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Reengineering mrna primary structure for enhanced protein production

A protein and protein expression technology, applied in DNA/RNA fragments, genetic engineering, recombinant DNA technology, etc., can solve problems such as inability to generate immune responses

Inactive Publication Date: 2012-04-25
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Poor protein expression can limit the large-scale application of some technologies, such as problems expressing enough antigen from DNA vaccines to generate an immune response for phase 3 clinical trials

Method used

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  • Reengineering mrna primary structure for enhanced protein production
  • Reengineering mrna primary structure for enhanced protein production
  • Reengineering mrna primary structure for enhanced protein production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Modification of multiple translation initiation sites within mRNA transcripts

[0076] The presence of multiple translation initiation sites within the 5′-UTR and coding regions of mRNA transcripts can reduce translation efficiency, for example by shifting ribosomes away from true or proven translation initiation codons. Alternatively or additionally, the presence of multiple translation initiation sites downstream of a true or proven translation initiation codon can induce translation of one or more protein isoforms that reduce the translation efficiency of the full-length protein. To increase translation efficiency of mRNA transcripts encoding commercially valuable human proteins, potential translation initiation sites in all reading frames upstream and downstream of true or proven translation initiation codons were mutated to eliminate these sites . In preferred aspects of this method, the mRNA sequence is altered, but the resulting amino acids encoded ...

Embodiment 2

[0151] Example 2: Modification of miRNA binding sites within mRNA transcripts

[0152] MicroRNAs (miRNAs) that bind to target mRNA transcripts reduce translation efficiency by inducing degradation of the target mRNA transcript or by preventing translation of the target mRNA transcript. To increase the translation efficiency of mRNA transcripts encoding commercially valuable human proteins, first identify the target mRNA within the 5' leader sequence, 5' untranslated region (UTR) sequence, coding sequence, and 3' untranslated region (UTR) sequence. All known or predicted miRNA binding sites are then mutated or altered in order to inhibit miRNA binding.

[0153] In a preferred aspect of the method, a seed sequence comprising the first 8 5'-nucleotides of the mature miRNA sequence is specifically targeted. The seed sequence comprises 5' nucleotides 1-7 or 2-8 of the mature miRNA sequence. Therefore, the seed sequences used in this method encompass both alternatives. The miRN...

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Abstract

Described herein are rules to modify natural mRNAs or to engineer synthetic mRNAs to increase their translation efficiencies. These rules describe modifications to mRNA coding and 3' UTR sequences intended to enhance protein synthesis by: 1) decreasing ribosomal diversion via AUG or non-canonical initiation codons in coding sequences, and / or 2) by evading miRNA-mediated down-regulation by eliminating one or more miRNA binding sites in coding sequences.

Description

[0001] Priority Document Citation [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61 / 155,049, filed February 24, 2009, entitled "Reengineering mRNA Primary Structure for Enhanced Protein Production." The subject matter of the above application is incorporated herein by reference in its entirety. Background technique [0003] Translation initiation in eukaryotes involves the recruitment of the 40S ribosomal subunit and other components of the translation machinery by the mRNA at the 5' cap structure or internal ribosome entry site (IRES). The 40S subunit moves to the start codon after being recruited. A widely accepted view of translation initiation postulates that the 40S subunit moves from the recruitment site to the initiation codon, which subunit scans past the 5' leader sequence in a 5' to 3' direction until it encounters the presence of the up to the first AUG codon in a good nucleotide background (Kozak "The Scannin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12P21/00
CPCC12N15/111C12N2320/50C12N2320/53C12N2310/141C12P21/02C12N15/67C12N15/63C12P21/00
Inventor V·P·莫罗S·A·查佩尔周伟G·M·伊德尔曼
Owner THE SCRIPPS RES INST
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