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Pharmaceutical composition for preventing or treating muscular disease or cachexia comprising, as active ingredient, mirna located in dlk1 -dio3 cluster or variant thereof

A technology for muscle diseases and active ingredients, which can be used in drug combinations, muscle system diseases, neuromuscular system diseases, etc., and can solve controversial issues

Pending Publication Date: 2020-12-04
AVENTI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the findings of these gene expression studies are also controversial due to conflicts with those provided by another research group et al.

Method used

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  • Pharmaceutical composition for preventing or treating muscular disease or cachexia comprising, as active ingredient, mirna located in dlk1 -dio3 cluster or variant thereof
  • Pharmaceutical composition for preventing or treating muscular disease or cachexia comprising, as active ingredient, mirna located in dlk1 -dio3 cluster or variant thereof
  • Pharmaceutical composition for preventing or treating muscular disease or cachexia comprising, as active ingredient, mirna located in dlk1 -dio3 cluster or variant thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1. Sample Preparation

[0124] Human skeletal muscle (gluteus maximus muscle) obtained from a patient undergoing total hip arthroplasty (THRA) at Seoul National University Bundang Hospital (Seoul National University Bundang Hospital; SNUBH) was immediately transferred to liquid nitrogen and stored at -70 ℃. The Institutional Review Board of SNUBH (B-1710-050-009) approved this experiment. Written consent from the participant or legal guardian was obtained and a total of 20 patient samples (25, 27, 32, 33 years old (2 patient samples), 41, 46 years old (2 patient samples), 50 years old (2 patient samples) patient samples), 51, 55, 66, 67, 70, 71, 75, 79 years (2 patient samples), and 80 years) were used to assess miRNA or Atrogin-1 protein expression.

[0125] All 20 samples were used for miRNA expression assays. However, only 8 samples were available for immunoblotting due to the limited amount dissolved. RNA and protein isolation and purification from human...

Embodiment 2

[0126] Example 2. Animal model

[0127] Young C57BL / 6 mice (3 months old) and old C57BL / 6 mice (24 months old) were purchased from the Laboratory Animal Resource Center (located at the Korea Research Institute of Bioscience and Biotechnology ; KRIBB)). BALB / c (6-week-old) mice were purchased from Damul Science (Daejeon, Korea). All mice in this study were maintained on a standard experimental diet (3.1 kcal / g) using feed purchased from Damul Science (Daejeon, Korea). To overexpress miRNA mimics in muscle tissue, 50 μl (10 8 CFU) of adenovirus, AdmiRa-376c-3p or control (Applied Biological Materials Inc, Canada) were injected into the TA muscle or its contralateral muscle in young and old mice, respectively.

[0128] Injections were performed weekly using a 29G (0.33mm) insulin syringe. Four weeks after injection, muscle tissue was isolated from adenovirus-injected mice and used for analysis. To generate a mouse model of cachexia, BABL / c mice were subcutaneously injected w...

Embodiment 3

[0130] Example 3. Cell culture

[0131] Primary myoblasts were isolated from the hind leg muscles of the mice in Example 2. Utilize scissors to finely section the muscle tissue, and then place a mixture containing dispase II (2.4U / mL, Roche), collagenase D (1%, Roche) and 2.5 μM CaCl 2 dissociation buffer, followed by incubation at 37 °C for 20 min. The slurry was triturated using a serological pipette and passed through a 70 μm nylon mesh (BD Biosciences) to remove debris.

[0132] Cells were harvested and cultured in Ham's F-10 (Gibco) with 20% FBS containing amphotericin B-penicillin-streptomycin and 5 ng / mL bFGF. To remove fibroblasts, spread cells on uncoated plates for 1 hour and transfer fixed cells to collagen-coated dishes. Differentiation of major muscle fibers was induced by culturing cells in DMEM (Gibco) differentiation medium containing antibiotics and 5% horse serum. C2C12 cells (ATCC) were cultured in DMEM (Gibco) containing amphotericin B-penicillin-strept...

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Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a muscular disease or cachexia, comprising, as an active ingredient, a miRNA located in Dlk1-Dio3 cluster or avariant thereof. In the present invention, it has been found that expression of miRNAs located in the Dlk1-Dio3 cluster is decreased as age increases. In particular, in a case where most of the miRNAsare over-expressed in fully differentiated myotubes, it has been confirmed that the diameter of the myotubes increases. In addition, also in a tumor-induced cachexia mouse model, it has been confirmed that cachexia was improved by inhibiting Atrogin-1 protein. Accordingly, the miRNA located in the Dlk1-Dio3 cluster or a variant thereof can be usefully utilized for the treatment and prevention ofan Atrogin-1 - dependent muscular disease and cachexia.

Description

technical field [0001] The present invention relates to a pharmaceutical composition for preventing or treating muscle disease or cachexia, the pharmaceutical composition comprising miRNA or a variant thereof located in Dlk1-Dio3 cluster as an active ingredient. Background technique [0002] Skeletal muscle mass and function progressively decline with age and are a major cause of death and poor quality of life in older adults. Aging skeletal muscle not only shows a decline in muscle mass but also a progressive decline in strength and function. This disease is called "aging-induced sarcopenia" (Jun-Won Heo et al., Aging-induced Sarcopenia and Exercise. The Official Journal of the Korean Academy of Kinesiology, 19(2). DOI: http: / / doi. org / 10.15758 / jkak.2017.19.2.43). Muscle mass decreases by about 1% per year in your thirties. The prevalence of age-induced sarcopenia is about 10% in older adults in their 60s and increases to about 50% in their 80s. Loss of muscle with agin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6883
CPCC12N15/1137C12N2310/11C12N2310/14C12N2310/141C12N2320/30C12N15/113C12Q1/6883C12Q2600/158C12Q2600/178A61P21/00
Inventor 权基善李光杓申女晋李宝拉李承敏
Owner AVENTI INC
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