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Compositions and processes for targeted delivery, expression and modulation of coding ribonucleic acids in tissue

A composition and delivery technology, applied in the field of messenger ribonucleic acid delivery

Pending Publication Date: 2020-05-29
UNITED THERAPEUTICS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although methods exist for introducing polynucleotides in vitro and ex vivo, they have the same limitations as discussed above

Method used

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  • Compositions and processes for targeted delivery, expression and modulation of coding ribonucleic acids in tissue
  • Compositions and processes for targeted delivery, expression and modulation of coding ribonucleic acids in tissue
  • Compositions and processes for targeted delivery, expression and modulation of coding ribonucleic acids in tissue

Examples

Experimental program
Comparison scheme
Effect test

example

[0181] general agreement

[0182] cell line

[0183] Human liver cancer (HCC) HepG2 ( HB-8065 TM ) and Hep3B ( HB-8064 TM ) cells were purchased from ATCC. Cells were cultured in Eagle's Minimal Essential Medium (EMEM) (Cellgro, USA), 10% FBS (HyClone, USA), streptomycin (100 μg / mL) and penicillin (100 U / mL -1 ) (Cellgro) at 37°C in 5% CO 2 Cultivated in a single-layer form in an atmosphere. HepG2 cells were treated at 5 μg / cm 2 The collagen concentration was grown on collagen (Gibco, USA) coated plates.

[0184] HMCPP5 (pooled cultured human hepatocytes; a mixture of cultured primary hepatocytes generated by pooling cells from 5 individual donors) was purchased from ThermoFisher Scientific, USA. The cells were inoculated in Williams' E medium ( Williams E Medium, WEM). 24 hours after inoculation, the WEM / Mix A medium was changed to supplemented with 0.1 μM dexamethasone and Mix B (penicillin / streptomycin, ITS (human recombinant insulin, human transferrin, selen...

example 1

[0221] Example 1: Tumor-specific gene expression regulated by miRNA-122

[0222] miRNA-122 is an abundant liver-specific miRNA whose expression is markedly reduced in human primary liver cancer (HCC) and HCC-derived cell lines such as Hep3B and HepG2. The purpose of this study was to demonstrate that by inserting the miRNA-122 target sequence (e.g., SEQ ID NO:2, as in image 3 modification of the 3' untranslated region (UTR) of the mRNA sequence described in variant 1) may allow translational repression and / or deadenylation followed by uncapping in normal hepatocytes but was not tested in Exogenous mRNA in HCC cell lines.

[0223] To examine endogenous miRNA-122 activity in healthy hepatocytes, using mCherry (red fluorescent protein) as the introduced gene of interest and following mCherry (red fluorescent protein) expression over time, mRNA-mCherry prepared according to the general protocol above or mRNA-mCherry-122 transfection of HMCPP5 cells (pooled cultured human hepato...

example 2

[0233] Example 2: Protein expression levels following tumor-specific gene expression

[0234] In another experiment, Western blot was used to determine the final protein expression level after transfection, as shown below.

[0235] Transfection and immunoblotting of cell lines - protein A

[0236] To assess tumor-specific expression levels of an exemplary 25kDa human protein (labeled 'Protein A'), hepatoma cells (HepG2 and Hep3B) and healthy hepatocytes (HMCPP5) were seeded into 12-well plates and treated with 0.5 μg / well Nanoformulated mRNA expressing human protein A, 25kDa (mRNA-A-DMP CTx ) or an mRNA expressing human protein A (a human protein of approximately 25 kDa) containing two miRNA122-binding sequences in the 3'UTR (SEQ ID NO:2), variant 1 (mRNA-A-miRNA122-DMP CTx ) transfection as described in Example 1 for mCherry transfection. Twenty-four hours after transfection, immunoblotting was performed after total protein extraction.

[0237] For immunoblotting, mediu...

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Abstract

A composition for expressing a polypeptide within a target organ, the composition comprising a delivery particle, and at least a first mRNA sequence complexed with, encapsulated by, or otherwise associated with the delivery particle. The mRNA sequence comprises a coding sequence which codes for the polypeptide, at least a first untranslated region (UTR) sequence, and at least one micro-RNA (miRNA)binding site sequence, wherein the miRNA binding site sequence is located within, immediately 5' to, or immediately 3' to, the first UTR sequence. The miRNA binding site sequence is selected so as toprovide for differential expression of the coding sequence between first and second cell types comprised within the target organ. The composition may be used in combination with or to supplement other therapeutic approaches, including chemotherapy, oncolytic viral therapy, and cellular therapies. Methods for making and using the composition are provided, particularly in treatment of disease, suchas cancer of the liver, brain, lung, breast and pancreas.

Description

[0001] related application [0002] This application claims the benefit of priority to UK Patent Application No. 1714430.4, filed 7 September 2017, and US Provisional Patent Application No. 62 / 632,056, filed 19 February 2018, which are hereby incorporated by reference in their entirety . technical field [0003] The present invention relates to messenger ribonucleic acid (mRNA) delivery technologies, generally nanoparticle-based delivery, and methods of making and using these mRNA delivery technologies in a variety of therapeutic, diagnostic, and prophylactic indications. Such delivery systems can be used as stand-alone interventions, or in combination with other therapeutic components. Background technique [0004] Gene therapy is the process of introducing an encoding polynucleotide into a patient's cells in order to treat a disease. For example, a mutated and / or non-functional gene can be replaced by an intact copy in the target cell. Gene therapy typically relies on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088C12N15/11A61K48/00
CPCA61K31/7088A61K35/763C12N15/86C12N2710/16632C12N2710/16643C12N15/88Y02A50/30A61K31/7105A61K31/713A61K45/06A61K48/00C12N15/63C12N2310/141C12N2330/10A61K2300/00C12N2800/107A61P35/00A61K48/0058
Inventor R·米科尔S·安托斯奇克
Owner UNITED THERAPEUTICS CORP
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