying an EGFP reporter gene (A) or recombinant lentivirus pseudotyped by VSV-G carrying an EGFP gene (B) was inoculated with the hepatocytes for 8hrs. Images were taken on day 6 post infection.
• AAV8 adenovirus-associated -virus 8
• B recombinant lentivirus pseudotyped by VSV-G carrying an EGFP gene
• FIG. 12 shows NTCP expression confers susceptibility to HDV and HBV infection.
• A NTCP mRNA expression level in the indicated cell lines and primary hepatocytes. The Huh-7 was used to normalize the relative expression levels in other cells.
• B Huh-7 cells, 24 hrs after transfection with hNTCP expression plasmid or vector control were infected with HDV at 500 genome equivalent copies/cell. On 8 dpi, HDV delta antigen was stained in green, nuclei were in blue.
• Huh-7 cells transfected with hNTCP were infected with HDV similarly as in panel B in the presence or absence of HBV entry inhibitors: HBIG (hepatitis B immune globulin), Myr-59, and anti-HBsAg mAb 17B9.
• HBV entry inhibitors HBIG (hepatitis B immune globulin), Myr-59, and anti-HBsAg mAb 17B9.
• HDV delta antigen specific mAb 4G5 was used as a control.
• HDV RNA copies of infected cells were quantified by real-time RT-PCR on 6 dpi.
• Huh-7 cells transfected with hNTCP were infected with HDV similarly as in panel B. The level of HDV viral RNAs in cells on indicated dpi was quantified by real-time RT-PCR.
• NTCP mRNA level in PTHs decreased rapidly over time during in vitro culturing.
• F PTH cells that were cultured for 13 days in vitro were transfected with hNTCP or a vector control and re-seeded onto supportive cells, followed by infection with HBV at 100 genomic copies/cell 24 hrs post cell re-seeding. On 8 dpi, intracellular HBsAg was stained with 17B9 in red, hepatocytes with anti-CYP3A4 in green, nuclei in blue.
• G-H HepG2 stable cell line expressing hNTCP was infected with HBV at 100 genomic copies/cell in the presence or absence of Myr-59 or HBIG. Secreted HBeAg at 4, 6 dpi (G) and HBV RNAs on 6 dpi were measured by ELISA and quantitative RT-PCR (H), respectively.
• Figure 15 shows surface expression of human NTCP mutants in Hu7 cells. 1, wt; 2, Q68A; 3, SN105/106AA; 4, S226A; 5, E257A.
• Figure 18 shows the structure of taurocholate (A) and taurolithocholate (B) and infection of HBV on PTH in the presence of taurocholate and taurolithocholate, respectively.
• Figure 19 shows HDV infection of primary mouse hepatocytes and primary rat hepatocytes transfected with human SLC10A1.
• PMH primary mouse hepatocytes
• PRH primary rat hepatocytes
• hAl human SLC10A1
• pcDNA6 expression vector
• Figure 20 shows that LY294002, a morpholine derivative of quercetin that inhibits phosphoinositide 3-kinases (PI3Ks), reduced HBV infection.
• PI3Ks phosphoinositide 3-kinases
• Figure 21 shows hepatoma cell line HepG2 transfected with NTCP was able to support HBV infection in vitro. HBV viral e antigens were examined as indicated on days 4, 7, and 10 post infection, viral specific RNAs were examined on day 12 after the infection.
• Figure 22 shows the functionality of NTCP in supporting the authentic HBV infection of human hepatocytes in vitro. Detailed Description of the Invention
• Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzvmology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al, eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Remington, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003).
• polypeptide oligopeptide
• peptide protein
• polymers of amino acids of any length e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more amino acids.
• the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
• the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
• polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
• variants are used in reference to polypeptides that have some degree of amino acid sequence identity to a parent polypeptide sequence.
• a variant is similar to a parent sequence, but has at least one substitution, deletion or insertion in their amino acid sequence that makes them different in sequence from a parent polypeptide.
• variants have been manipulated and/or engineered to include at least one substitution, deletion, or insertion in their amino acid sequence that makes them different in sequence from a parent.
• a variant may retain the functional characteristics of the parent polypeptide, e.g. , maintaining a biological activity that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of that of the parent polypeptide.
• an "antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule, and can be an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE.
• IgY which is the major antibody type in avian species such as chicken, is also included within the definition.
• the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the
• immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
• the term "antigen" refers to a target molecule that is specifically bound by an antibody through its antigen recognition site.
• the antigen may be monovalent or polyvalent, i.e., it may have one or more epitopes recognized by one or more antibodies.
• Examples of kinds of antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc.
• epitopes refers to a peptide sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000 amino acids (or any integer there between), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence.
• the length of the fragment may, for example, comprise nearly the full-length of the antigen sequence, or even a fusion protein comprising two or more epitopes from the target antigen.
• An epitope for use in the subject invention is not limited to a peptide having the exact sequence of the portion of the parent protein from which it is derived, but also encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and
• the term "specifically binds" refers to the binding specificity of a specific binding pair. Recognition by an antibody of a particular target in the presence of other potential targets is one characteristic of such binding. Specific binding involves two different molecules wherein one of the molecules specifically binds with the second molecule through chemical or physical means. The two molecules are related in the sense that their binding with each other is such that they are capable of
• a "tag” or an “epitope tag” refers to a sequence of amino acids, typically added to the N- and/or C- terminus of a polypeptide.
• tags fused to a polypeptide can facilitate polypeptide purification and/or detection.
• a tag or tag polypeptide refers to polypeptide that has enough residues to provide an epitope recognized by an antibody or can serve for detection or purification, yet is short enough such that it does not interfere with activity of chimeric polypeptide to which it is linked.
• the tag polypeptide typically is sufficiently unique so an antibody that specifically binds thereto does not substantially cross-react with epitopes in the polypeptide to which it is linked.
• Suitable tag polypeptides generally have at least 5 or 6 amino acid residues and usually between about 8-50 amino acid residues, typically between 9-30 residues.
• the tags can be linked to one or more chimeric polypeptides in a multimer and permit detection of the multimer or its recovery from a sample or mixture.
• Such tags are well known and can be readily synthesized and designed.
• Exemplary tag polypeptides include those used for affinity purification and include His tags, the influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody.
• Polynucleotide or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
• the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
• phosphoamidates, cabamates, etc. and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s).
• proteins e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.
• intercalators e.g., acri
• any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports.
• the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping groups moieties of from 1 to 20 carbon atoms.
• Other hydroxyls may also be derivatized to standard protecting groups.
• Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0- methyl-2'-0- allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
• One or more phosphodiester linkages may be replaced by alternative linking groups.
• linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S("thioate”), P(S)S ("dithioate”), "(O)NR 2 ("amidate”), P(0)R, P(0)OR' , CO or CH 2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—0--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
• substantially complementary or substantially matched means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, “substantially complementary or substantially matched” means that two nucleic acid sequences can hybridize under high stringency condition(s).
• Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS, at 42°C.
• High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x
• RNA interference refers generally to a process in which a double-stranded RNA molecule or a short hairpin RNA molecule reducing or inhibiting the expression of a nucleic acid sequence with which the double- stranded or short hairpin RNA molecule shares substantial or total homology.
• short interfering RNA or “siRNA” or “RNAi agent” refers to an RNA (or RNA analog) sequence comprising between about 10-50 nucleotides (or nucleotide analogs) that elicits RNA interference. See Kreutzer et al, WO 00/44895; Zernicka-Goetz et al, WO
• vector refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well known within the skill of the artisan.
• An expression vector includes vectors capable of expressing DNA's that are operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments.
• an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.
• Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
• a promoter region or promoter element refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked.
• the promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter.
• the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include the bacteriophage T7 and T3 promoters, and the like.
• operatively linked or operationally associated refers to the functional relationship of DNA with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
• operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
• Treating” or “treatment” or “alleviation” refers to therapeutic treatment wherein the object is to slow down (lessen) if not cure the targeted pathologic condition or disorder or prevent recurrence of the condition.
• a subject is successfully “treated” if, after receiving a therapeutic amount of a therapeutic agent, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of the particular disease. Reduction of the signs or symptoms of a disease may also be felt by the patient. A patient is also considered treated if the patient experiences stable disease.
• treatment with a therapeutic agent is effective to result in the patients being disease-free 3 months after treatment, preferably 6 months, more preferably one year, even more preferably 2 or more years post treatment.
• prediction or “prognosis” is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, or the likely outcome of a disease.
• the prediction relates to the extent of those responses or outcomes.
• the prediction relates to whether and/or the probability that a patient will survive or improve following treatment, for example treatment with a particular therapeutic agent, and for a certain period of time without disease recurrence.
• the predictive methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
• the predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as a given therapeutic regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc.
• pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
• the use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.
• Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates,
• dihydrogenphosphates metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene - 1 -sulfonates, naphthalene-2-sulfonates, phenylacetate
• phenylbutyrates citrates, lactates, ⁇ -hydroxybutyrates, glycolates, tartrates, and mandelates.
• the term "therapeutically effective amount” or “effective amount” refers to an amount of a therapeutic agent that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate the infection or the progression of the infection or an associated disease associated with the infection.
• a therapeutically effective dose further refers to that amount of the therapeutic agent sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
• a therapeutically effective dose refers to that ingredient alone.
• a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
• an effective amount is an amount that inhibits or reduces viral entry into a cell.
• combination refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a compound and a combination partner (e.g., another drug as explained below, also referred to as
• therapeutic agent or “co-agent”
• combination partners may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect.
• coadministration or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
• pharmaceutical combination as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
• level or “levels” are used to refer to the presence and/or amount of protein or polynucleotide, and can be determined qualitatively or quantitatively.
• a “qualitative” change in the protein or polynucleotide level refers to the appearance or disappearance of a protein or polynucleotide that is not detectable or is present in samples obtained from normal controls.
• a “quantitative” change in the levels of one or more proteins or polynucleotides refers to a measurable increase or decrease in the protein or polynucleotide levels when compared to a healthy control.
• a "healthy control” or "normal control” is a biological sample taken from an individual who does not suffer from an HBV and/or HDV infection or a disease associated with the infection.
• a “negative control” is a sample that lacks any of the specific analyte the assay is designed to detect and thus provides a reference baseline for the assay.
• NTCP is a multiple transmembrane glycoprotein, presumably spans cellular membrane up to 10 times with small
• NTCP expression level in diseased livers is significantly lower than that of normal livers.
• NTCP suitable for the embodiments of the present disclosure may be produced with recombinant DNA technology in various host cells.
• pcDNA6 may be used to express the recombinant NTCP proteins from human, tupaia, rat and mouse (Fig. 3).
• the proteins have an EGFP or a nine-amino acid sequence of the rhodopsin carboxyl tail (TETSQVAPA, C9) tag at the C-terminus.
• the host cells are selected from bacterial, fungal, plant, yeast, insect or mammalian cells.
• the term host cell includes both the cells, progeny of the cells and protoplasts created from the cells that are used to produce a NTCP according to the disclosure.
• the host cells are prokaryotic cells and typically bacteria host cells.
• inducible promoter may refer to a promoter that is active under environmental or developmental regulation.
• An expression vector comprising a DNA construct with a polynucleotide encoding the NTCP may be any vector which is capable of replicating autonomously in a given host organism or of integrating into the DNA of the host.
• the expression vector can be a plasmid.
• two types of expression vectors for obtaining expression of genes are contemplated.
• the first expression vector may comprise DNA sequences in which the promoter, NTCP -coding region, and terminator all originate from the gene to be expressed.
• gene truncation can be obtained by deleting undesired DNA sequences (e.g., DNA encoding unwanted domains) to leave the domain to be expressed under control of its own transcriptional and translational regulatory sequences.
• the second type of expression vector may be preassembled and contains sequences needed for high-level transcription and a selectable marker.
• the coding region for the NTCP gene or part thereof can be inserted into this general-purpose expression vector such that it is under the transcriptional control of the expression construct promoter and terminator sequences.
• genes or part thereof may be inserted downstream of a strong promoter.
• Methods used to ligate the DNA construct comprising a polynucleotide encoding the NTCP, a promoter, a terminator and other sequences and to insert them into a suitable vector are well known in the art. Linking can be generally accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic
• oligonucleotide linkers are used in accordance with conventional practice (Bennett & Lasure, More Gene Manipulations In Fungi, Academic Press, San Diego (1991) pp 70- 76). Additionally, vectors can be constructed using known recombination techniques (e.g., Invitrogen Life Technologies, Gateway Technology).
• Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, (e.g., lipofection mediated and DEAE-Dextrin mediated transfection); incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion.
• General transformation techniques are known in the art (see, e.g., Campbell et al, (1989) Curr. Genet. 16:53-56).
• Any agent that may prevent or reduce the interaction between the NTCP protein and HBV/HDV virus is contemplated by the present invention.
• a reduction refers to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the interaction between the NTCP protein and HBV/HDV virus, or of the infection activity of the HBV and/or HDV virus.
• Interaction refers the binding of the HBV and/or HDV virus with the NTCP protein, which may lead to a conformational change to the NTCP protein.
• One type of agent that prevents or reduces interaction between NTCP and HBV and/or HDV is an antibody of the NTCP protein, such as an antibody that interferes the interaction between the NTCP protein and HBV and/or HDV.
• Antibodies that specifically bind to an epitope at the extracellular domain of the NTCP may be used as such an agent.
• antibodies that do not bind to an epitope on the NTCP protein that would inhibit its transporter function Any techniques well known in the art may be used to screen for antibodies with the desired properties.
• Monoclonal antibodies, recombinant antibodies and humanized/human antibodies, and epitope- binding fragments thereof, are all contemplated for the present invention.
• Another type of agent that prevents or reduces interaction between NTCP and HBV and/or HDV is a variant of the NTCP protein, such as a mutant form of NTCP.
• Preliminary data indicates the NTCP mutations that affected its physiological function could also interfere with HBV infection. Mutations of residues in the extracellular domains of NTCP may be especially useful as the extracellular domains may be involved in the interaction with HBV/HDV interaction.
• Q68A is a mutation that knocks out taurocholate uptake, locates at transmembranes (TM3), highly conserved among
• SN 105/106AA is a mutation that disrupts the Nal binding site, locates at TM4b, conserved in SLC10A1, A2, A4, and A6.
• S226A is a mutation on phosphorylation of S226 significantly enhanced surface expression and uptake of taurocholate, it locates at TM8, unique to SLC10A1.
• E257A is a mutation at a site critical for both Nal and Na2 binding, locates in TM9a, highly conserved in
• NTCP variants contemplated include a fragment of NTCP and a soluble NTCP polypeptide. Fragments of NTCP which comprise an extracellular domain may be desirable agents that may bind to HBV and/or HDV and prevent them from interacting with the endogenous NTCP. Variant forms of NTCP that do not support HBV and/or HDV interaction while function as the transporter, for example, the mouse or rat NTCP, may also be useful in treating and/or preventing infection.
• NTCP is a major hepatic Na + -bile acid symporter and responsible for cotransport of sodium and bile acids across cellular membranes to maintain the enterohepatic circulation of bile acids.
• Bile acids are made in the liver by the cytochrome P450- mediated oxidation of cholesterol. They are conjugated with taurine or the amino acid glycine, or with a sulfate or a glucuronide.
• Bile salts constitute a large family of molecules, composed of a steroid structure with four rings, a five- or eight-carbon side- chain terminating in a carboxylic acid, and the presence and orientation of different numbers of hydroxyl groups.
• Bile acids that may be used include but not limited to: taurolithocholate, cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid and hyodeoxycholic acid.
• Their derivatives in conjugation with taurine or the amino acid glycine, or with a sulfate or a glucuronide, may also be used for blocking HBV/HDV infection.
• Agents that increase, prevent or reduce the production/function of the NTCP protein through any mechanism are contemplated by the present invention, e.g., transcription, translation, post-translational modification, etc.
• the function of the NTCP protein refers to the Na + -bile acid symporter function, or its function as the HBV/HDV receptor.
• An increase or reduction refers to a change of at least 10%, 20%, 30%>, 40%>, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the production/function of the NTCP protein, or of the infection activity of the HBV and/or HDV virus.
• the function of the NTCP protein as the HBV/HDV receptor may be assessed by the interaction between NTCP and HBV/HDV virus, or by the infection by HBV/HDV virus of cells, tissues or animals expressing the NTCP protein, as described herein. Any known methods of measuring viral polypeptides and
• polynucleotides such as ELISA, quantitative RT-PCR, etc.
• ELISA quantitative RT-PCR
• NTCP function Sequences of siRNA and shRNA against NTCP
• Double-stranded RNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
• RNAi RNA interference
• the process is known to occur in a wide variety of organisms, including embryos of mammals and other vertebrates.
• the use of these dsRNAs (or recombinantly produced or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of the SLC10A1 mRNAs for degradation in mammalian cells.
• Use of long dsRNAs in mammalian cells to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response.
• Specific targeting of a particular gene function which is possible with 19-23 nt dsRNAs of the present invention, is useful in functional genomic and therapeutic applications.
• the present invention also relates to small interfering RNA (siRNA) sequences, RNA interfering vectors, and RNA interfering lentiviruses that are directed at the SLC10A1 gene, e.g., the tupaia or the human SLC10A1 gene. Therefore, in another aspect, the present invention provides an isolated double-stranded ribonucleic acid
• dsRNA molecule that inhibits expression of a human or tupaia NTCP, wherein a first strand of the dsRNA is substantially identical to at least 19 consecutive nucleotides of the human or tupaia SLC10A1 gene, and a second strand of the dsRNA is substantially complementary to the first strand.
• an siRNA target sequence may be designed on the basis of the tupaia or the human SLC10A1 gene, preferably 15 to 27, more preferably 19 to 23, and optimally 19, 20 or 21, consecutive bases.
• siRNAs targeting tupaia SLC10A1 gene [0112] siRNAs targeting tupaia SLC10A1 gene:
• siRNA- 1 5 * -CUAUGUAGGCAUUGUGAUAdTdT-3 * ,
• siRNA-2 5 * -GUGUUAUCCUGGUGGUUAUdTdT-3 * ,
• siRNA-3 5 * -GGACAUGAAUCUCAGCAUUdTdT-3 * ,
• siRNA-4 5 '-GGGC AAGAGC AUC AUGUUUdTdT-3 ',
• siRNAs were designed based on the coding sequence of the tupaia SLC10A1 gene shown below:
• siRNAs targeting human SLC1 OA1 gene [0113] siRNAs targeting human SLC1 OA1 gene :
• siRNA-1 1 5'-GGGAAAUGGCACCUACAAAdTdT-3',
• siRNA-405 5'-CACAAGUGCUGUAGAAUUAdTdT-3',
• siRNA-406 5'-CUAUAAAGGCAUCGUGAUAdTdT-3',
• siRNA-pool(4) 5*-GGAUCGUCCUCAAAUCCAAdTdT-3*, (from Qiagen) siRNA-pool(5): 5 '-GGAGUC AGCCGGAGAAC AAdTdT-3 ', (from Qiagen) siRNA-pool(6): 5'-GGACAAGGUGCCCUAUAAAdTdT-3', (from Qiagen) siRNA-pool(7): 5'-GGUGCUAUGAGAAAUUCAAdTdT-3', (from Qiagen)
• siRNAs were designed based on the cDNA of the human SLCIOAI gene shown below:
• the present invention relates to siRNA molecules that mediate RNAi.
• the siRNA molecules of the present invention can also comprise a 3' hydroxyl group.
• the siRNA molecules can be single-stranded or double stranded; such molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3').
• the siRNA molecule is double stranded and either blunt ended or comprises overhanging ends.
• At least one strand of the siRNA molecule has a 3' overhang from about 1 to about 6 nucleotides (e.g., pyrimidine nucleotides, purine nucleotides) in length.
• the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length.
• the siRNA molecule is double stranded, one strand has a 3' overhang and the other strand can be blunt-ended or have an overhang.
• the siRNA molecule can be double stranded and both strands comprise an overhang
• the length of the overhangs may be the same or different for each strand.
• the siRNA of the present invention comprises 21 nucleotide strands which are paired and which have overhangs of from about 1 to about 3, particularly about 2, nucleotides on both 3' ends of the siRNA.
• the 3' overhangs can be stabilized against degradation.
• the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
• substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine 2 nucleotide 3' overhangs by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi.
• the absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium.
• the siRNA molecules of the present invention can be obtained using a number of techniques known to those of skill in the art.
• the siRNA can be chemically synthesized or recombinantly produced using methods known in the art.
• the siRNA can also be obtained using an in vitro system.
• the in vitro system can also be used to obtain siRNA of about 19 to about 23 nucleotides in length which mediates RNA interference of the mRNA of the SLC10A1 gene.
• the method of obtaining the siRNA sequence using the in vitro system can further comprise isolating the RNA sequence from the combination.
• the siRNA molecules can be isolated using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to separate siRNA from the combination, gel slices comprising the RNA sequences removed and RNAs eluted from the gel slices. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to isolate the RNA produced. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to isolate siRNAs.
• the RNA-protein complex isolated from the in vitro system can also be used directly in the methods described herein (e.g., method of mediating RNAi of mRNA of the SLC10A1 gene).
• the siRNAs described herein can be used in a variety of ways.
• the siRNA molecules can be used to mediate RNA interference of mRNA of a gene in a cell or organism.
• the siRNA is introduced into human cells or a human in order to mediate RNA interference in the cells or in cells in the individual, such as to prevent or treat a disease or undesirable condition.
• a gene or genes that cause or contribute to the disease or undesirable condition is targeted and the corresponding mRNA (the transcriptional product of the targeted gene) is degraded by RNAi.
• an siRNA that targets the corresponding mRNA (the mRNA of the targeted gene) for degradation is introduced into the cell or organism.
• siRNA produced by other methods (e.g., chemical synthesis, recombinant DNA production) to have a composition the same as or sufficiently similar to an siRNA known to mediate RNAi can be similarly used to mediate RNAi.
• siRNAs can be altered by addition, deletion, substitution or modification of one or more nucleotides and/or can comprise non-nucleotide materials.
• the shRNA may comprise a sense fragment, which comprises a nucleotide sequence substantially identical to a target sequence in the SLCIOAI gene, and an antisense fragment, wherein the sense and antisense fragments are separated by a loop fragment, wherein the loop fragments may comprise a sequence selected from the group consisting of UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.
• the present invention also discloses a SLCIOAI RNAi lentivirus and the preparation and application thereof.
• a nucleic acid construct that expresses the above-described siRNA may be constructed by means of gene cloning and packaged with a lentivirus that expresses the above-described siRNA.
• Cell experiments prove that the above-described siRNA sequence can specifically silence the expression of endogenous SLCIOAI genes in liver cells.
• a DNA sequence encoding the above-described siRNA may be contained in a lentivirus vector.
• the lentivirus vector may further comprise a promoter sequence.
• the lentivirus vector may further comprise a nucleotide sequence encoding a detectable marker in the liver cell, wherein the detectable marker may be a green fluorescent protein (GFP).
• the lentivirus vector may be selected from the group consisting of pLKO. l-puro, pLKO. l-CMV-tGFP, pLKO. l-puro-CMV-tGFP, pLKO. l-CMV-Neo, pLKO.
• the siR A lentiviruses designed for SLC10A1 stably and specifically lower SLC10A1 expression and effectively inhibit the infection of HBV and/or HDV virus.
• NTCP neurotrophic factor
• Stieger Handb Exp Pharmacol, 205 (201 1); Meier & Stieger, Annu Rev Physiol 64, 635 (2002).
• Any molecules which regulate its expression, modification, or subcellular distribution may be used for the prevention/treatment of HBV/HDV infection and the infection related diseases.
• NTCP is a phosphorylated protein
• the kinases responsible for NTCP phosphorylation have not yet been fully elucidated, nonetheless, phosphorylation status strongly affect its functionality (Anwer et al, J Biol Chem 280, 33687 (2005); Webster et al, J Biol Chem 277, 28578 (2002); Schonhoff et al, Am J Physiol
• kinase inhibitors may be used to inhibit and/or modulate the function of NTCP.
• NTCP is a glycoprotein, glycosylation status is critical for its functionality (including HBV infection). Therefore, glycosylation inhibitors are also contemplated as agents that can inhibit and/or modulate the function of NTCP.
• Transcription of NTCP may be modulated by activating/inhibiting nuclear factors, such as RARa-RXRa, etc., that regulate transcription of the SLC10A1 gene.
• NTCP expression by targeting histone or genomic DNA modifications is also contemplated.
• HBV is a DNA virus and it has a circular genome of partially double-stranded DNA.
• the viruses replicate through an RNA intermediate form by reverse transcription, which practice relates them to retroviruses.
• the genome of HBV is made of circular DNA, but it is unusual because the DNA is not fully double-stranded.
• One end of the full length strand is linked to the viral DNA polymerase.
• the genome is about 3020-3320 nucleotides long (for the full-length strand) and about 1700-2800 nucleotides long (for the short length-strand).
• the virus particle (virion) consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein.
• the nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity.
• the outer envelope contains embedded proteins that are involved in viral binding of, and entry into, susceptible cells.
• the virus has a virion diameter of about 42 nm, but pleomorphic forms exist, including filamentous and spherical bodies lacking a core. These particles are not infectious and are composed of the lipid and protein that forms part of the surface of the virion, which is called the surface antigen (HBsAg), and is produced in excess during the life cycle of the virus.
• Acute infection with HBV is associated with acute viral hepatitis. It has been noted that itchy skin has been an indication as a possible symptom of all hepatitis virus types.
• HBV hepatocellular carcinoma
• HDV is considered to be a subviral satellite because it can propagate only in the presence of the HBV. Transmission of HDV can occur either via simultaneous infection with HBV (coinfection) or superimposed on chronic hepatitis B or hepatitis B carrier state (superinfection). It has an outer coat containing three HBV envelope proteins (called large, medium, and small hepatitis B surface antigens, and host lipids surrounding an inner nucleocapsid. The nucleocapsid contains single-stranded, circular RNA of about 1679 nucleotides and about 200 molecules of hepatitis D antigen (HDAg) for each genome.
• HDAg hepatitis D antigen
• the HDV genome exists as an enveloped negative sense, single- stranded, closed circular RNA nucleotide sequence is 70% self-complementary, allowing the genome to form a partially double stranded RNA structure that is described as rodlike.
• the present invention provides pharmaceutical compositions for the prevention or treatment of HBV and/or HDV infection or diseases associated with the infection in a mammal, comprising any agent that prevents or reduces
• NTCP production/function of NTCP in said mammal, and/or an agent that prevents or reduces interaction between NTCP in said mammal and HBV and/or HDV.
• the invention also relates to pharmaceutically acceptable prodrugs of the agents, and treatment methods employing such pharmaceutically acceptable prodrugs.
• prodrug means a precursor of a designated compound that, following administration to a subject, yields the compound in vivo via a chemical or physiological process such as solvolysis or enzymatic cleavage, or under physiological conditions (e.g., a prodrug on being brought to physiological pH is converted to the agent).
• prodrug is a prodrug that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to the subject.
• the present invention also relates to pharmaceutically active metabolites of the agents, and uses of such metabolites in the methods of the invention.
• “pharmaceutically active metabolite” means a pharmacologically active product of metabolism in the body of a compound or salt thereof.
• Prodrugs and active metabolites of a compound may be determined using routine techniques known or available in the art. See, e.g., Bertolini et al., J. Med. Chem. 1997, 40, 2011-2016; Shan et al., J. Pharm. Sci. 1997, 86 (7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv. Drug Res. 1984, 13, 255-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development
• Any suitable formulation of the compounds described herein can be prepared. See, generally, Remington's Pharmaceutical Sciences, (2000) Hoover, J. E. editor, 20th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa., pages 780- 857.
• a formulation is selected to be suitable for an appropriate route of administration. Some routes of administration are oral, parenteral, by inhalation, topical, rectal, nasal, buccal, vaginal, via an implanted reservoir, or other drug administration methods. In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate.
• Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate.
• Suitable inorganic salts may also be formed, including
• hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts are obtained using standard procedures well known in the art, for example, by a sufficiently basic compound such as an amine with a suitable acid, affording a physiologically acceptable anion.
• a sufficiently basic compound such as an amine with a suitable acid, affording a physiologically acceptable anion.
• Alkali metal e.g., sodium, potassium or lithium
• alkaline earth metal e.g., calcium
• contemplated compounds are administered in a pharmacological composition
• the compounds can be formulated in admixture with a pharmaceutically acceptable excipient and/or carrier.
• contemplated compounds can be administered orally as neutral compounds or as pharmaceutically acceptable salts, or intravenously in a physiological saline solution.
• Conventional buffers such as phosphates, bicarbonates or citrates can be used for this purpose.
• one of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration.
• contemplated compounds may be modified to render them more soluble in water or other vehicle, which for example, may be easily accomplished with minor modifications (salt formulation, esterification, etc.) that are well within the ordinary skill in the art. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in a patient.
• the agents as described herein are generally soluble in organic solvents such as chloroform, dichloromethane, ethyl acetate, ethanol, methanol, isopropanol, acetonitrile, glycerol, N,N-dimethylformamide, N,N-dimetheylaceatmide,
• the present invention provides formulations prepared by mixing an agent with a pharmaceutically acceptable carrier.
• the formulation may be prepared using a method comprising: a) dissolving a described agent in a water-soluble organic solvent, a non-ionic solvent, a water-soluble lipid, a cyclodextrin, a vitamin such as tocopherol, a fatty acid, a fatty acid ester, a phospholipid, or a combination thereof, to provide a solution; and b) adding saline or a buffer containing 1-10% carbohydrate solution.
• the carbohydrate comprises dextrose.
• water soluble organic solvents for use in the present methods include and are not limited to polyethylene glycol (PEG), alcohols, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or a combination thereof.
• PEG polyethylene glycol
• alcohols include but are not limited to methanol, ethanol, isopropanol, glycerol, or propylene glycol.
• Illustrative examples of water soluble non-ionic surfactants for use in the present methods include and are not limited to CREMOPHOR® EL, polyethylene glycol modified CREMOPHOR® (polyoxyethyleneglyceroltriricinoleat 35), hydrogenated CREMOPHOR® RH40, hydrogenated CREMOPHOR® RH60, PEG-succinate, polysorbate 20, polysorbate 80, SOLUTOL® HS (polyethylene glycol 660 12- hydroxystearate), sorbitan monooleate, poloxamer, LABRAFIL® (ethoxylated persic oil), LABRASOL® (capryl-caproyl macrogol-8-glyceride), GELUCIRE® (glycerol ester), SOFTIGEN® (PEG 6 caprylic glyceride), glycerin, glycol-polysorbate, or a combination thereof.
• CREMOPHOR® EL polyethylene glycol modified CREMOPHOR® (polyoxy
• Illustrative examples of water soluble lipids for use in the present methods include but are not limited to vegetable oils, triglycerides, plant oils, or a combination thereof.
• lipid oils include but are not limited to castor oil, polyoxyl castor oil, corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, a triglyceride of coconut oil, palm seed oil, and hydrogenated forms thereof, or a combination thereof.
• Illustrative examples of fatty acids and fatty acid esters for use in the present methods include but are not limited to oleic acid, monoglycerides, diglycerides, a mono- or di-fatty acid ester of PEG, or a combination thereof.
• Illustrative examples of cyclodextrins for use in the present methods include but are not limited to alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta- cyclodextrin, or sulfobutyl ether-beta-cyclodextrin.
• Illustrative examples of phospholipids for use in the present methods include but are not limited to soy phosphatidylcholine, or distearoyl phosphatidylglycerol, and hydrogenated forms thereof, or a combination thereof.
• One of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration.
• the compounds may be modified to render them more soluble in water or other vehicle. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in a patient.
• an agent that increase, prevents or reduces production/function of NTCP, and/or an agent that prevents or reduces interaction between NTCP and HBV and/or HDV, and pharmaceutical compositions thereof may be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or other drug
• parenteral includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
• a sterile injectable composition such as a sterile injectable aqueous or oleaginous suspension, may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
• acceptable vehicles and solvents include mannitol, water, Ringer's solution and isotonic sodium chloride solution.
• Suitable carriers and other pharmaceutical composition components are typically sterile.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides).
• Fatty acids such as oleic acid and its glyceride derivatives
• injectables are useful in the preparation of injectables, as are pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
• oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
• These oil solutions or suspensions can also contain a long- chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents.
• Various emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purpose of formulation.
• a composition for oral administration may be any orally acceptable dosage form including, but not limited to, tablets, capsules, emulsions and aqueous suspensions, dispersions and solutions.
• commonly used carriers include lactose and corn starch.
• Lubricating agents such as magnesium stearate, can also be added.
• useful diluents include lactose and dried corn starch.
• a nasal aerosol or inhalation compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in, for example saline, employing suitable preservatives (for example, benzyl alcohol), absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents known in the art.
• suitable preservatives for example, benzyl alcohol
• absorption promoters to enhance bioavailability
• other solubilizing or dispersing agents known in the art.
• an agent that increases, prevents or reduces production/function of NTCP, and/or an agent that prevents or reduces interaction between NTCP and HBV and/or HDV may be administered alone or in combination with other anti-infection agents for the treatment of various infections or conditions.
• Combination therapies according to the present invention comprise the administration of at least one compound of the present invention or a functional derivative thereof and at least one other pharmaceutically active ingredient.
• the active ingredient(s) and pharmaceutically active agents may be administered separately or together.
• administration will be selected in order to achieve the desired combined therapeutic effect.
• the agent that modulates NTCP production and/or interaction may be co-administered with a known drug for treating HBV and/ or HDV infection, wherein the drug is selected from the group consisting of an interferon, a nucleoside analogue, a non-nucleoside antiviral and a non-interferon immune enhancer.
• Intron A Interferon Alpha
• Pegasys Pegasys (Pegylated Interferon)
• Other drugs include: Clevudine (L-FMAU), Emtricitabine (FTC), MIV-210, Amdoxovir (DAPD), NOV-205 (BAM 205), LB80380 (ANA380), Myrcludex B, HAP Compound Bay 41-4109, REP 9AC, Alinia (Nitazoxanide), and Zadaxin (Thymosin alpha- 1).
• Myrcludex B Another drug which may be used is the entry inhibitor Myrcludex B, which was originated from the preSl of HBV envelope and is now in clinical trial. Myrcludex B has been shown to inhibit HBV/HDV infection in vitro and in animal model(s) by addressing an unknown cellular component, most likely a receptor. The idea for Myrcludex B was developed within two renowned research institutions in Europe, the French National Institute of Health and Medical Research (INSERM) and University of Heidelberg in Germany. The lead compound blocks the penetration of HBV and HDV into hepatocytes. By treating with an entry inhibitor, new infection of constantly- evolving, healthy hepatocytes may be blocked and the number of infected cells should significantly decline within a few months.
• Cell lines non-susceptible to HBV and/or HDV infection e.g., HepG2 or Huh7, or other liver derived cells, or cells with hepatic function
• stably expression human/tree shrew or other NTCP that support HBV/HDV infection may be constructed by transfecting the cell line with a vector encoding the NTCP.
• Other cells include no- longer-susceptible primary hepatocytes. Expression of the human/tree shrew NTCP results in the susceptibility of the transfected cell line to infection.
• non-human transgenic animal models Any transgenic methods well known in the art may be used to generate the animal models, e.g., knockout and/or knockin mouse models or humanized animal models whose SLC10A1 gene is replaced by the human counterpart.
• the endogenous SLC10A1 gene expression may be eliminated by ZFN- and TALEN-mediated gene knockout or other techniques.
• An SLC10A1 knockout mouse may be further engineered to express the SLC10A1 gene from another species, such as human or tree shrew. Further, a knockin mouse expressing an exogenous SLC10A1 gene at the SLC10A1 locus may be generated.
• the cell lines and non-human transgenic animal models may be used for the screening of drug candidates for HBV and/or HDV infection or a disease associated with said infection.
• HBV infection remains a major public health problem, about one million people die from HBV-related liver diseases each year. Individuals co-infected with HBV and its satellite HDV have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins with critical contributions of a receptor-binding- region on the pre-Sl domain of the large (L) envelope protein. However the identity of the cellular receptor(s) is unknown. It has been identified that NTCP, a liver bile acids transporter, specifically binds to the pre-Sl of L protein of the viruses. Silencing NTCP by small interference R As inhibits HBV and HDV infection of primary hepatocytes.
• NTCP expression renders Huh-7, an otherwise non-susceptible hepatocellular carcinoma cell line, permissive for HDV entry, and rescues the susceptibility of no-longer- susceptible primary hepatocytes to HBV infection.
• NTCP is a functional receptor for HBV and HDV.
• NTCP being an entry receptor for HBV and HDV raises interesting questions regarding its involvement in the pathogenesis of the viral infection related diseases.
• Available treatments against HBV are inadequate or noneffective to those who are chronically infected, and are at high risk of cirrhosis and hepatocellular carcinoma (Lai & Yuen, N Engl J Med 359, 2488 (2008)).
• Identification of NTCP as a functional receptor for HBV and HDV will lead to a better understanding of HBV and HDV infection and NTCP presents a potential target for developing new prevention and treatment against the viruses and their related diseases.
• PTHs primary tupaia hepatocytes
• Adult tree shrews (Tupaia belangeri chinensis) were purchased from Kunming Institute of Zoology, Chinese Academy of Sciences and housed in a tupaia animal facility at the National Institute of Biological Science, Beijing. All studies were performed in accordance with institutionally approved protocols and adherent to guidelines of the National Institute of Biological Sciences Guide for the care and use of laboratory animals.
• PTH cells were obtained from anesthetized tupaia (100-150g) with a two-step perfusion method as previously described (Walter et al, Hepatology 24, 1 (1996)). Cell suspensions after perfusion were filtered through a 70 ⁇ cell strainer and centrifuged at 50g for 3 minutes.
• the cell pellet containing PTHs was resuspended in plating medium that is Williams E medium supplemented with 10% FBS, 5 ⁇ g/ml transferrin, 5ng/ml sodium selenite, 2mM L-glutamine, lOOU/ml penicillin and 100 ⁇ g/ml streptomycin. Cells were then plated on collagen-coated cell culture dishes or plates.
• PMM primary hepatocytes maintenance medium
• PMM Williams E medium supplemented with 5 ⁇ g/ml transferrin, lOng/ml EGF, 3 ⁇ g/ml insulin, 2 L-glutamine, 18 ⁇ g/ml hydrocortisone, 40ng/ml dexamethasone, 5ng/ml sodium selenite, 2% DMSO, lOOU/ml penicillin and 100 ⁇ / ⁇ 1 streptomycin.
• Cells were maintained in a 5% C0 2 humidified incubator at 37°C with regular medium change every 2 days.
• PHHs Primary human hepatocytes (PHHs) and primary tupaia fibroblast (PTF) cells.
• PHHs were purchased from Becton Dickinson (NJ, USA) or Shanghai RILD Inc.
• the cells were cultured similarly as PTHs using the same plating medium and maintaining PMM medium as described above.
• Primary fibroblasts cells were isolated from adult tree shrews and used as supportive cells for culturing
• hepatocytes (Khetani et al, Hepatology 40, 545 (2004); Huang et al, Manuscript in preparation (Briefly, PTF cells, which are primary fibroblasts isolated from skin of adult tree shrew, were used as supportive cells for co-culturing with hepatocytes. PTFs were plated onto plates at confluence of about 80%. 24 hours later, isolated hepatocytes were seeded on top of pre-seeded PTFs. Cells were co-cultured in PMM and the medium were replenished every two days.)).
• HDV A plasmid containing a head to tail trimer of 1.0 X HDV cDNA of a genotype I HDV virus (Genbank accession number: AF425644.1) under the control of a CMV promoter was constructed with de novo synthesized HDV cDNA for the production of HDV RNPs.
• a pUC18 plasmid containing 2431-1990 HBV Genotype D, Genbank accession number: U95551.1
• the same plasmid bearing mutation(s) generated by site-directed mutagenesis was used for expressing HBV envelope proteins under the control of endogenous HBV promoter.
• HDV virions were produced in Huh-7 as previously described (Sureau et al, / Virol 66, 1241 (1992)).
• HBV HBV viruses were obtained by ultracentrifugation of sera from HBV patients with written consent. The genotypes of HBV used in the study is B or C (sequences were deposited to Genbank, accession numbers JQ412089 and JQ412091).
• Recombinant adeno-associated virus 8 (AAV8) carrying EGFP reporter gene was produced similarly as described (Xiao et al, / Virol 72, 2224 (1998)) by co-transfection of 293 cells with plasmids for AAV8 packaging, EGFP gene transduction and adenovirus helper, respectively; AAV8-HBV recombinant carrying 1.3 copies of HBV genome (Dong et al, Bing Du Xue Bao 26, 425 (2010)) was purchased from Five-plus Inc.
• Lenti-VSV-G an HIV-1 genome-based lentivirus pseudotyped by glycoprotein of vesicular stomatitis virus and carrying a firefly luciferase or an EGFP reporter gene, was produced by co-transfection of 293 T cells with plasmids for HIV genome packaging, gag/pol expression and luciferase (or EGFP) reporter, respectively, as described (Sui et al, / Virol 79, 5900 (2005)).
• Antibodies. 2D3 is a mouse monoclonal antibody (mAb) (subtype IgGl) specifically targeting the 19-33 amino acids of the pre-Sl domain of HBV L protein.
• 4G5 is a mouse mAb (IgGl) recognizing HDV delta antigen. Both were generated and produced using conventional hybridoma technique. 17B9, a mouse mAb specific to HBV S protein, was kindly provided by Prof. Lin Jiang at Lanzhou Institute of Biological Products, China. Secondary antibodies for immunofluorescence staining and western blot were purchased from Invitrogen (CA, USA) or Sigma-Aldrich (MO, USA).
• Hepatitis B immune globulin was from the National Institutes for Food and Drug control, Beijing, China.
• Peptides, chemicals, ELISA and other kits Peptides with non-natural amino acid L-2-amino-4,4-azipentanoic acid (L-photo-leucine) were synthesized by American Peptide Company Inc. (CA, USA). Other peptides corresponding to the N-terminal of pre-Sl domain of HBV L protein (genotype C, strain S472, GenBank: EU554535.1) were synthesized by SunLight Peptides (Beijing, China). ELISA kits for HBsAg and HBeAg measurement were from Wantai Pharm Inc. (Beijing, China). Quantitative real time PCR kit and Reverse Transcriptase reagents were from Takara Inc. (Beijing, China).
• Streptavidin-coupled magnetic beads (Dynabeads® MyOneTM Streptavidin Tl) and magnetic beads coated in glycidyl ether (Epoxy) groups (Dynabeads® M-270 Epoxy) were purchased from Life Technologies. 2D3 magnetic beads were prepared by covalently crosslinking 2D3 to Dynabeads® M-270 Epoxy following manufacturer's instructions. Other reagents were purchased from New England Biolabs (MA, USA), Life Technologies (CA, USA) or Sigma-Aldrich (MO, USA).
• HDV strand-specific real time RT-PCR HDV strand-specific real time RT-PCR.
• HDV strand-specific PCR was performed as previously described by Freitas et al.
• primer HDV398R 5 - CGCTTCGGTCTCCTCTAACT-3'
• primer HDV288F 5 * - GCAGACAAATCACCTCCAGA -3 *
• TaqMan probe was 5'- AGAGCTCTGACGCGCGAGGAGTAAGC-3 '. All quantitative PCR (qPCR) assays were analyzed with an ABI Fast 7500 Real Time PCR instrument (Applied Biosystems).
• RNA from HBV infected cells was isolated with Trizol reagent following manufacturer's instructions. The total RNA was reverse transcripted into cDNA by PrimeScriptTM RT kit from Takara. Levels of HBV specific RNAs were determined using real time RT-PCR.
• HBV2270F (5'- GAGTGTGGATTCGCACTCC-3') and HBV2392R (5 * -GAGGCGAGGGAGTTCTTCT- 3 * ) were used for HBV 3.5Kb transcripts
• HBV1805F (5 * -TCACCAGCACCATGCAAC- 3 * )
• HBV1896R (5 * -AAGCCACCCAAGGCACAG-3 * ) for all HBV specific transcripts (Fig.SlOB).
• Real time PCR was performed on an ABI Fast 7500 real time system instrument (Applied Biosystems, USA). The viral RNA copies were calculated with a standard curve generated from known nucleic acid quantities. [0166] HDV virion binding and inhibition assays.
• HDV binding assay was performed by incubating 5xl0 7 copies of genome equivalent HDV with lxl 0 5 target cells at 16°C for 4h in the presence of 4% PEG8000, followed by extensive wash with cold PBS (pH 7.4) for 4 times. The cells were then lysed directly with Trizol reagent for RNA isolation and followed by reverse transcription. RNA copy numbers of viral genome and internal control glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) mRNA were determined by real time PCR. For binding inhibition assay, peptides or other testing reagents were pre-incubated with target cells at various concentrations at 16°C for lh before incubating with the virus.
• GPDH glyceraldehyde-3 -phosphate dehydrogenase
• Viral infection and inhibition assays Viral infections of HDV and HBV were conducted with PTH cells in 24 or 48 well plates after 48-72 hours (or otherwise indicated) of cell plating and at multiplicities of genome equivalents of 500 and 100, respectively. Normally 5xl0 7 copies of genome equivalent HDV or lxl 0 7 copies of genome equivalent HBV were inoculated with lxl 0 5 cells and incubated for 16 hours except otherwise indicated. Cells were then washed with medium for three times and maintained in PMM with regular medium change every 2 days. For HDV infection, 4% PEG was present during the 16 hours viral inoculation period (Barrera et al, / Virol 78, 5233 (2004)).
• peptides or other reagents were pre- incubated with cells at 37°C for lh before virus inoculation. Infection of HDV was assessed by RT-PCR to quantify viral genomic RNA as described above or by intracellular staining of HDV delta antigen with a monoclonal antibody 4G5 on indicated days post infection.
• HBV infection secreted viral antigens, HBsAg and HBeAg were examined with commercial ELISA kits and intracellular HBsAg was stained with a monoclonal antibody 17B9 that is specific to HBsAg at indicated times post infection.
• HBsAg and HBeAg were examined using 50 ⁇ supernatants with commercial ELISA Kits (Wantai Pharm) following manufacturer's instructions. In some cases, HBsAg level was normalized with WHO HBsAg reference serum (Kindly provided by Dr. Zhenglun Liang from the National Institutes for Food and Drug control, Beijing, China).
• cDNA library construction for deep sequencing of tupaia transcriptome Primary tupaia hepatocytes were isolated as described above.
• PTH mR A was purified from 10 ⁇ g of total RNA using Oligo-dT magnetic beads. The mRNA was fragmented into small pieces by incubation with divalent cations at 94°C for exactly 5 minutes. The first strand cDNA was synthesized using random primers and Superscript II reverse transcriptase (Invitrogen) with fragmented mRNAs. RNA template was then removed by RNase H and double-stranded cDNA was prepared with DNA polymerase I. cDNA with blunt ends was created by T4 DNA polymerase and Klenow DNA polymerase and an "A" base was subsequently added to the 3' end of the blunt phosphorylated DNA fragments by Klenow fragment (3' to 5' exo minus).
• the cDNA was then ligated with adapters and then ran on a 2% agarose gel.
• the fragments with a size range from 200 ⁇ 25bp were purified, followed by amplification using the manufacture's primers.
• the PCR products were then purified using QIAquick PCR purification Kit (Qiagen), quantified and diluted for cluster generation and deep sequencing.
• Qiagen QIAquick PCR purification Kit
• the 72-cycle pair-end sequencing was performed with Sequencing Kits (Version 5) on an Illumina Genome Analyzer IIx (Illumina, San Diego, USA).
• Illumina CASAVA pipeline vl .8.1 was used for sequence extraction and filtering.
• transcriptome of primary tupaia hepatocytes De novo reconstruction of transcriptome from cDNA library deep sequencing data: 253,919,616-pair 72nt sequences from the sequencing results of the hepatocyte cDNA library described above were fed to Trinity (Grabherr et al, Nat Biotechnol 29, 644 (2011)) r20110519 with default parameters, with which 209,063 transcripts of average length of l,421nt (minimum 300nt, maximum 21,043nt and scaffold N50 of 3,674nt) were generated.
• NCBI Biotechnology Information
• Reference Sequence (RefSeq) project human protein sequences (release date 27 Jun 2011; ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/rnRNA_Prot/), Universal Protein Resource (UniProt) UniProt Knowledgebase (UniProtKB) human proteome (release 2011 06: ftp ://ftp .uniprot.org/pub/ databases/uniprot/ current_release/knowledgebase/proteomes/) and NCBI nonredundant protein sequence database (release date 6 Jul 2011 :
• blastx (Camacho et al. , BMC
• Annotation of protein sequences Protein sequences from the transcripts and protein sequences that passed the criteria of quality control were chosen for functional annotation. Each chosen protein sequence was first annotated with its corresponding blastp (Camacho et al., BMC Bioinformatics 10, 421 (2009)) matches from NCBI human protein sequences. Those not annotated in the first step were then submitted for similar annotation process with UniprotKB human proteome and NCBI non-redundant protein sequence database. Protein sequences that were not annotated by previous steps were submitted for annotation with their corresponding transcripts.
• Protein sequences with their corresponding transcripts that can be annotated by the blastx (Camacho et al., BMC Bioinformatics 10, 421 (2009)) hits of NCBI human protein sequences, UniProtKB human proteome or NCBI non-redundant protein sequence database were annotated with these hits from transcripts.
• Generation of tupaia hepatocyte protein sequences database All identified protein sequences were included in the hepatocyte protein sequences database. The protein sequences were labeled with corresponding functional annotation results. Any identified protein sequences that were not successfully annotated are labeled with "Uncharacterized protein".
• L-photo-leucine-bearing wild type bait (WTb) or control bait peptide (N9Kb) was dissolved in DMSO in dark and diluted to working concentrations with Williams E medium.
• L-photo-leucine contains a photo activatable diazirine ring, irradiation of UV light at 365nm induces a loss of nitrogen of the diazirine ring and yields a reactive carbine group with short half-life for covalent crosslinking at nearly zero-distance.
• bait peptides were applied to -1X10 7 primary hepatocytes plated on collagen-coated dishes.
• UVP UV 365nm light
• a CL- 1000 Ultraviolet crosslinker UVP, CA, USA
• Cells were then washed one more time after the crosslink to remove residual free peptides and subsequently lysed with 1 ml radioimmunoprecipitation assay (RIPA, pH 7.4) buffer containing 50mM Tris, 150mM NaCl, 0.1 % SDS; 0.5 % sodium deoxycholate, 1% NP40 and IX protease inhibitor cocktail (Roche).
• the cell lysates were precipitated with ⁇ streptavidin Tl magnetic beads, followed by extensive wash for at least six times with 1ml RIPA buffer each time, and then eluted by boiling for 5 minutes with 50 ⁇ 1 non-reducing SDS-PAGE loading buffer containing lOOmM Tris-HCl (pH6.8), 2% SDS, 10% glycerol.
• the eluted sample was then diluted with cold RIPA buffer to a final volume of 1ml and was precipitated with ⁇ (1X10 8 ) 2D3 -conjugated M-270 dynal beads for 6 hours at 4°C, washed for 5 times with 1ml RIPA buffer, then eluted by boiling 5min in 100 ⁇ non-reducing loading buffer.
• the UPLC separation gradient included a 30 min gradient from 0 to 30% acetonitrile, followed by a 10-min gradient to 80% acetonitrile, then 10 min of 80% acetonitrile and back to 0% acetonitrile within 5 min.
• the mass spectrometer was operated in the data-dependent mode.
• Survey MS scans were acquired in the orbitrap with the resolution set to a value of 60000. Each survey scan (300-2000 m/z) was followed by 4 data-dependent CID tandem mass (MS/MS) scans at 35% normalized collision energy and 4 data-dependent HCD tandem mass (MS/MS) scans at 40% normalized collision energy with 15000 resolution in orbitrap.
• AGC target values were 500,000 for the survey scan, 10,000 for the ion trap MS/MS scan and 50,000 for the orbitrap MS/MS scan.
• Target ions already selected for MS/MS were dynamically excluded for 30 seconds.
• Tandem mass spectra were searched against the Illumina-deep sequencing- determined tupaia hepatocyte protein database that was concatenated with reversed sequences to estimate false positives and was supplemented with the sequence of bait peptide under the Linux operating system using the ProLuCID (Xu et al, Mol Cell Proteomics 5, SI 74 (2006)) protein database search algorithm with peptide mass tolerance of ⁇ 100 ppm, fragment ion mass tolerance of ⁇ 400 ppm, half tryptic specificity and a static modification of 57.0215 on Cys due to carboxyamidomethylation.
• ProLuCID Xu et al, Mol Cell Proteomics 5, SI 74 (2006)
• ProLuCID search results were then filtered with DTA Select 2.0 (Tabb et al., J Proteome Res 1, 21 (2002)) using a cutoff of 1%> for peptide false identification rate (— fp 0.01). Peptides with DeltaMass > 10 ppm (-DM 10) were rejected; the minimum number of peptides to identify a protein was set to 1 (-p 1).
• tsNTCP-si2 5 * -GUGUUAUCCUGGUGGUUAUdTdT-3 * ,
• tsNTCP-si4 5 '-GGGC AAGAGC AUC AUGUUUdTdT-3 '
• PTH cells were trypsinized one day after transfection and plated on collagen-coated 48-well plates with pre-seeded primary tupaia fibroblast (PTF) cells that were isolated from adult tupaia and serve as supportive cells for hepatocytes (Khetani et al, Hepatology 40, 545 (2004); Huang et al, Manuscript in preparation (Briefly, PTF cells, which are primary fibroblasts isolated from skin of adult tree shrew, were used as supportive cells for co-culturing with hepatocytes.
• PTF primary tupaia fibroblast
• PTFs were plated onto plates at confluence of about 80%. 24 hours later, isolated hepatocytes were seeded on top of pre-seeded PTFs. Cells were co-cultured in PMM and the medium were replenished every two days.)). Three days later, the cells were inoculated for 8 hours with HBV, HDV, AAV8-HBV or Lenti-VSV-G (4 days after siRNA transfection).
• NTCP gene knockdown in the siRNA transfected PTHs a fraction of the same transfected PTHs used for the above described infection assay was trypsinized and plated on collagencoated 48-well plates one day after transfection and cultured for three more days, then NTCP mRNA was quantified by real time RT-PCR.
• Huh-7 cells were transfected with plasmids expressing NTCPs or a vector control using Lipofectamine 2000. 24 hours after transfection, HDV inoculation was conducted in the presence of 4% PEG8000. 1X10 5 cells were incubated with 5X10 7 genome equivalent copies of HDV for 24 hours in the presence or absence of entry inhibitors. Culture medium was changed every two days. Cells were fixed on day 6 post infection and stained for intracellular HDV delta antigen with mAb 4G5. HDV viral genomic RNA copies in cell lysates were quantified by RT-PCR.
• hepatocytes were trypsinized and seeded onto collagen-coated 12- well plate on day 13th after liver perfusion. On the following day, the hepatocytes were transfected with hNTCP expression plasmid or a vector plasmid with Lipofectamine 2000 (Invitrogen). One day after transfection, the PTH cells were trypsinized and reseeded onto PTFs that were pre-seeded in 48-well plate. The next day, the PTHs were inoculated with HBV for 8 hours, remaining virus was removed by washing for three times with PMM and cell culture medium was refreshed every two days.
• HBsAg and HBeAg from culture supernatant were examined with commercial ELISA kits.
• cells were fixed with 4% PFA and permeabilized with 0.5% TritionX-100, and then stained with mAb 17B9 for HBsAg and anti-CYP3A4 antibody (Sigma) for cytochrome P450 3A4 enzyme in the hepatocytes.
• Leul 1 is 100% conserved among HBV genotypes and the 14th residue is a phenylalanine in most genotypes but a leucine in some HBV strains of genotypes F and G.
• the bait peptide named Myr-47/WTb or WTb hereinafter
• WTb the bait peptide
• This N9K peptide named Myr-47/N9Kb (or N9Kb hereinafter) was used as a bait control for WTb.
• Both WTb and N9Kb peptides were myristoylated at N- terminus and conjugated with a biotin tag on a C-terminal lysine residue (Fig. 1 A).
• WTb or N9Kb bait peptide was then applied to primary tupaia hepatocytes (PTHs) and crosslinked by irradiation of UV light.
• Crosslinked bait peptide and associated molecules were precipitated by streptavidin Tl beads and separated on SDS-PAGE.
• Western blot using 2D3 as a probe revealed several bands, including a prominent, smear band around ⁇ 65KDa in both wildtype- and N9K (2 ⁇ ) crosslinked samples (Fig. ID).
• the WTb crosslinked protein(s) has no intermolecular disulphide bonds as it migrated similarly under both non-reducing and reducing conditions (Fig. IE). Interestingly, the WTb crosslinked protein bands appeared only when crosslinking was performed on plate-attached hepatocytes but not with lysates from the same cells (data not shown), indicating a native conformation of the target protein(s) is essential for efficient crosslinking by the bait peptide. Noteworthy, the target protein(s) decreased in abundance rapidly over time of cell culture in vitro (Fig. 6B).
• PHHs Human primary hepatocytes
• non-HBV susceptible human liver cancer cell lines were also examined for crosslinking, only with PHH cells, a band with slightly smaller molecular weight than that seen in PTH cells was observed in WTb crosslinked sample. Similar to PTHs, the protein crosslinked in PHH cells is also glycosylated and the crosslink can be competed by Myr-47/WT peptide (Fig. 6C).
• the identity of the target protein(s) was revealed with affinity purification followed by mass spectrometry analysis.
• the purification was conducted under stringent conditions and mainly included three tandem steps: capturing all biotin labeled proteins with streptavidin Tl beads; sorting out the target protein(s) by 2D3 immobilized beads; and followed by purifying with streptavidin Tl beads again to remove residual molecules that were not covalently crosslinked with the bait peptide.
• the purified samples were eluted by boiling and subsequently separated with SDS-PAGE and then silver stained. A ⁇ 65kDa band was visible and at the same size as that visualized by Western blot and it shifted to around 43kDa upon PNGase F treatment (Fig. 7A).
• NTCP human sodium taurocholate cotransporting polypeptide
• NTCP is the protein specifically interacted with the WTb bait peptide.
• NTCP is mainly expressed in liver (Stieger, Handb Exp Pharmacol, 205 (2011)), consistent with the liver tropism of HBV and HDV. It localizes to the sinusoidal (basolateral) plasma membrane of hepatocytes (Stieger et al, Gastroenterology 107, 1781 (1994)), a location fits well with a receptor role for the blood-borne HBV and HDV. Whereas virus may first attach to hepatocytes via heparin sulfate (Schulze et al.,
• HCC hepatocellular carcinoma
• NTCP hepatocellular carcinoma
• NTCPs from both human and tupaia were cloned and first validated for binding with WT b bait peptide and an N-terminal myristolated pre-Sl peptide of native residues. Both NTCPs can be efficiently crosslinked by the WTb when overexpressed on 293T cells (Fig. 9A). WTb but not the control N9Kb peptide bound to 293 T cells expressing tupaia NTCP
• tsNTCP with a green fluorescence tag (tsNTCP-EGFP) and colocalized with tsNTCP- EGFP on cell surface, and free Myr-47/WT peptide competed the binding (Fig. 9B).
• hNTCP human NTCP
• Fig. 9C a native pre-Sl peptide specifically recognized human NTCP (hNTCP) transfected 293T cells
• Virus binding to cells expressing NTCP was then examined.
• Both tsNTCP and hNTCP promoted specific HDV virion binding.
• non-infectious mutant HDV virus bearing a pN9K mutation in the pre-S 1 domain of its L envelope protein bound to neither hNTCP nor tsNTCP expressing Huh-7 cells Fig. 9F.
• NTCP small interfering RNAs
• siRNAs small interfering RNAs
• HBV infection of NTCP-silenced hepatocytes decreased markedly compared with that of cells treated with a control siRNA, both intracellular and secreted viral antigens were greatly reduced.
• the reduction correlated well with the efficiency of NTCP mRNA silencing, more than 85 % viral infection was blocked by knocking NTCP mRNA down to 16% (Fig. 10A).
• NTCP is a key cellular component necessary for HBV and HDV infection on primary hepatocytes.
• NTCP NTCP mRNA is low in human hepatocarcinoma cell lines that are not susceptible to HBV or HDV infection, the mRNA level of NTCP is about more than 10,000 times lower in Huh-7 cells than that in primary human and tupaia hepatocytes (Fig. 12A). It was examined if NTCP overexpression renders Huh-7 cells susceptible to viral infection.
• Huh-7 cells transfected with vector plasmid allowed no HDV infection
• hNTCP-transfected Huh-7 cells supported efficient HDV infection comparable to PTH cells, 5-10% of cells being infected as shown by staining of HDV delta antigen, which mainly locates in cell nuclei (Fig. 12B), and HDV viral genomic RNA
• HBV entry inhibitors including Myr-47/WT peptide and Hepatitis B Immune Globulin (HBIG), demonstrating a genuine infection of HDV mediated by HBV envelopes on the hNTCP-transfected Huh-7 cells (Fig. 12C).
• HBV envelopes a hepatoma cell line HepaRG
• Huh-7 complemented with NTCP provides a valuable system with easy access for studying viral entry mediated by HBV envelope proteins.
• HBV infection on Huh-7 and HepG2 cells complemented with NTCP was also examined. With optimized conditions, considerable de novo viral infections, as evidenced by increasing amount of secreted HBV e antigen and substantial accumulation of the HBV 3.5Kb RNA and total HBV mRNA during the testing period, were observed when the cells were transfected with hNTCP and subsequently infected by HBV virions. Therefore, NTCP substantially contributes to efficient HBV infection although other factors may also be involved in the infection process which usually requires hepatic functions of well differentiated hepatocytes (Seeger et al, in Field's Virology, D. M. Knipe, P. M. Howley, Eds. (Lippincott, Williams, and Wilkins, Philadelphia, 2007), vol. 2, pp. 2977).
• Hepatoma cells and primary hepatocytes transfected with NTCP were able to support
• Huh-7 cells regularly maintained in DMEM with 10% FBS, were transfected with plasmids expressing NTCPs or a vector control using Lipofectamine 2000. Cells were cultured in DMEM with 10% FBS after transfection for 12 hours, and then the medium was changed to PMM. 48 hours after transfection, cells were inoculated for 16hours with HBV and cells were co-cultured with primary fibroblast from tupaia. The cells were maintained in PMM and the medium was changed every two days. Viral S antigens were examined as indicated on days 4, 6, 9 after the infection (Fig. 13). A different hepatoma cell line HepG2 was also tested with similar result. HBV viral e antigens were examined as indicated on days 4, 7, and 10 post infection, viral specific R As were examined on day 12 after the infection (Fig. 21).
• NTCP decreased in primary cultured PTH at both m NA level (Fig. 12D) and protein level (Fig. 6). It was hypothesized that the short-lived susceptibility of PTH to HBV infection (Fig. 14) could be largely due to the rapid loss of NTCP expression in the cells, and exogenous expressing NTCP may rescue the susceptibility of these cells to HBV infection. To test this, PTH cells that lost NTCP mRNA expression and no longer susceptible to viral infection, were transfected with human NTCP and subsequently tested for HBV infection.
• hNTCP- but not control plasmid transfected PTH cells markedly regained susceptibility to HBV infection with 3 ⁇ 5% of total hepatocytes being infected (Fig. 12E).
• the level of secreted viral antigen from NTCP-transfected cells was also significantly increased compared with that in the control (Fig. 12F).
• the infectivity is not yet comparable to that on short-time cultured PTH, considering the relative low efficiency for transfection ( ⁇ 15- 20%) of the primary PTH cells, the infectivity conferred by NTCP transfection is remarkable.
• NTCP mutations that affected its physiological function could also interfere with HBV infection
• Four mutations, which are to interfere with NTCP physiological functions, have been made and tested for their surface expression, binding activities with FITC labeled preSl peptide (myr-59-FITC), and functionalities as HDV receptor.
• Huh7 cells were transfected with wildtype- and mutant human NTCP, all the constructs bear a C9 tag at their C-terminus. 36h post transfection, cells were detached and cell surface proteins were biotinylated with sulfo-NHS-LC-Biotin, cells were then lysed with 1% CHAPS in PBS and subsequently precipitated with anti-C9 antibody and detected by Western blot. Surface expression of NTCP mutants: wildtype and mutant NTCP show comparable surface expression level (except Q68A) (Fig. 15).
• Huh7 cells were transfected with wildtype- and mutant human NTCP, 36h post transfection, cells were stained with myr-59-FITC (Fig. 16).
• Huh7 cells were transfected with wildtype- and mutant human NTCP, 36h post transfection, cells were infected by HDV virus, 5 days post infection, the HDV genomic in infected Huh7 cells was quantified by RT-PCR. HDV infection on NTCP mutants Q68A,SN105/106AA, E257A was clearly reduced (Fig. 17).
• NTCP transgenic mice or rat as model for HBV/HDV infection in vivo
• NTCP from human or tupaia could support HDV entry. It has been known that mouse hepatocytes could support robust HBV and HDV replication, the only restriction for HBV/HDV infection on these cells is at the viral entry level.
• Primary hepatocytes from mouse or rat were isolated, and transfected with human NTCP expressor, and then inoculated with HDV virus. The human NTCP transfected cells could be infected with HDV (Fig. 19).
• NTCP transgenic mice or rat will be a very useful model for studying the HBV/HDV infection in vivo.
• LY294002 a morpholine derivative of quercetin that inhibits
• PBKs phosphoinositide 3-kinases
• Fig. 20 reduced HBV infection
• 50 uM LY294002 was used for a time-course experiments: the results show that the earlier the chemical was applied to the cells, the stronger effect on inhibition of HBV infection, indicating it works at entry step of HBV infection, most likely through inhibits NTCP
• a plasmid containing a head to tail trimer of 1.0 X HDV cDNA of HDV (Genotype I, Genbank accession number: AF425644.1) under the control a CMV promoter was constructed with de novo synthesized HDV cDNA for the production of HDV RNPs.
• HDV virions were produced in Huh-7 as previously described.
• Stable HepG2 cell line was established by transfect human NTCP/pcDNA3.1 vector into HepG2 cells and selected with puromycin. Cells were grown in culture conditions, with defined ingredients.
• the Cell culture medium hepatocytes maintenance medium (PMM) which is Williams E medium supplemented with 5 ⁇ g/ml transferrin, lOng/ml EGF, 3 ⁇ g/ml insulin, 2 L-glutamine, 18 ⁇ g/ml hydrocortisone, 40ng/ml dexamethasone, 5ng/ml sodium selenite, 2% DMSO, lOOU/ml penicillin and 100 ⁇ g/ml streptomycin. Cells were maintained in a 5% C0 2 humidified incubator at 37°C with regular medium change every 2 days.
• Readout is obtained from immunofluorescences detection of intranuclear delta antigen of HDV using mouse monoclonal antibody 4G5.
• NTCP sodium-taurocholate cotransporting polypeptide
• BSEP bile salt export pump

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