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PHARMACEUTICAL COMPOSITION CONTAINING A STABILISED mRNA OPTIMISED FOR TRANSLATION IN ITS CODING REGIONS

a technology of coding region and pharmaceutical composition, which is applied in the direction of dna/rna fragmentation, genetic material administration regime, virus, etc., can solve the problems of affecting the function of endogenous genes, affecting the efficacy of therapeutic agents, and bringing with it significant risk of gene therapy, so as to overcome the disadvantages and increase the effectiveness of therapeutic agents

Inactive Publication Date: 2010-09-23
CUREVAC AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The modified mRNA system improves the stability and translation efficiency of RNA, reducing integration risks and immune responses, while providing a safer and more effective RNA-based therapeutic and vaccine solution.

Problems solved by technology

Despite this precautionary measure, however the risk of uncontrolled propagation of the introduced therapeutic gene as well as viral genes remains due to the possibility of recombination events.
On the other hand the integration into the genome brings with it a significant risk for gene therapy.
Such integration events may, for example, involve an insertion of the incorporated DNA into an intact gene, which produces a mutation that interferes with or completely ablates the function of the endogenous gene.
Alternatively, there is also the risk of inducing transformation of the transfected cell if the integration site occurs in a gene that is critical for regulating cell growth.
The integration of such promoters into the genome of the treated cell may, however, lead to undesirable changes in the regulation of the gene expression in the cell.
A further disadvantage of the use of DNA as a therapeutic agent or vaccine is the induction of pathogenic anti-DNA antibodies in the patient, resulting in a potentially fatal immune response.
Indeed, anti-RNA antibodies have not been detected to date.
However, some basic problems still have to be solved before medical applications based on RNA expression systems can be widely employed.
One of the problems in the use of RNA is the reliable, cell-specific and tissue-specific efficient transfer of the nucleic acid.
Since RNA is normally found to be very unstable in solution, up to now RNA could not be used or used only very inefficiently as a therapeutic agent or inoculating agent in the conventional applications designed for DNA use.

Method used

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  • PHARMACEUTICAL COMPOSITION CONTAINING A STABILISED mRNA OPTIMISED FOR TRANSLATION IN ITS CODING REGIONS
  • PHARMACEUTICAL COMPOSITION CONTAINING A STABILISED mRNA OPTIMISED FOR TRANSLATION IN ITS CODING REGIONS
  • PHARMACEUTICAL COMPOSITION CONTAINING A STABILISED mRNA OPTIMISED FOR TRANSLATION IN ITS CODING REGIONS

Examples

Experimental program
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example 1

[0091]As an exemplary embodiment of the process for determining the sequence of a modified mRNA according to the invention, a computer program was established that modifies the nucleotide sequence of an arbitrary mRNA in such a way as to maximise the G / C content of the nucleic acid, and maximise the presence of codons recognized by abundant tRNAs present in a particular cell(s). The computer program is based on an understanding of the genetic code and exploits the degenerative nature of the genetic code. By this means a modified mRNA having desirable properties is obtained, wherein the amino acid sequence encoded by the modified mRNA is identical to that of the unmodified mRNA sequence. Alternatively, the invention may encompass alterations in either the G / C content or codon usage of an mRNA to produce a modified mRNA.

[0092]The source code in Visual Basic 6.0 (program development environment employed: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is given in the Appendi...

example 2

[0093]An RNA construct with a sequence of the lac-Z gene from E. coli optimised with regard to stabilisation and translational efficiency was produced with the aid of the computer program of Example 1. A G / C content of 69% (compared to the wild type sequence of 51%; C. F. Kalnins et al., EMBO J. 1983, 2(4): 593-597) was achieved in this manner. Through the synthesis of overlapping oligonucleotides that comprise the modified sequence, the optimised sequence was produced according to methods known in the art. The terminal oligonucleotides have the following restriction cleavage sites: at the 5′ end an EcoRV cleavage site, and at the 3′ end a BglII cleavage site. The modified lacZ sequence was incorporated into the plasmid pT7Ts (GenBank Accession No. U26404; C. F. Lai et al., see above) by digestion with EcoRV / BglII. pT7Ts contains untranslated region sequences from the β-globin gene of Xenopus laevis at the 5′ and 3′ ends. The plasmid was cleaved with the aforementioned restriction e...

example 3

[0095]The gene for the influenza matrix protein (wild type sequence, see FIG. 1A; derived amino acid sequence, see FIG. 1B) was optimised with the aid of the computer program according to the invention of Example 1. The G / C-rich sequence variant shown in FIG. 1C (SEQ ID NO: 3) was thereby formed. A G / C-rich sequence coding for a secreted form of the influenza matrix protein, which includes an N-terminal signal sequence was also determined (see FIG. 1D; SEQ ID NO: 4). The secreted form of the influenza matrix protein has the advantage of increased immunogenicity as compared to that of the non-secreted form.

[0096]Corresponding mRNA molecules were designed starting from the optimised sequences. The mRNA for the influenza matrix protein, optimised with regard to G / C content and codon usage, was additionally provided with stabilising sequences in the 5′ region and 3′ region (the stabilisation sequences derive from the 5′-UTRs and 3′-UTRs of the β-globin-mRNA of Xenopus laevis; pT7Ts-Vekt...

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Abstract

The present invention relates to a pharmaceutical composition containing an mRNA that is stabilised by sequence modifications in the translated region and is optimised for the translation. The pharmaceutical composition according to the invention is particularly suitable as an inoculating agent as well as a therapeutic agent for tissue regeneration. In addition a process is described for determining sequence modifications that serve for the stabilisation and translation optimisation of mRNA.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This Application is a Continuation of application Ser. No. 10 / 729,830 filed on Dec. 5, 2003, which is a continuation-in-part of PCT Application No. PCT / EP02 / 06180 filed Jun. 5, 2002, which in turn, claims priority from German Application 10127283.9, filed Jun. 5, 2001. Applicants claim benefit under 35 U.S.C. §120 as to the U.S. application and the PCT application and under 35 U.S.C. §119 to the German application, and the disclosures of all of said applications are incorporated herein by reference.SUBMISSION OF SEQUENCE LISTING[0002]The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_Listing—22122—00009_CON2. The size of the text file is 19 KB, and the text file was created on May 19, 2010.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[00...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/21C07H21/02A61K31/7105A61K39/12A61K38/19A61K39/29A61K39/245A61K39/145A61P31/18A61P31/12A61P37/04A61P31/14A61P31/16A61P31/20A61P31/22A61K38/00A61K39/00A61K48/00A61P35/04C07K14/11C12N15/67G16B20/50G16B30/00
CPCA61K38/19A61K39/00G06F19/22A61K39/0011A61K39/145A61K48/00A61K48/005A61K48/0066A61K2039/53C07K14/005C07K14/4748C12N15/67C12N2740/16022G06F19/18A61K2300/00A61K47/542A61K47/6455G16B20/00G16B30/00A61K38/1735A61K38/193A61K38/1816A61K38/28A61P11/00A61P25/28A61P31/00A61P31/04A61P31/12A61P31/14A61P31/16A61P31/18A61P31/20A61P31/22A61P35/00A61P35/04A61P37/04A61P43/00A61K39/001184A61K39/001194A61K39/00117A61K39/001188A61K39/001191A61K39/001106A61K39/001197A61K39/001156A61K39/001189A61K39/001192A61K39/001153A61K39/001186G16B20/50Y02A50/30A61K39/0258C07K14/245C12N7/00C12N15/11C12N2310/334C12N2310/336C12N2760/16034C12N2760/16071A61K48/0075A61K39/12A61K48/0083C12N2770/24122C12N2770/24134C12N2760/14122C12N2760/14134A61K39/21C12N2740/16034C12N2760/16022C07K14/4727
Inventor VON DER MULBE, FLORIANHOERR, INGMARPASCOLO, STEVE
Owner CUREVAC AG
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