Novel 3' terminal sequence of hepatitis C virus genome and diagnostic and therapeutic uses thereof

Abstract

The invention relates to the discovery of a novel RNA sequence at the 3′ terminal sequence of hepatitis C virus (HCV) genome RNA. Included in the invention are the 3′ sequence, its complement, and their use for nucleic-acid based diagnostics and for developing and evaluating novel anti-HCV therapies. This sequence element, which is conserved among HCV genotypes, is likely to be essential for viral replication, and required for construction of full-length HCV cDNA clones capable of yielding infectious RNA, progeny virus or replication-competent HCV replicons. Such functional clones are useful tools for evaluation of therapeutic approaches and as substrates for developing candidate attenuated or inactivated HCV derivatives for vaccination against HCV.

What is claimed is:  
     
         1 . A nucleic acid molecule or degenerate variant thereof, which encodes a 3′ terminal sequence element, or a fragment thereof, selected from the group consisting of: 
 (A) the nucleotide sequences of SEQ ID NOS:14;  
 (B) the nucleotide sequences of FIG. 3 (SEQ ID NOS:20-24);  
 (C) the nucleotide sequences of FIG. 6 (SEQ ID NOS:28-31);  
 (D) the nucleotide sequence of FIG. 8 (SEQ ID NO:33-36);  
 (E) nucleotide sequences that hybridize to any of the foregoing sequences under standard hybridization conditions; and  
 (F) nucleotide sequences having a secondary structure substantially similar to the structure of FIG. 4.  
 
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the 3′ terminal sequence element is present in HCV.  
     
     
         3 . A recombinant DNA or RNA molecule comprising a nucleotide sequence or degenerate variant thereof, which encodes a 3′ terminal sequence element, or a fragment thereof, selected from the group consisting of: 
 (A) the nucleotide sequences of SEQ ID NOS:14;  
 (B) the nucleotide sequences of FIG. 3 (SEQ ID NOS:20-24);  
 (C) the nucleotide sequences of FIG. 6 (SEQ ID NOS:28-31);  
 (D) the nucleotide sequences of FIG. 8 (SEQ ID NOS:33-36);  
 (E) nucleotide sequences that hybridize to any of the foregoing sequences under standard hybridization conditions; and  
 (F) nucleotide sequences having a secondary structure substantially homologous to the structure of FIG. 4.  
 
     
     
         4 . The nucleic acid molecule or recombinant DNA or RNA of claims  1 , 2 or 3, wherein said 3′ terminal sequence element is operatively linked to an expression control sequence.  
     
     
         5 . A probe capable of screening for the 3′ terminal sequence element of alternate strains of HCV prepared from the nucleotide sequence of  claim 2 .  
     
     
         6 . The probe of  claim 5  labeled with a detectable marker.  
     
     
         7 . A host cell transformed with a recombinant DNA or RNA molecule comprising a DNA or RNA sequence or degenerate variant thereof, which encodes a 3′ terminal sequence element, or a fragment thereof, selected from the group consisting of: 
 (A) the nucleotide sequences of SEQ ID NOS:1-4;  
 (B) the nucleotide sequences of FIG. 3 (SEQ ID NOS:2024);  
 (C) the nucleotide sequences of FIG. 6 (SEQ ID NOS:28-31);  
 (D) the nucleotide sequences of FIG. 8 (SEQ ID NOS:33-36);  
 (E) nucleotide sequences that hybridize to any of the foregoing sequences under standard hybridization conditions; and  
 (F) nucleotide sequences having a secondary structure substantially similar to the structure of FIG. 4.  
 
     
     
         8 . The host cell of  claim 7  wherein the host is a human cell in tissue culture.  
     
     
         9 . The host cell of  claim 8 , wherein the host cell is a liver cell.  
     
     
         10 . A method for detecting the presence or activity of the 3′ terminal sequence element of  claim 1 , comprising: 
 A. contacting a biological sample from a mammal in which the presence or activity of a virus containing the 3′ terminal sequence element is suspected with a binding partner of said 3′ terminal sequence element under conditions that allow binding of said 3′ terminal sequence element to said binding partner to occur; and  
 B. detecting whether binding has occurred between said 3′ terminal sequence element from said sample and the binding partner; 
 wherein the detection of binding indicates that presence or activity of said virus in said sample.  
 
 
     
     
         11 . The method of  claim 10 , wherein the virus is HCV.  
     
     
         12 . A method for detecting the presence and activity of a virus in mammals comprising detecting the presence or activity of a 3′ terminal sequence element according to the method of  claim 10 , wherein detection of the presence or activity of the 3′ terminal sequence element indicates the presence and activity of in said mammals.  
     
     
         13 . A method for detecting binding sites for the 3′ terminal sequence element of  claim 1 , comprising: 
 A. placing a labeled binding partner which specifically binds to said 3′ terminal sequence element in contact with a biological sample from a mammal in which binding sites for said 3′ terminal sequence element are suspected, and  
 B. examining said biological sample in binding studies for the presence of said labeled binding partner; 
 wherein the presence of said labeled binding partner indicates a binding site for said 3′ terminal sequence element.  
 
 
     
     
         14 . A method of testing the ability of a drug or other entity to modulate the activity of the 3′ terminal sequence element of claim I which comprises: 
 A. culturing test cells which are infected with a virus or transfected with a nucleic acid containing said 3′ terminal sequence element;  
 B. adding the drug under test; and  
 C. measuring the replication of said virus or nucleic acid in said test cells.  
 
     
     
         15 . The method of  claim 14 , wherein the virus is HCV or the nucleic acid is an HCV RNA replicon.  
     
     
         16 . An assay system for screening drugs and other agents for ability to modulate the replication of a virus containing the 3′ terminal sequence element of  claim 1 , comprising: 
 A. culturing observable test cells inoculated with a drug or agent directed to the 3′ terminal sequence element;  
 B. harvesting a supernatant from said test cells; and  
 C. examining said supernatant for the presence of said virus wherein an increase or a decrease in a level of said virus indicates the ability of a drug to modulate the replication of said virus.  
 
     
     
         17 . The assay system of  claim 16 , wherein the virus is HCV.  
     
     
         18 . An assay system for screening drugs and other agents for ability to modulate the replication of a virus containing the 3′ terminal sequence element of  claim 1 , comprising: 
 A. culturing observable test cells inoculated with a drug or agent directed to the 3′ terminal sequence element;  
 B. contacting said test cells with a labelled binding partner of or a primer which will amplify the genome of said virus; and  
 C. determining the level of viral genome in the test cells wherein an increase or a decrease in a level of said genome indicates the ability of a drug to modulate the replication of said virus.  
 
     
     
         19 . The assay system of  claim 18 , wherein the virus is HCV.  
     
     
         20 . A test kit for the demonstration of a virus in a eukaryotic cellular sample, comprising: 
 A. a predetermined amount of a detectably labelled specific binding partner of the 3′ terminal sequence element of  claim 1;     B. other reagents; and    C. directions for use of said kit.    
     
     
         21 . The test kit of  claim 20 , wherein the virus is HCV.  
     
     
         22 . A method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals caused by a virus containing the 3′ terminal sequence element of  claim 1 , comprising administering to a mammal a therapeutically effective amount of a material selected from the group consisting of the 3′ terminal sequence element, its complement or a fragment thereof, an agent capable of promoting the production and/or activity of said 3′ terminal sequence element, an agent capable of mimicking the activity of said 3′ terminal sequence element, an agent capable of inhibiting the activity of said 3′ terminal sequence element, and mixtures thereof, or a specific binding partner thereto.  
     
     
         23 . The method of  claim 22 , wherein the virus is HCV.  
     
     
         24 . The method of  claim 22  wherein said disease states include hepatitis, cirrhosis, and hepatocellular carcinoma.  
     
     
         25 . The method of  claim 22  wherein said 3′ terminal sequence element, its complement or a fragment thereof, an agent capable of promoting the production and/or activity of said 3′ terminal sequence element, an agent capable of mimicking the activity of said 3′ terminal sequence element, an agent capable of inhibiting the activity of said 3′ terminal sequence element, and mixtures thereof, or a specific binding partner thereto is administered to modulate the course of therapy wherein a primary therapeutic agent is also being administered.  
     
     
         26 . The method of  claim 22  wherein said 3′ terminal sequence element, its complement or a fragment thereof, an agent capable of promoting the production and/or activity of said 3′ terminal sequence element, an agent capable of mimicking the activity of said 3′ terminal sequence element, an agent capable of inhibiting the activity of said 3′ terminal sequence element, and mixtures thereof, or a specific binding partner thereto is administered to modulate the course of therapy where interferon is being administered as the primary therapeutic agent.  
     
     
         27 . The method of  claim 22  wherein said 3′ terminal sequence element, its complement or a fragment thereof, an agent capable of promoting the production and/or activity of said 3′ terminal sequence element, an agent capable of mimicking the activity of said 3′ terminal sequence element, an agent capable of inhibiting the activity of said 3′ terminal sequence element, and mixtures thereof, or a specific binding partner thereto is administered to modulate the course of therapy where two or more additional therapeutic agents are being co-administrated.  
     
     
         28 . The method of  claim 22  wherein said 3′ terminal sequence element, its complement or a fragment thereof, an agent capable of promoting the production and/or activity of said 3′ terminal sequence element, an agent capable of mimicking the activity of said 3′ terminal sequence element, an agent capable of inhibiting the activity of said 3′ terminal sequence element, and mixtures thereof, or a specific binding partner thereto is administered to modulate the course of therapy where interferon is being co-administered with one or more additional therapeutic agents.  
     
     
         29 . A pharmaceutical composition for the treatment of cellular debilitation, derangement and/or dysfunction in mammals, comprising: 
 A. a therapeutically effective amount of a material selected from the group consisting of the 3′ terminal sequence element of  claim 1 , its complement or a fragment thereof, an agent capable of promoting the production and/or activity of said 3′ terminal sequence element, an agent capable of mimicking the activity of said 3′ terminal sequence element, an agent capable of inhibiting the activity of said 3′ terminal sequence element, and mixtures thereof, or a specific binding partner thereto; and    B. a pharmaceutically acceptable carrier.    
     
     
         30 . An attenuated virus comprising the 3′ terminal sequence element of  claim 1  in combination with a sequence which is substantially homologous to that of HCV-1, HC-J1, HC-J, HCV-BK, HCV-H, HC-J6, HC-J8, HC-J483, HC-J491, HC-C2, HCV-JK, HCV-N, HCV-T, HCV-JT, HC-G9, HCV-K3a, NZL1, or HCV-Tr, which virus is replication-impaired.  
     
     
         31 . An antisense nucleic acid against a viral genome RNA or its complement comprising a nucleic acid sequence hybridizing to one of the nucleic acid molecules of SEQ ID NOS:1-12, 20-24, 28-31 and 33-36 or their complements.  
     
     
         32 . The antisense nucleic acid of  claim 31  which comprises an oligonucleotide modified to enhance stability, uptake or targeted intracellular degradation.  
     
     
         33 . The antisense nucleic acid of  claim 31  which is RNA.  
     
     
         34 . The antisense nucleic acid of  claim 31  which is DNA.  
     
     
         35 . A recombinant DNA or RNA molecule having a DNA or RNA sequence which, on transcription, produces an antisense ribonucleic acid against a viral genome RNA or its complement, said antisense ribonucleic acid comprising a nucleic acid sequence which hybridizes to one of the nucleic acid molecules of SEQ ID NOS:14, 20-24, 28-31 and 33-36 or their complements.  
     
     
         36 . A virus-producing cell line transfected with the recombinant DNA or RNA molecule of  claim 35 .  
     
     
         37 . A ribozyme that cleaves the viral mRNA of  claim 35 .  
     
     
         38 . The ribozyme of  claim 37  which is a Tetrahymena-type ribozyme.  
     
     
         39 . The ribozyme of  claim 37  which is a Hammerhead-type ribozyme.  
     
     
         40 . A recombinant DNA molecule having a DNA sequence which, upon transcription, produces the ribozyme of  claim 37 .  
     
     
         41 . A virus-expressing cell line transfected with a recombinant DNA or RNA molecule comprising a replication competent HCV RNA comprising the 3′ terminal sequence element of  claim 1 .  
     
     
         42 . A virus-producing cell line transfected with a recombinant DNA or RNA molecule comprising the 3′ terminal sequence element of  claim 1  in combination with a sequence which is substantially homologous to that of HCV-1, HC-J1, HC-J, HCV-BK, HCV-H, HC-J6, HC-J8, HC-J483, HC-J491, HC-C2, HCV-JK, HCV-N, HCV-T, HCV-JT, HC-G9, HCV-K3a, NZL1, or HCV-TR.