Chimeric nucleic acid molecule with non-AUG translation initiation sequences
The present disclosure relates to nucleic acid vaccine compositions and methods for preventing or treating pathological conditions, such as cancer or infectious disease. Further, the disclosure provides methods for more efficient production of antigens via mRNA containing one or more non-conventional start codons to promote multiplex initiation of translation in eukaryotic cells.
1. A chimeric nucleic acid molecule, comprising a multiplex translation initiation (MTI) sequence comprising from two to about five translation initiation sites operatively linked in frame to a nucleic acid molecule encoding a fusion protein comprising from two to about ten eukaryotic folate receptor-alpha (FRα) antigenic peptides, wherein at least one of the MTI translation initiation sites is a non-AUG translation initiation site and the MTI allows the production of more than one mole of fusion protein per mole of mRNA.
2. The chimeric nucleic acid molecule of claim 1 , wherein the MTI comprises one, two, three, or four non-AUG translation initiation sites.
3. The chimeric nucleic acid molecule of claim 2 , wherein the non-AUG translation initiation sites are CUG translation initiation sites.
4. The chimeric nucleic acid molecule of claim 3 , wherein the MTI comprises an AUG translation initiation site downstream of the CUG translation initiation sites.
5. The chimeric nucleic acid molecule of claim 4 , wherein the MTI comprises one or two nuclear localization domains located upstream of the AUG translation initiation site and downstream of at least one CUG translation initiation site.
6. The chimeric nucleic acid molecule of claim 1 , wherein the MTI comprises a 5′-portion of a human FGF2 gene, wherein the 5′-portion of the human FGF2 gene contains the FGF2 AUG translation initiation site and about 123 nucleotides to about 385 nucleotides upstream of the FGF2 AUG translation initiation site that is in frame with the nucleic acid molecule encoding the fusion protein.
7. The chimeric nucleic acid molecule of claim 6 , wherein the MTI further comprises from about 15 nucleotides to about 45 nucleotides downstream of the FGF2 AUG translation initiation site.
8. The chimeric nucleic acid molecule of claim 6 , wherein the 5′-portion of the human FGF2 gene encodes a polypeptide having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.:8-12, or encodes a polypeptide as set forth in any one of SEQ ID NOS.:8-12.
9. The chimeric nucleic acid molecule of claim 1 , wherein the MTI sequence has at least 90% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOS.:1-6, 95, or 96.
10. The chimeric nucleic acid molecule of claim 1 , wherein the fusion protein comprises from two to about five different or same antigenic FRα peptides, or the fusion protein comprises five different antigenic FRα peptides.
11. The chimeric nucleic acid molecule of claim 1 , wherein one or more of the FRα antigenic peptides are an HLA Class I antigenic FRα peptide, an HLA Class II antigenic FRα peptide, an HLA Class II antigenic FRα peptide comprising an embedded HLA Class I antigenic FRα peptide, or any combination thereof.
12. The chimeric nucleic acid molecule of claim 1 , wherein two or more of the encoded FRα antigenic peptides of the fusion protein are separated by one to about ten junction amino acids, a spacer comprising from two to about 35 amino acids, a natural cleavage site comprising from one to about ten amino acids, a self-cleaving amino acid sequence, or combinations thereof.
13. The chimeric nucleic acid molecule of claim 1 , wherein two or more of the FRα antigenic peptides of the fusion protein are separated by a spacer comprising a (Gly 4 Ser) n , wherein n is an integer from 1 to 5.
14. The chimeric nucleic acid molecule of claim 1 , wherein the encoded fusion protein further comprises a secretion signal amino acid sequence, a membrane localization amino acid sequence, a dendritic cell targeting amino acid sequence, or any combination thereof.
15. The chimeric nucleic acid molecule of claim 1 , wherein each encoded FRα antigenic peptide of the fusion protein has at least 90% amino acid sequence identity to any one of SEQ ID NOS.:69-93 or to any one of SEQ ID NOS.:69-93 comprising from one to about five spacer amino acids on the N-terminus, C-terminus or both.
16. The chimeric nucleic acid molecule of claim 1 , wherein each encoded FRα antigenic peptide of the fusion protein has at least 90% amino acid sequence identity to any one of SEQ ID NOS.:69-78 or to any one of SEQ ID NOS.:69-78 comprising from one to about five spacer amino acids on the N-terminus, C-terminus or both.
17. The chimeric nucleic acid molecule of claim 16 , wherein the encoded fusion protein further comprises a secretion signal amino acid sequence, a membrane localization amino acid sequence, a dendritic cell targeting amino acid sequence, or any combination thereof.
18. The chimeric nucleic acid molecule of claim 16 , wherein the encoded fusion protein further comprises: (a) an VSVG signal amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:43 that is operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the fusion protein; (b) a VSVG trafficking amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:47 or 48 that is operably linked in frame to the MTI sequence; (c) a VSVG membrane localization amino acid sequence as encoded by nucleotides 457 to 516 of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence; or (d) a VSVG trafficking amino acid sequence and membrane localization amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence.
19. The chimeric nucleic acid molecule of claim 18 , wherein the fusion protein further comprises a dendritic cell targeting amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:45.
20. The chimeric nucleic acid molecule of claim 1 , wherein each encoded FRα antigenic peptide of the fusion protein has at least 90% amino acid sequence identity to any one of SEQ ID NOS.:79-93 or to any one of SEQ ID NOS.:79-93 comprising from one to about five spacer amino acids on the N-terminus, C-terminus or both.
21. The chimeric nucleic acid molecule of claim 1 , wherein each encoded FRα antigenic peptide of the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOS.:69-93 or as set forth in any one of SEQ ID NOS.:69-93 comprising from one to about five spacer amino acids on the N-terminus, C-terminus or both.
22. The chimeric nucleic acid molecule of claim 1 , wherein each encoded FRα antigenic peptide of the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOS.:69-78 or as set forth in any one of SEQ ID NOS.:69-78 comprising from one to about five spacer amino acids on the N-terminus, C-terminus or both.
23. The chimeric nucleic acid molecule of claim 22 , wherein the encoded fusion protein further comprises: (a) an VSVG signal amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:43 that is operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the fusion protein; (b) a VSVG trafficking amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:47 or 48 that is operably linked in frame to the MTI sequence; (c) a VSVG membrane localization amino acid sequence as encoded by nucleotides 457 to 516 of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence; or (d) a VSVG trafficking amino acid sequence and membrane localization amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence.
24. The chimeric nucleic acid molecule of claim 23 , wherein the fusion protein further comprises a dendritic cell targeting amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:45.
25. The chimeric nucleic acid molecule of claim 1 , wherein each encoded FRα antigenic peptide of the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NOS.:79-93 or as set forth in any one of SEQ ID NOS.:79-93 comprising from one to about five spacer amino acids on the N-terminus, C-terminus or both.
26. The chimeric nucleic acid molecule of claim 1 , wherein the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:49, 67, or 68.
27. The chimeric nucleic acid molecule of claim 1 , wherein the encoded fusion protein comprises a polypeptide having at least 90% sequence identity with any one of the polypeptides set forth in SEQ ID NOS.:53-55 or 59-61.
28. The chimeric nucleic acid molecule of claim 1 , wherein the chimeric nucleic acid molecule is an mRNA.
29. The chimeric nucleic acid molecule of claim 1 , wherein the chimeric nucleic acid molecule is DNA, the chimeric DNA molecule contained in an expression vector and operably linked to an expression control sequence.
30. A composition, comprising a chimeric nucleic acid molecule of claim 1 and a therapeutically acceptable carrier or excipient.