Artificial alphavirus-derived rna replicon expression systems

Abstract

Synthetic alphavirus-derived replicon expression systems comprising nucleic acid sequences encoding at least one modified nonstructural protein, and synthetic nucleic acid sequences encoding at least one heterologous protein are described. Methods of producing at least one heterologous protein in a cell, or of inducing an immune response in a subject by administering and/or expressing the synthetic alphavirus-derived replicon expression systems are provided.

What is claimed is: 
     
         1 . A method of preventing, inhibiting, or treating the symptoms of a disease or condition in a subject comprising:
 providing a synthetic alphavirus-derived replicon nucleic acid molecule comprising: (i) a first nucleic acid encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4, and comprising at least one silent mutation introduced within a region from nt 503 to nt 658, nt 658 to nt 1620, nt 1620 to nt 2560, nt 2560 to nt 3954, nt 3954 to nt 4120, nt 6381 to nt 7083, and nt 6966 to nt 7526 in the sequence of the alphavirus genome as set forth in SEQ ID NO: 17; and (ii) a second nucleic acid comprising a modified subgenomic open reading frame (ORF);   and   administering a therapeutically effective amount of the synthetic alphavirus-derived replicon nucleic acid molecule to a subject.   
     
     
         2 . The method of  claim 1 , wherein the modified subgenomic ORF comprises a sequence encoding a first heterologous protein. 
     
     
         3 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding an altered nsP4 that comprises a second heterologous protein. 
     
     
         4 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence with at least 90% identity to the reference sequence forth in SEQ ID NO: 21, or 25. 
     
     
         5 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding a junction of nsP1 and nsP2 and comprising a sequence with at least 90% identity to the reference sequence selected from the group consisting of SEQ ID NOS: 24, 22, and 26. 
     
     
         6 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding a junction of nsP2 and nsP3 and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO:23. 
     
     
         7 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding nsP4 and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 19 or 20. 
     
     
         8 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 25, and a polynucleotide encoding nsP2 and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 26. 
     
     
         9 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding nsP2 and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 22, and a polynucleotide encoding nsP4 and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 20. 
     
     
         10 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding nsP1, and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 21; a polynucleotide encoding nsP1, and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 25, a polynucleotide encoding a junction of nsP1 and nsP2, and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 24; a polynucleotide encoding nsP2, and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 26; a polynucleotide encoding a junction of nsP2 and nsP3, and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 23; and a polynucleotide encoding nsP4, and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 20. 
     
     
         11 . The method of  claim 1 , wherein the first nucleic acid comprises a polynucleotide encoding the nonstructural polyprotein and comprising a polynucleotide sequence with at least 90% identity to the reference sequence as set forth in SEQ ID NO: 30. 
     
     
         12 . The method of  claim 1 , the first nucleic acid molecule comprises a polynucleotide sequence with at least 90% identity to the reference sequence as set forth in SEQ ID NO: 18. 
     
     
         13 . The method of  claim 3 , wherein the first heterologous protein or the second heterologous comprises an immunogenic protein. 
     
     
         14 . The method of  claim 3 , wherein the first heterologous protein or the second heterologous protein comprises an antigenic protein isolated or derived from a viral pathogen. 
     
     
         15 . The method of  claim 14 , wherein the viral pathogen is picornavirus. 
     
     
         16 . The method of  claim 3 , wherein the second heterologous protein comprises a 3Cprotease protein, and the altered nsP4 comprises an amino acid sequence with at least 90% identity to the reference sequence as set forth in SEQ ID NO: 40. 
     
     
         17 . The method of  claim 16 , wherein the altered nsP4 is encoded by a polynucleotide sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 32. 
     
     
         18 . The method of  claim 3 , wherein the second nucleic acid comprises a polynucleotide encoding an 01 Manisa P1 protein and comprising a sequence with at least 90% identity to the reference sequence set forth in SEQ ID NO: 33. 
     
     
         19 . The method of  claim 3 , wherein the first heterologous protein comprises an 01 Manisa P1 protein and the second heterologous protein comprises a 3Cprotease. 
     
     
         20 . The method of  claim 3 , wherein the second nucleic acid comprises a polynucleotide encoding an antigenic protein. 
     
     
         21 . The method of  claim 3 , wherein the second heterologous protein comprises a STING protein, and the altered nsP4 comprises an amino acid sequence with at least 90% identity to the reference sequence as set forth in SEQ ID NO: 42. 
     
     
         22 . The method of  claim 3 , wherein the first heterologous protein comprises an antigenic protein and the second heterologous protein comprises a STING protein. 
     
     
         23 . The method of  claim 1 , wherein the step of administering results in preventing, treating, reducing the severity or slowing the progression of the disease in the subject. 
     
     
         24 . The method of  claim 1 , wherein the disease is cancer. 
     
     
         25 . The method of  claim 24 , wherein the cancer is melanoma or epithelial tumor. 
     
     
         26 . The method of  claim 1 , wherein the disease is FMD. 
     
     
         27 . The method of  claim 1 , wherein the mammal subject is a cloven-hooved animal or a human.