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Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids

a technology of lyophilization and mannose, which is applied in the direction of antibody medical ingredients, drug compositions, immunological disorders, etc., can solve the problems of reducing the transfection efficiency, reducing the biological activity reducing the expression of the encoded protein in vitro, so as to increase the transfection efficiency increase the in vivo expression of the encoded protein, and improve the stability of the nucleic acid

Inactive Publication Date: 2012-10-11
CUREVAC SE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019]The present invention is summarized as (the use of) a solution and uses thereof containing at least one nucleic acid (sequence) and free mannose for lyophilization, transfection and / or injection, particularly of RNA and mRNA. The inventive solution exhibits a positive effect on stabilization of the nucleic acid (sequence) during lyophilization and storage but also leads to a considerable increase of the transfection efficiency of a nucleic acid. It thus also increases in vivo expression of a protein encoded by such a nucleic acid upon increased transfection rate. The present invention is furthermore directed to a method of lyophilization using the mannose-containing solution, to pharmaceutical compositions, vaccines, kits, first and second medical uses applying such a mannose-containing solution and / or a nucleic acid (sequence) lyophilized or resuspended with such a solution.

Problems solved by technology

In contrast, reviewing the prior art a skilled person would have rather suggested that—considering its teaching—addition of mannose as lyoprotectant diminishes transfection efficiency and even more problematic, may lead to a decrease of biological activity of the nucleic acid, or, if a protein is encoded, to a decrease of the expression of the encoded protein in vitro or in vivo.
Free mannose was never considered as suitable.
Particularly Sun et al. only examined in vitro transfection assays with free sugar containing solutions which are very difficult to transfer to the in vivo situation due to the dilution effect.

Method used

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  • Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
  • Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
  • Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Plasmids

[0733]For the present examples DNA sequences encoding Photinus pyralis luciferase as well as DNA sequences encoding Ovalbumin were prepared and used for subsequent in vitro transcription reactions and expression studies.

[0734]According to a first preparation, the DNA sequence corresponding to pCV19-Ppluc(GC)-muag-A70-C30 was prepared, which encodes the Photinus pyralis luciferase coding sequence. The constructs were prepared by modifying the wild type Photinus pyralis luciferase encoding DNA sequence by introducing a GC-optimized sequence for a better codon usage and stabilization, stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)), a stretch of 70× adenosine at the 3′-terminal end (poly-A-tail) and a stretch of 30× cytosine at the 3′-terminal end (poly-C-tail), corresponding to SEQ ID NO: 1 (see FIG. 5). The sequence of the final DNA construct had a length of 1857 nucleotides. The corresponding mRNA sequence was termed...

example 2

In Vitro Transcription

[0741]The respective DNA plasmids prepared according to Example 1 were transcribed in vitro using T7-Polymerase (T7-Opti mRNA Kit, CureVac, Tibingen, Germany) following the manufactures instructions. Subsequently the mRNA was purified using PureMessenger® (CureVac, Tubingen, Germany).

example 3

Lyophylisation

[0742]The PureMessenger® purified and precipitated mRNA obtained according to Examples 1 and 2 coding for Photinus pyralis luciferase (Luc mRNA) (SEQ ID NO: 1) or Ovalbumin (SEQ ID NO: 2) were prepared for transfection and expression tests.

[0743]The PureMessenger® purified and precipitated mRNA obtained according to Examples 1 and 2 coding for Photinus pyralis luciferase (Luc mRNA) (SEQ ID NO: 1) or Ovalbumin (SEQ ID NO: 2) was dissolved in water for injection (WFI) to 5 g / l. Subsequently the mRNA was diluted with WFI (water for injection) or salt solution (see FIG. 2), with addition of glucose, trehalose, mannite or mannose. Aliquots of these solutions were lyophilized (Controls were frozen in liquid nitrogen or kept in solution). The locked cups were stored for the indicated time at 60° C. The resuspension was conducted with WFI.

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Abstract

The present invention is directed to (the use of) a solution containing at least one nucleic acid (sequence) and free mannose for lyophilization, transfection and / or injection, particularly of RNA and mRNA. The inventive solution exhibits a positive effect on stabilization of the nucleic acid (sequence) during lyophilization and storage but also leads to a considerable increase of the transfection efficiency of a nucleic acid. It thus also increases in vivo expression of a protein encoded by such a nucleic acid upon increased transfection rate. The present invention is furthermore directed to a method of lyophilization using the mannose-containing solution, to pharmaceutical compositions, vaccines, kits, first and second medical uses applying such a mannose-containing solution and / or a nucleic acid (sequence) lyophilized or resuspended with such a solution.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application represents the U.S. National Stage Application of International Application No. PCT / EP2010 / 006788, filed Nov. 8, 2010, which claims priority to International Application No. PCT / EP2009 / 008804, filed Dec. 9, 2009, all of which are incorporated herein by reference in their entirety for all purposes.FIELD OF THE INVENTION[0002]The present invention is directed to (the use of) a solution containing at least one nucleic acid (sequence) and free mannose for lyophilization, transfection and / or injection, particularly of RNA and mRNA. The inventive solution exhibits a positive effect on stabilization of the nucleic acid (sequence) during lyophilization and storage but also leads to a considerable increase of the transfection efficiency of a nucleic acid. It thus also increases in vivo expression of a protein encoded by such a nucleic acid upon increased transfection rate. The present invention is furthermore directed to a method ...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61K31/713A61P37/04C07H21/04C07H21/02C12N15/63A61K49/00
CPCC12N15/87A61K31/7088A61P37/04A61K39/0011A61K47/12A61K47/26A61K48/00A61K2039/53
Inventor MUTZKE, THORSTEN
Owner CUREVAC SE
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