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RNA encoding protein

A technology of protein coding, applied in the field of mRNA

Pending Publication Date: 2021-09-28
维萨梅布有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite all the progress made in the field, the efficient expression, especially efficient secretion (recombinant expression) of encoded proteins in cell-free systems, cells or organisms remains a challenging problem

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0475] Embodiment 1 methods and materials

[0476] Cloning of IGF1 and replacement of signal peptide

[0477] IGF1 is a 70 amino acid polypeptide synthesized in the endoplasmic reticulum and secreted through the Golgi apparatus, which acts as an extracellular growth factor in an autocrine and paracrine manner. To ensure correct mRNA-induced IGF1 expression and secretion from transfected cells, the mRNA sequence included the native N-terminal pre-pro-IGF1 sequence (pre-pro-IGF1 ) of human IGF1. This sequence consists of a sequence (nucleotides 1-63) encoding a human IGF1 prodomain (signal peptide) of 21 amino acids and a sequence encoding a human prodomain of 27 amino acids (nucleotides 64-144). In addition, this construct contained a sequence (nucleotides 145-354) encoding the entire coding sequence of mature human IGF1 having 70 amino acids. In Cpd.2-7, the predomain (signal peptide, nucleotides 1-63) was replaced by each predomain of IGF2, ALB, BDNF, CXCL12 or a synthetic ...

Embodiment 2

[0561]To test the efficacy of topically applied IGF-I mRNA in a mouse model of skeletal muscle injury, tibialis anterior (TA) muscles of 8-10 week-old male C57BL6 / J mice were treated with notexin (notexin) on day 0. ) induced myotoxic injury. Vehicle or 1 μg mRNA (Cpd.4) was applied to the injured muscle by intramuscular injection on day 1 post-injury and repeated on day 4 post-injury. TA muscle function was measured on days 1, 4, 7, 10, 14, 21, and 28 after injury. A subset of the contralateral TA muscle was also assessed throughout the study to assess healthy control levels of TA muscle function.

[0562] Methods and materials

[0563] Cloning of IGF-1 and in vitro transcription of IGF-1 mRNA

[0564] Cloning of IGF-1 and in vitro transcription of IGF-1 mRNA were performed as described in Example 1. Using codon-optimized Cpd.4DNA ( Figure 4 ), cloned in the pMA-T vector to provide as Figure 11 Constructs indicated. This construct was used to generate in vitro transc...

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Abstract

The present invention relates to a mRNA comprising a nucleic acid sequence encoding a protein and a signal peptide and a transcription unit, an expression vector or a gene therapy vector comprising a nucleic acid encoding the protein and a signal peptide. Also disclosed herein is a therapeutic composition comprising the mRNA, transcription unit, expression vector or gene therapy vector and use of the therapeutic composition in treating a disease or a condition.

Description

technical field [0001] The present invention relates to the mRNA that comprises the nucleic acid sequence of coding protein and signal peptide, wherein the amino acid 1-9 of the N-terminus of the aminoacid sequence of signal peptide has the average hydrophobicity score greater than 2, and wherein signal peptide is selected from: [0002] i) a signal peptide heterologous to said protein, wherein the signal peptide heterologous to said protein is optionally modified by insertion, deletion and / or substitution of at least one amino acid, provided that said protein is not an oxidoreductase; [0003] ii) a signal peptide homologous to said protein, wherein the signal peptide homologous to said protein is modified by insertion, deletion and / or substitution of at least one amino acid; and [0004] iii) A naturally occurring amino acid sequence which essentially does not have a signal peptide function, wherein said naturally occurring amino acid sequence is optionally modified by inser...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62
CPCC12N15/625A61K48/005A61K48/00A61P21/00C07K14/475C07K2319/02
Inventor J·A·塞尔瓦拉吉H·沙夫豪瑟F·梅茨格
Owner 维萨梅布有限公司
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