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RNA particles comprising polysarcosine

A technology of polysarcosine and particles, applied in the field of RNA particles, can solve problems such as high risk

Pending Publication Date: 2021-05-28
JOHANNES GUTENBERG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Here, the risk may be particularly high due to the potential inherent immunogenicity of RNA

Method used

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  • RNA particles comprising polysarcosine
  • RNA particles comprising polysarcosine
  • RNA particles comprising polysarcosine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0269] Example 1: Materials and methods

[0270] Material

[0271] Encodes luciferase or secreted The mRNA of luciferase (secNLuc) was provided by the RNABiochemistry unit (BioNTech RNA Pharmaceuticals, Mainz, Germany) (concentration of mRNA was 2 to 5 mg / mL in water or 10 mM Hepes; 0.1 mM EDTA; pH 7.0).

[0272] The ionizable cationic lipid DODMA (1,2-dioleyloxy-N,N-dimethyl-3-aminopropane) and the helper lipid DOPE (1,2-dioleoyl-sn-glycerol-3 - phosphoethanolamine) was purchased from Merck. The helper lipid DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) was obtained from Avanti Polar Lipids. Cholesterol was from Sigma Aldrich. Sodium dodecyl sulfate (SDS) was obtained from Sigma Adlrich.

[0273] Prior to preparation, lipids were dissolved in absolute ethanol (Carl Roth) to concentrations ranging from 5 to 100 mM, and ethanolic lipid solutions were stored at -20°C.

[0274] Ethanolic lipid solution, sterile citrate buffer 100 mM pH 5.4, and RNA were equilibrated at...

Embodiment 2

[0306] Example 2: Generation of RNA lipid nanoparticles comprising pSarc

[0307] Amino acid-based polypeptide lipid-polysarcosine conjugated lipids were used to prepare mRNA lipid nanoparticles.

[0308] By mixing the ethanol phase containing the lipids DODMA, cholesterol, DSPC and C14pSarc20 in a molar ratio of 40:50-X:10:X with 3 volumes of 0.15 mg / mL RNA in citrate buffer (100 mM pH 5.4) Mix to prepare LNP.

[0309] Table 1: Physicochemical characterization of RNA lipid nanoparticles prepared with different mole fractions (%) of C14pSarc20.

[0310]

[0311] figure 1 The relationship between particle size and mole fraction of polysarcosinated LNP is shown. Lipid nanoparticles were fabricated using lipid mixtures containing increased mole fractions of C14PSarc20. Under suitable conditions, colloidally stable particles can be obtained. Although no measurable sized particles formed at very low PSarc fractions (0.5 and 1%), at 2.5 mol% and above 2.5 mol%, particles wit...

Embodiment 3

[0312] Example 3: Particles of pSarc-lipids with different sarcosine polymerized unit lengths

[0313] By mixing one volume of the ethanol phase containing lipid DODMA, cholesterol, DSPC and C14pSarcX (X=11, 20, 34 or 65) with different polymer lengths in a molar ratio of 40:45:10:5 with 3 volumes of LNPs were prepared by mixing 0.15 mg / mL RNA in citrate buffer (100 mM pH 5.4).

[0314] Table 2: Physicochemical characterization of RNA lipid nanoparticles prepared with 5 mole fraction (%) of C14pSarc with different aggregate lengths.

[0315]

[0316] figure 2 Shown is the relationship between polysarcosine length (polymerization unit) of PSarc lipids used for LNP formation and in vitro protein expression of luciferase-encoding mRNA LNPs in different cell lines. LNP formulated with mRNA encoding luciferase was tested in lung tumor cells (TC-1 ) myocytes (C2C12), hepatocytes (Hep-G2) and macrophages (RAW 264.7). Twenty-four hours after transfection, the bioluminescent sig...

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Abstract

The present disclosure relates to RNA particles for delivery of RNA to target tissues after administration, in particular after parenteral administration such as intravenous, intramuscular, subcutaneous or intratumoral administration, and compositions comprising such RNA particles. The RNA particles in one embodiment comprise single-stranded RNA such as mRNA which encodes a peptide or protein of interest, such as a pharmaceutically active peptide or protein. The RNA is taken up by cells of a target tissue and the RNA is translated into the encoded peptide or protein, which may exhibit its physiological activity.

Description

technical field [0001] The present disclosure relates to RNA particles for the delivery of RNA to target tissues after administration, particularly after parenteral administration such as intravenous, intramuscular, subcutaneous or intratumoral administration, and compositions comprising such RNA particles. In one embodiment, the RNA particle comprises single-stranded RNA, eg, mRNA encoding a peptide or protein of interest (eg, a pharmaceutically active peptide or protein). The RNA is taken up by the cells of the target tissue, and the RNA is translated into an encoded peptide or protein that can exhibit its physiological activity. Background technique [0002] The use of RNA to deliver foreign genetic information into target cells offers an attractive alternative to DNA. Advantages of using RNA include transient expression and nontransformation characteristics. The RNA does not need to enter the nucleus for expression, and in addition the RNA does not integrate into the h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/34A61K48/00A61K31/195C07K1/06C08G69/10
CPCC08G69/10A61K47/6911A61K47/6929A61K47/62A61K47/543C12N15/88A61K47/645A61K47/6935A61K47/6939A61K48/0025A61K48/0041C12N15/85A61K9/5146A61K47/18A61K47/24A61K47/28
Inventor 马蒂亚斯·巴尔茨本亚明·韦伯海因里希·哈斯菲利普·赫勒萨拉·诺盖拉安妮·施莱格尔
Owner JOHANNES GUTENBERG UNIV