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US 11,761,009 B2Granted
Artificial nucleic acid molecules for improved protein expression
AssigneeCureVac AG
InventorsAndreas Thess, Thomas Schlake, Stefanie Grund
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Abstract
The invention relates to an artificial nucleic acid molecule or a vector comprising an open reading frame and a 3′-UTR comprising at least two poly(A) sequences. The invention also relates to a method for increasing protein production from an artificial nucleic acid molecule and to the use of a 3′-UTR for a method for increasing protein production from an artificial nucleic acid molecule. Moreover, the invention concerns the use of the artificial nucleic acid molecule, the vector, the kit or the pharmaceutical composition as a medicament, as a vaccine or in gene therapy.
Claims60
An artificial RNA molecule comprising:
- a) a 5′-cap structure;
- b) at least one open reading frame (ORF) encoding a protein;
- and
- c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
- (i) the first poly(A) sequence comprises at least 20 adenine nucleotides;
- and
- (ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,
- wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
- wherein the RNA molecule comprises at least one nucleotide analogue, which is a modified form of uridine.
6 more elements
An artificial RNA molecule comprising:
- a) a 5′-cap structure;
- b) at least one open reading frame (ORF) encoding an antigen;
- and
- c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
- (i) the first poly(A) sequence comprises at least 20 adenine nucleotides;
- and
- (ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,
- wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
- wherein the ORF encoding the antigen has a G/C content that is increased by at least 7% relative to a corresponding reference ORF encoding the antigen.
6 more elements
A pharmaceutical composition comprising an artificial RNA molecule and one or more pharmaceutically acceptable vehicles, said artificial RNA comprising:
- a) a 5′-cap structure;
- b) at least one open reading frame (ORF) encoding an antigen;
- and
- c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
- (i) the first poly(A) sequence comprises at least 20 adenine nucleotides;
- and
- (ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,
- wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
- wherein the RNA molecule is complexed with a cationic carrier or a polycationic carrier,
- wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation, and
- wherein when the RNA molecule is expressed in an organism, the RNA molecule yields increased expression of the antigen encoded by the open reading frame in comparison to a reference RNA comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
8 more elements
A pharmaceutical composition comprising an artificial RNA molecule and one or more pharmaceutically acceptable vehicles, said artificial RNA comprising:
- a) a 5′-cap structure;
- b) at least one open reading frame (ORF) encoding a viral antigen;
- and
- c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
- (i) the first poly(A) sequence comprises at least 20 adenine nucleotides;
- and
- (ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,
- wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
- wherein the RNA molecule is complexed with a cationic lipid carrier or a polycationic lipid carrier,
- wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation, and
- wherein, when administered to an organism by intramuscular injection, the composition yields an increased immune response to the antigen in comparison to an intramuscular injection of a reference composition comprising a reference RNA molecule having an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
8 more elements
A pharmaceutical composition comprising an artificial RNA molecule and one or more pharmaceutically acceptable vehicles, said artificial RNA comprising:
- a) a 5′-cap structure;
- b) at least one open reading frame (ORF) encoding a viral antigen;
- and
- c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
- (i) the first poly(A) sequence comprises at least 20 adenine nucleotides;
- and
- (ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,
- wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
- wherein the RNA molecule is complexed with a cationic lipid carrier or a polycationic lipid carrier,
- wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation, and
- wherein, when administered to an organism by intramuscular injection, the composition yields an increased neutralizing antibody response to the antigen in comparison to an intramuscular injection of the reference composition comprising the reference RNA having an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
8 more elements
A pharmaceutical composition comprising an artificial RNA molecule and one or more pharmaceutically acceptable vehicles, said artificial RNA comprising:
- a) a 5′-cap structure;
- b) at least one open reading frame (ORF) encoding an antigen;
- and
- c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
- (i) the first poly(A) sequence comprises at least 20 adenine nucleotides;
- and
- (ii) the second poly(A) sequence comprises at least 60 adenine nucleotides,
- wherein the first and the second poly(A) sequences are separated by a linker nucleic acid sequence comprising at least 3 consecutive nucleotides that are not adenine nucleotides,
- wherein when the RNA molecule is administered intramuscularly to an organism, the RNA molecule yields increased expression of the antigen encoded by the at least one open reading frame in comparison to the expression from a reference nucleic acid molecule comprising an identical nucleic acid sequence as the artificial RNA molecule but lacking the linker nucleic acid sequence, and
- wherein the ORF encoding the antigen has a G/C content that is increased by at least 15% relative to a corresponding reference ORF encoding the antigen.
7 more elements
Dependencies are inferred from the claim text.