Attenuated virus mutated at sites of evolutionarily conserved RNA structure
Inventors
Tuller, Tamir • Goz, Eli • ATAR, Shimshi • Zur, Hadas
Assignees
Synvaccine Ltd • Ramot at Tel Aviv University Ltd • Yeda Research and Development Co Ltd
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Publication Number
US-11795440-B2
Publication Date
2023-10-24
Expiration Date
Abstract
Attenuated viruses and methods of designing them are disclosed. In one embodiment, there is disclosed an attenuated form of a virulent virus comprising an RNA encoding a viral protein or a nucleic acid sequence transcribable to said RNA, wherein the folding energy or structure of the RNA is changed at positions of evolutionarily conserved RNA structures with respect to that of said RNA encoding said viral protein in the virulent virus so as to bring about attenuation of the virus.
Core Innovation
The invention relates to making an attenuated form of a virulent virus genome by synonymously substituting at least one nucleotide in a region of evolutionarily conserved local RNA folding energy. The substitutions are made on an RNA sequence encoding a viral protein or on a nucleic acid sequence transcribable to that RNA, with the encoded viral protein amino-acid sequence preserved as identical to the virulent virus protein.
The local RNA folding energy of the conserved region is used to guide attenuation through a predetermined threshold derived from the average local folding energy of a randomized sequence of the virulent virus. When the region comprises folding energy below the predetermined threshold, the substitution increases folding energy; when the region comprises folding energy above the predetermined threshold, the substitution decreases folding energy.
The method is further constrained so that the at least one substitution decreases replicative fitness of the attenuated form of a virus compared to the virulent virus. Thus, attenuation is achieved by using synonymous nucleotide substitutions that shift evolutionarily conserved local RNA folding energy in the specified direction while maintaining an identical viral protein amino-acid sequence.
Claims Coverage
The document excerpt provides two independent claims (clm-00001 and clm-00020). Each independent claim uses synonymous substitutions in evolutionarily conserved local RNA folding energy regions with a threshold derived from randomized sequences, while requiring an identical encoded viral protein amino-acid sequence and decreased replicative fitness versus the virulent virus.
Synonymous substitutions guided by evolutionarily conserved local RNA folding energy threshold
Receiving a sequence of an RNA of the virulent virus encoding a viral protein or a nucleic acid sequence transcribable to the RNA, and synonymously substituting at least one nucleotide in a region of evolutionarily conserved local RNA folding energy to another nucleotide, where the substitution increases folding energy when the region is below a predetermined threshold and decreases folding energy when the region is above the predetermined threshold.
Threshold derived from randomized sequence with identical amino-acid sequence
Defining the predetermined threshold as derived from the average local folding energy of a randomized sequence of the virulent virus, where the randomized sequence encodes an amino acid sequence identical to the corresponding non-randomized amino acids sequence of the virulent virus.
Preserved viral protein amino-acid sequence and decreased replicative fitness
Ensuring that the viral protein encoded by the synonymously substituted RNA comprises an amino acid sequence identical to the amino acid sequence of the viral protein of the virulent virus, and requiring that the at least one substitution decreases replicative fitness of the attenuated form of a virus compared to the virulent virus.
RNA or DNA genome mutagenesis with synonymous folding-energy substitutions
Mutating the RNA genome of the virulent virus to synonymously substitute at least one nucleotide in a region of evolutionarily conserved local RNA folding energy, or mutating the DNA genome to synonymously substitute at least one nucleotide in a region encoding an evolutionarily conserved local RNA folding energy region, using the specified increase/decrease relationships relative to a predetermined threshold.
Randomized threshold with identical amino-acid sequence for DNA- or RNA-based method
Using a predetermined threshold derived from the average local folding energy of a randomized sequence of the virulent virus, where the randomized sequence encodes an amino acid sequence identical to the corresponding non-randomized amino acids sequence of the virulent virus.
Mutated genome encodes identical viral protein sequence with reduced replicative fitness
Requiring that a viral protein encoded by the mutated genome comprises an amino acid sequence identical to the amino acid sequence of the viral protein encoded by the genome of the virulent virus, and that the at least one substitution decreases replicative fitness of the attenuated form of a virus compared to the virulent virus.
Across clm-00001 and clm-00020, the inventive concept is attenuation via synonymous substitutions that shift evolutionarily conserved local RNA folding energy relative to a randomized-sequence-derived threshold, while preserving the encoded viral protein amino-acid sequence and reducing replicative fitness versus the corresponding virulent virus.
Stated Advantages
Reduces replicative fitness of an attenuated form of a virus compared to the virulent virus.
Documented Applications
A vaccine comprising an attenuated virus.
Immunizing and protective immune response methods using an attenuated virus.
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