Construct system and uses therefor

Abstract

The present invention discloses construct systems and methods for comparing different iso-accepting codons according to their preference for translating RNA transcripts into proteins in cell or tissues of interest or for producing a selected phenotype in an organism of interest or part thereof. The codon preference comparisons thus obtained are particularly useful for modifying the translational efficiency of protein-encoding polynucleotides in cells or tissues of interest or for modulating the quality of a selected phenotype conferred by a phenotype-associated polypeptide upon an organism of interest or part thereof.

Description

FIELD OF THE INVENTION[0001]The present invention relates generally to gene expression. More particularly, the present invention relates to construct systems and methods for comparing different iso-accepting codons according to their preference for translating RNA transcripts into proteins in cell or tissues of interest or for producing a selected phenotype in an organism of interest or part thereof. The codon preference comparisons thus obtained are particularly useful for modifying the translational efficiency of protein-encoding polynucleotides in cells or tissues of interest or for modulating the quality of a selected phenotype conferred by a phenotype-associated polypeptide upon an organism of interest or part thereof.BACKGROUND OF THE INVENTION[0002]The expression of foreign heterologous genes in transformed cells is now commonplace. A large number of mammalian genes, including, for example, murine and human genes, have been successfully expressed in various host cells, includ...

AI Technical Summary

Benefits of technology

[0009]The present invention is predicated in part on the discovery that the sensitivity of determining the translational efficiency or phenotypic preference of different synonymous codons can be improved using a construct system that employs different reporter polynucleotides that encode the same amino acid sequence, wherein individual reporter polynucleotides use the same codon (also referred to herein as “an interrogating codon”) to code for a particular amino acid at one or more positions of the amino acid sequence, and wherein the interrogating codon of one reporter polynucleotide is different to but synonymous with the interrogating codon of another reporter polynucleotide. In specific embodiments, the sensitivity is improved further by incorporating two or more interrogating codons to code for the particular amino acid in the amino acid sequence.

[0015]In some embodiments, the reporter polynucleotide of individual synthetic constructs further comprises an ancillary coding sequence that encodes a detectable tag, which is suitably a member of a specific binding pair, which includes for example, antibody-antigen (or hapten) pairs, ligand-receptor pairs, enzyme-substrate pairs, biotin-avidin pairs, and the like. In illustrative examples of this type, the ancillary coding sequence of one reporter polynucleotide encodes a different tag than the ancillary coding sequence of another reporter polynucleotide. In these examples, it is possible to detectably distinguish the polypeptide products of different reporter polynucleotides in the same cell or organism of interest or part thereof, thereby permitting simultaneous determination of the translational efficiencies of different interrogating codons in the same cell or organism or part.

[0088]In some embodiments, the chimeric construct is in the form of a pharmaceutical composition that optionally comprises a pharmaceutically acceptable excipient and / or carrier. Accordingly, in another aspect, the invention provides pharmaceutical compositions that are useful for modulating an immune response to a target antigen in a mammal, which response is conferred by the expression of a parent polynucleotide that encodes a polypeptide corresponding to at least a portion of the target antigen. These compositions generally comprise a chimeric construct and a pharmaceutically acceptable excipient and / or carrier, wherein the chimeric construct comprises a synthetic polynucleotide that is operably connected to a regulatory sequence and that is distinguished from the parent polynucleotide by the replacement of a first codon in the parent polynucleotide with a synonymous codon that has a different immune response preference than the first codon and wherein the first and synonymous codons are selected according to any one of TABLES 2, 3, 5 and 6. In some embodiments, the compositions further comprise an adjuvant that enhances the effectiveness of the immune response. In some embodiments, the composition is formulated for transcutaneous or dermal administration, e.g., by biolistic or microneedle delivery or by intradermal injection. Suitably, in embodiments in which a stronger or enhanced immune response to the target antigen is desired, the first and synonymous codons are selected according to TABLES 2 or 3. Conversely, in embodiments in which a weaker or reduced immune response to the target antigen is desired, the first and synonymous codons are selected according to TABLES 5 or 6.

[0090]In a related aspect, the invention encompasses methods of enhancing the quality of an immune response to a target antigen in a mammal, which response is conferred by the expression of a parent polynucleotide that encodes a polypeptide corresponding to at least a portion of the target antigen. These methods generally comprise: introducing into the mammal a chimeric construct comprising a synthetic polynucleotide that is operably connected to a regulatory sequence and that is distinguished from the parent polynucleotide by the replacement of a first codon in the parent polynucleotide with a synonymous codon that has a higher immune response preference than the first codon, wherein the first and synonymous codons are selected according to TABLES 2 or 3. In these methods, expression of the synthetic polynucleotide typically results in a stronger or enhanced immune response than the one obtained through expression of the parent polynucleotide under the same conditions.

[0091]In another related aspect, the invention extends to methods of reducing the quality of an immune response to a target antigen in a mammal, which response is conferred by the expression of a parent polynucleotide that encodes a polypeptide corresponding to at least a portion of the target antigen. These methods generally comprise: introducing into the mammal a chimeric construct comprising a synthetic polynucleotide that is operably connected to a regulatory sequence and that is distinguished from the parent polynucleotide by the replacement of a first codon in the parent polynucleotide with a synonymous codon that has a lower immune response preference than the first codon, wherein the first and synonymous codons are selected according to TABLES 5 or 6. In these methods, expression of the synthetic polynucleotide typically results in a weaker or reduced immune response than the one obtained through expression of the parent polynucleotide under the same conditions.

[0092]Yet a further aspect of the present invention embraces methods of enhancing the quality of an immune response to a target antigen in a mammal, which response is conferred by the expression of a first polynucleotide that encodes a polypeptide corresponding to at least a portion of the target antigen. These methods generally comprise: co-introducing into the mammal a first nucleic acid construct comprising the first polynucleotide in operable connection with a regulatory sequence; and a second nucleic acid construct comprising a second polynucleotide that is operably connected to a regulatory sequence and that encodes an iso-tRNA corresponding to a codon of the first polynucleotide, wherein the codon has a low or intermediate immune response preference and is selected from the group consisting of AlaGCA, AlaGCG, AlaGCC, ArgAGG, ArgCGG, AsnAAT, AspGAT, CysTGT, GluGAG, GlyGGG, GlyGGT, GlyGGC, IleATA, IleATT, LeuTTG, LeuTTA, LeuCTA, LeuCTT, PheTTC, ProCCA, ProCCG, ProCCT, SerAGC, SerAGT, SerTCT, SerTCA, SerTCC, ThrACA, ThrACT, TyrTAT, ValGTA and ValGTT. In specific embodiments, the codon has a ‘low’ immune response preference, and is selected from the group consisting of AlaGCA, AlaGCG, ArgCGG, AsnAAT, AspGAT, CysTGT, GluGAG, GlyGGG, GlyGGT, GlyGGC, IleATA, LeuTTG, LeuTTA, PheTTC, ProCCA, ProCCG, SerAGC, SerAGT, ThrACT, TyrTAT and ValGTA.

Problems solved by technology

Nevertheless, despite the burgeoning knowledge of expression systems and recombinant DNA technology, significant obstacles remain when one attempts to express a foreign or synthetic gene in a selected host cell.

This lower than expected translation efficiency is often due to the protein coding regions of the gene having a codon usage pattern that does not resemble those of highly expressed genes in the host cell.

It is widely known in this regard that translation of “rare codons”, for which the corresponding iso-tRNA is in low abundance relative to other iso-tRNAs, may cause a ribosome to pause during translation which can lead to a failure to complete a nascent polypeptide chain and an uncoupling of transcription and translation.

Thus, the expression of an exogenous gene may be impeded severely if a particular host cell of an organism or the organism itself has a low abundance of iso-tRNAs corresponding to one or more codons of the exogenous gene.

In other words, it was discovered that different cells of an organism can exhibit different translational efficiencies for the same codon and that it was not possible to predict which codons would be preferred, less preferred or non preferred in a selected cell type.

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