Chimeric nucleic acid molecules with non-aug translation initiation sequences and uses thereof

Abstract

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 method of eliciting an immune response, comprising administering to a human subject an effective amount of a population of immune cells comprising immune cells containing a chimeric nucleic acid molecule,
 wherein the chimeric nucleic acid molecule comprises 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 comprised of a plurality of 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,   thereby eliciting an immune response.   
     
     
         2 . The method of  claim 1 , wherein the MTI comprises one, two, three, or four non-AUG translation initiation sites. 
     
     
         3 . The method of  claim 2 , wherein one, two, three, or four of the non-AUG translation initiation sites are CUG translation initiation sites. 
     
     
         4 . The method of  claim 3 , wherein the MTI comprises an AUG translation initiation site downstream of the one, two, three, or four CUG translation initiation sites. 
     
     
         5 . The method 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 method 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 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 fusion protein. 
     
     
         7 . The method 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 method 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 method 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 method of  claim 1 , wherein the encoded fusion protein comprises from two to about ten antigenic peptides. 
     
     
         11 . The method of  claim 10 , wherein two to about ten of the antigenic peptides are each different from each other. 
     
     
         12 . The method of  claim 1 , wherein the antigenic peptide comprises a tumor-associated antigen or a pathogen-related antigen. 
     
     
         13 . The method of  claim 12 , wherein the tumor-associated antigen comprises an antigen associated with breast cancer, triple negative breast cancer, inflammatory breast cancer, ovarian cancer, uterine cancer, colorectal cancer, colon cancer, primary peritoneal cancer, testicular cancer, renal cancer, melanoma, glioblastoma, lung cancer, prostate cancer, or any combination thereof. 
     
     
         14 . The method of  claim 12 , wherein the pathogen-related antigen comprises an antigen from a virus, parasite, or bacteria. 
     
     
         15 . The method of  claim 10 , wherein one or more of the antigenic peptides are an HLA Class I antigenic peptide, an HLA Class II antigenic peptide, an HLA Class II antigenic peptide comprising an embedded HLA Class I antigenic peptide, or any combination thereof. 
     
     
         16 . The method of  claim 1 , wherein the encoded fusion protein comprises an amino acid cleavage sequence amino-terminal to one or more of the encoded polypeptide components of the fusion protein, wherein the amino acid cleavage sequence comprises a 2A peptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), or foot-and-mouth disease virus (F2A). 
     
     
         17 . The method of  claim 1 , wherein the encoded fusion protein 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. 
     
     
         18 . The method of  claim 1 , wherein the encoded fusion protein comprises:
 (a) a 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, optionally comprising a nucleic acid molecule encoding an amino acid cleavage sequence operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the VSVG signal amino acid sequence;   (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;   (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;   (e) any one of (a)-(d) further comprising a dendritic cell targeting amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:45; or   any one of (a) or (c)-(e) further comprising an intracellular trafficking sequence.   
     
     
         19 . The method of  claim 1 , wherein the chimeric nucleic acid molecule is:
 (a) an mRNA molecule;   (b) a DNA molecule;   (c) a DNA or an RNA molecule contained in a vector and operably linked to an expression control sequence.   
     
     
         20 . The method of  claim 19 , wherein the vector of subpart (c) of  claim 21  comprises:
 (a) a plasmid vector comprised of DNA, wherein the chimeric nucleic acid molecule is a DNA molecule contained in the DNA plasmid vector, 
 (b) a viral vector comprised of DNA, wherein the chimeric nucleic acid molecule is a DNA molecule contained in the DNA viral vector, or 
 (c) a viral vector comprised of RNA, wherein the chimeric nucleic acid molecule is an RNA molecule contained in the RNA viral vector. 
 
     
     
         21 . The method of  claim 20 , wherein the vector comprises a viral vector selected from a rhabdoviral, adenoviral, herpesviral, poxviral, or retroviral vector. 
     
     
         22 . The method of  claim 1 , wherein the chimeric nucleic acid molecule is formulated as a composition comprising a therapeutically acceptable carrier or excipient. 
     
     
         23 . The method of  claim 1 , wherein the elicited immune response comprises a cellular immune response. 
     
     
         24 . The method of  claim 1 , further comprising administering an effective amount of an antigenic peptide immunization composition. 
     
     
         25 . The method of  claim 24 , wherein the chimeric nucleic acid molecule encodes one or more of the same antigenic peptides of the antigenic peptide composition. 
     
     
         26 . The method of  claim 25 , wherein:
 (a) the population of immune cells comprising immune cells containing the chimeric nucleic acid molecule and the antigenic peptide immunization compositions are administered simultaneously;   (b) the population of immune cells comprising immune cells containing the chimeric nucleic acid molecule and the antigenic peptide immunization compositions are administered sequentially;   (c) the population of immune cells comprising immune cells containing the chimeric nucleic acid molecule is administered from about 1 hour to about 8 weeks after administering the antigenic peptide immunization composition;   (d) the antigenic peptide immunization composition is administered from about 1 hour to about 8 weeks after administering the population of immune cells comprising immune cells containing the chimeric nucleic acid molecule;   (e) any one of (a)-(d), wherein the immune cells are autologous to the human subject or allogeneic to the human subject; or   (f) any one of (a)-(e), wherein the immune cells are T cells.   
     
     
         27 . The method of  claim 26 , further comprising one or more additional administrations of an effective amount of the antigenic peptide immunization composition and/or the chimeric nucleic acid molecule after the first administration of the antigenic peptide immunization composition and/or the chimeric nucleic acid molecule. 
     
     
         28 . The method of  claim 1 , wherein the population of immune cells comprising the immune cells containing the chimeric nucleic acid molecule is administered intradermally. 
     
     
         29 . The method of  claim 12 , wherein before or after administering the population of immune cells comprising the chimeric nucleic acid molecule encoding a fusion protein comprised of a plurality of tumor-associated antigens, the human subject is treated with surgery, chemotherapy, radiation therapy, antibody therapy, immunosuppressive therapy, or any combination thereof. 
     
     
         30 . The method of  claim 12 , wherein before or after administering the population of immune cells comprising the chimeric nucleic acid molecule encoding a fusion protein comprised of a plurality of tumor-associated antigens, the human subject is administered cyclophosphamide, trastuzumab, anti-PD1, anti-PDL1, anti-CTLA4, or any combination thereof.