RNA containing modified nucleosides and methods of use thereof
AssigneeThe Trustees of the University of Pennsylvania
InventorsKatalin Kariko, Drew Weissman
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Abstract
This invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside, gene therapy vectors comprising same, methods of synthesizing same, and methods for gene replacement, gene therapy, gene transcription silencing, and the delivery of therapeutic proteins to tissue in vivo, comprising the molecules. The present invention also provides methods of reducing the immunogenicity of RNA, oligoribonucleotide, and polyribonucleotide molecules.
Claims27
A composition comprising an in vitro-synthesized modified RNA comprising an open reading frame that encodes a protein of interest for translation in a mammalian cell, wherein said in vitro-synthesized modified RNA comprises a modified nucleoside selected from the group consisting of pseudouridine (Ψ), 1-methylpseudouridine (m1Ψ), 5-methyluridine (m5U), 2-thiouridine (s2U), 2′-O-methyluridine (Um), and 5-methoxyuridine (mo5U) in place of uridine, and/or N6-methyladenosine (m6A) in place of adenosine.
A method for inducing a mammalian cell to produce a protein of interest comprising:
- contacting said mammalian cell with in vitro-synthesized modified RNA encoding a protein of interest, wherein said in vitro-synthesized modified RNA comprises at least one modified nucleoside selected from the group consisting of pseudouridine (Ψ), 1-methylpseudouridine (m1Ψ), 5-methyluridine (m3U), 2-thiouridine (s2U), 2′-O-methyluridine (Um), and 5-methoxyuridine (mo5U) in place of uridine, and N6-methyladenosine (m6A) in place of adenosine;
- wherein said significantly lower immunogenicity of said in vitro-synthesized modified RNA compared to its in vitro-synthesized unmodified counterpart mRNA is detectable by at least the following method, wherein the method comprises the steps of:
- a) transfecting a murine or human dendritic cell with the in vitro-synthesized modified mRNA comprising Ψ, m1Ψ, m5U, s2U, Um or mo5U in place of uridine, or m6A in place of adenosine and, separately, contacting a murine or human dendritic cell with the in vitro-synthesized unmodified counterpart mRNA not comprising Ψ, m1Ψ, m5U, s2U, Um, or mo5U in place of uridine, or m6A in place of adenosine;
- and
- b) measuring the quantity of IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1ß, IL-6, IFN-ß or IL-8 secreted in response to contacting the murine or human dendritic cell with the in vitro-synthesized modified mRNA comprising Ψ, m1Ψ, m5U, s2U, Um or mo5U in place of uridine, or m6A in place of adenosine, and, separately, measuring the quantity of IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1ß, IL-6, IFN-ß or IL-8 secreted in response to contacting the murine or human dendritic cell with the in vitro-synthesized unmodified counterpart mRNA not comprising Ψ, m1Ψ, m5U, s2U, Um or mo5U or m6A in place of adenosine,
- wherein the quantity of IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1ß, IL-6, IFN-ß or IL-8 secreted from the murine or human dendritic cell in response to said contacting with the
- in vitro-synthesized modified mRNA comprising Ψ, m1Ψ, m5U, s2U, Um or mo5U in place of uridine, or m6A in place of adenosine is less than the respective quantity of IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1ß, IL-6, IFN-ß or IL-8 secreted from the murine or human dendritic cell in response to said contacting with the in vitro-synthesized unmodified counterpart mRNA not comprising Ψ, m1Ψ, m5U, s2U, Um or mo5U in place of uridine, or m6A in place of adenosine.
Dependencies are inferred from the claim text.