Compositions and methods for enhancing gene expression
AssigneeJanssen Pharmaceuticals, Inc.
InventorsKurt Iver Kamrud, Maung Nyan Win, Nathaniel Stephen Wang, Jason L. DeHart
Life of this patent
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Abstract
The present disclosure generally relates to nucleic acid molecules for use in regulating gene expression. Disclosed herein include nucleic acid molecules containing one or more structural elements of the viral capsid enhancer operably linked to a coding sequence of a gene of interest. In some embodiments, the viral capsid enhancer comprises a Downstream Loop (DLP) from a viral capsid protein, or a variant of the DLP.
Claims50
A nucleic acid molecule, comprising a modified viral RNA replicon, wherein the modified viral RNA replicon comprises:
- a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer comprising one or more RNA stem-loops, wherein the viral capsid enhancer is heterologous to the viral RNA replicon;
- and a second nucleic acid sequence comprising a sequence encoding for at least one viral nonstructural protein of a corresponding unmodified viral RNA replicon, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19.
A nucleic acid molecule comprising a modified viral RNA replicon, wherein the modified viral RNA replicon comprises, ordered from the 5′- to 3′-end,
- (1) a 5′ untranslated region (5′-UTR),
- (2) a nucleotide sequence encoding an amino-terminal fragment of the rispl of the VEEV,
- (3) a downstream loop (DLP) motif derived from Sindbis virus (SINV),
- (4) a nucleotide sequence encoding a 2A protease sequence (P2A), and
- (5) a nucleotide sequence encoding a polyprotein comprising the sequences of the non-structural proteins nsp1, nsp2, nsp3 and nsp4 of the VEEV.
A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:
- 19.
A nucleic acid molecule comprising a modified non-alphavirus RNA replicon, wherein the modified non-alphavirus RNA replicon comprises a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer or a variant thereof comprising one or more RNA stem-loops, and a second nucleic acid sequence comprising a sequence encoding for at least one viral nonstructural protein of a corresponding unmodified viral RNA replicon, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19.
A method for producing a polypeptide of interest in a cell, comprising introducing a modified viral RNA replicon into the cell, wherein the modified viral RNA replicon comprises:
- a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer comprising one or more RNA stem-loops, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and a second nucleic acid sequence comprising a sequence encoding for at least one nonstructural protein of a corresponding unmodified viral RNA replicon, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, and further wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19;
- and the modified RNA replicon further comprises one or more expression cassettes, wherein at least one of the one or more expression cassettes comprises a promoter operably linked to a coding sequence for the polypeptide of interest, thereby producing the polypeptide of interest in the cell.
A method for conferring a resistance to the innate immune system in a subject, comprising administering to the subject a nucleic acid molecule comprising a nucleic acid sequence which encodes a modified viral RNA replicon, wherein the modified viral RNA replicon comprises:
- a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer comprising one or more RNA stem-loops, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and a second nucleic acid sequence comprising a sequence encoding for at least one viral nonstructural protein of a corresponding unmodified viral RNA replicon and one or more expression cassettes, wherein at least one of the one or more expression cassettes comprises a promoter operably linked to a coding sequence for the polypeptide of interest, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, and further wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19.
A method for producing a polypeptide of interest in a subject, comprising administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a modified non-alphavirus RNA replicon, wherein the modified non- alphavirus RNA replicon comprises a first nucleic acid sequence encoding one or more structural elements of an alphavirus capsid enhancer comprising one or more RNA stem-loops, a second nucleic acid sequence comprising a sequence encoding for at least one viral nonstructural protein of a corresponding unmodified viral RNA replicon and one or more expression cassettes, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, wherein at least one of the one or more expression cassettes comprises a promoter operably linked to a coding sequence for the polypeptide of interest, and further wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19.
A method for producing a polypeptide of interest, comprising culturing a host cell comprising a nucleic acid molecule which comprises a nucleic acid sequence encoding a modified non-alphavirus RNA replicon, wherein the modified non-alphavirus RNA replicon comprises a first nucleic acid sequence encoding one or more structural elements of an alphavirus capsid enhancer comprising one or more RNA stem-loops, a second nucleic acid sequence comprising a sequence encoding for at least one viral nonstructural proteins of a corresponding unmodified viral RNA replicon and one or more expression cassettes, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, wherein at least one of the one or more expression cassettes comprises a promoter operably linked to a coding sequence for the polypeptide of interest, and further wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19.
A method for producing a messenger RNA (mRNA) in a cell, comprising introducing into the cell a nucleic acid molecule comprising a modified viral RNA replicon, wherein the modified viral RNA replicon comprises a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer comprising one or more RNA stem-loops, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and a second nucleic acid sequence comprising a sequence encoding for at least one viral nonstructural protein of a corresponding unmodified viral RNA replicon, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, further wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19, and wherein the modified RNA replicon further comprises one or more expression cassettes, wherein at least one of the one or more expression cassettes comprises a promoter operably linked to a coding sequence for a gene of interest (GOI), thereby producing a mRNA of the GOI.
A method for conferring a resistance to the innate immune system in a subject, comprising administering to the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a modified non-alphavirus RNA replicon, wherein the modified non-alphavirus RNA replicon comprises a first nucleic acid sequence encoding one or more structural elements of an alphavirus capsid enhancer comprising one or more RNA stem-loops, and a second nucleic acid sequence comprising a sequence encoding for at least one viral nonstructural proteins of a corresponding unmodified viral RNA replicon, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19, and wherein expression of the modified non-alphavirus RNA replicon encoded by the nucleic acid molecule confers a resistance to innate immune response in the subject.
A method for conferring a resistance to the innate immune system in a subject, comprising administering to the subject a nucleic acid molecule comprising a nucleic acid sequence which encodes a modified viral RNA replicon, wherein the modified viral RNA replicon comprises:
- a first nucleic acid sequence encoding one or more structural elements of a viral capsid enhancer comprising one or more RNA stem-loops, wherein the viral capsid enhancer is heterologous to the viral RNA replicon, and a second nucleic acid sequence comprising a sequence encoding for at least one viral nonstructural protein of a corresponding unmodified viral RNA replicon, wherein the first nucleic acid sequence is operably linked upstream to the second nucleic acid sequence, and wherein expression of the modified replicon RNA encoded by the nucleic acid molecule confers a resistance to innate immune response in the subject, and further wherein the nucleic acid molecule comprises a 5′ UTR comprising a corresponding RNA for nucleotides 1-45 of SEQ ID NO:
- 19.
Dependencies are inferred from the claim text.