CHIMERIC NUCLEIC ACID MOLECULES WITH NON-AUG TRANSLATION INITIATION SEQUENCES AND USES THEREOF
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 .- 30 . (canceled)
31 . A T cell comprising 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 polypeptide comprising one or more cytokine, 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 polypeptide per mole of mRNA,
wherein the chimeric nucleic acid molecule is an mRNA molecule or an mRNA molecule contained in a vector and operably linked to an expression control sequence.
32 . The T cell of claim 31 , wherein the MTI comprises one, two, three, or four non-AUG translation initiation sites.
33 . The T cell of claim 32 , wherein the non-AUG translation initiation sites are CUG translation initiation sites.
34 . The T cell of claim 33 , wherein the MTI comprises an AUG translation initiation site downstream of the CUG translation initiation sites.
35 . The T cell of claim 31 , wherein the MTI comprises (a) a nucleic acid molecule encoding one or two nuclear localization domains located downstream of two or three CUG translation initiation sites and upstream of an AUG translation initiation site, or (b) two or three CUG translation initiation sites upstream of a nucleic acid molecule encoding one or two nuclear localization domains and no AUG translation initiation site.
36 . The T cell of claim 31 , wherein the MTI comprises a 5′-portion of a human FGF2 gene, wherein the 5′-portion of the human FGF2 gene contains an 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 polypeptide.
37 . The T cell of claim 36 , wherein the MTI further comprises from about 15 nucleotides to about 45 nucleotides downstream of the FGF2 AUG translation initiation site.
38 . The T cell of claim 36 , 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.
39 . The T cell of claim 31 , 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.
40 . The T cell of claim 31 , wherein the encoded polypeptide comprises a fusion protein.
41 . The T cell of claim 40 , wherein the fusion protein comprises from two to about ten polypeptide components.
42 . The T cell of claim 41 , wherein one or more of each encoded polypeptide component of the fusion protein further comprises on the N-terminus and/or C-terminus: (a) from one to about ten junction amino acids; (b) a spacer comprising from two to about 35 amino acids; (c) a spacer comprising a (Gly 4 Ser) n wherein n is an integer from 1 to 5; (d) a natural cleavage site comprising from one to about ten amino acids; (e) a self-cleaving amino acid sequence; (f) an intracellular trafficking sequence; or (g) any combination thereof.
43 . The T cell of claim 31 , wherein the encoded polypeptide comprises a secretion signal amino acid sequence, a membrane localization amino acid sequence, an endosome targeting sequence, a dendritic cell targeting amino acid sequence, or any combination thereof.
44 . A T cell comprising a multiplex translation initiation (MTI) sequence-comprising a 5′-portion of a human FGF2 gene, wherein the 5′-portion of the human FGF2 gene comprises (i) an AUG translation initiation site and (ii) a sequence comprising from about 123 nucleotides to about 385 nucleotides upstream of the AUG translation initiation site, wherein the sequence upstream of the AUG translation initiation site comprises one to four translationally active non-AUG translation initiation sites in frame with the AUG translation initiation site, wherein the MTI is operatively linked in frame to a nucleic acid molecule encoding a polypeptide comprising one or more cytokine and the MTI allows the production of more than one mole of polypeptide per mole of mRNA,
wherein the chimeric nucleic acid molecule is an mRNA molecule or an mRNA molecule contained in a vector and operably linked to an expression control sequence.
45 . The T cell of claim 44 , wherein the MTI comprises one, two, or three non-AUG translation initiation sites.
46 . The T cell of claim 45 , wherein the MTI comprises (a) a nucleic acid molecule encoding one or two nuclear localization domains located downstream of two or three non-AUG translation initiation sites and upstream of the FGF2 AUG translation initiation site, or (b) two or three non-AUG translation initiation sites upstream of a nucleic acid molecule encoding one or two nuclear localization domains and no FGF2 AUG translation initiation site.
47 . The T cell of claim 44 , wherein the MTI further comprises from about 15 nucleotides to about 45 nucleotides downstream of the FGF2 AUG translation initiation site.
48 . The T cell of claim 44 , 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.
49 . The T cell of claim 44 , 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.
50 . A method comprising administering the T cell of claim 31 to a subject.
51 . A method comprising administering the T cell of claim 44 to a subject.
52 . A method comprising introducing, into T cells from a subject, mRNA 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 polypeptide comprising one or more cytokine, 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 polypeptide per mole of mRNA.
53 . The method of claim 52 , further comprising optionally expanding the T cells, and reintroducing the T cells to the subject.