Methods for modulation of autophagy through the modulation of autophagy-enhancing gene products

A technology of gene products and genes, applied in the field of regulating autophagy by enhancing gene products to regulate autophagy, can solve the problem of few autophagy regulators

Inactive Publication Date: 2012-08-15
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the genes and pathways regulating autophagy in mammals are still poorly understood, there are still few autophagy regulators that have been proven to be targets for the development of new drugs and therapeutic methods for the treatment of the disease.

Method used

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  • Methods for modulation of autophagy through the modulation of autophagy-enhancing gene products
  • Methods for modulation of autophagy through the modulation of autophagy-enhancing gene products
  • Methods for modulation of autophagy through the modulation of autophagy-enhancing gene products

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preparation example Construction

[0214] Methods of preparation of such formulations or compositions may comprise the step of bringing into association an agent of the invention with the carrier and, optionally, one or more excipients.

[0215] Liquid formulation forms of the compounds of this invention for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid preparations may also contain inert diluents widely used in the art, for example, water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzene Methanol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, and castor oil), glycerin, THF, polyethylene glycol, and sorbitan Fatty acid esters, or mixtures thereof.

[0216] Besides diluents, the oral compositions can also include adjuvants such as wetting agents, emuls...

Embodiment 1

[0301] Example 1. High-throughput image-based siRNA screening of genes involved in autophagy regulation

[0302] Genes involved in the regulation of autophagy in mammals were identified using human neuroblastoma H4 cells stably expressing the LC3-GFP reporter. Under normal growth conditions, LC3-GFP in the cells is diffusely distributed in the cytosol. When autophagy is induced, LC3-GFP is recruited in the cytosol and can be observed to present spots corresponding to autophagosomes. To confirm this system, cells were transfected with siRNA against the essential autophagy mediator ATG5 or against mTOR, a suppressor gene of starvation-induced autophagy. After culturing for 72 h under normal nutritional conditions, the cells were transfected with ATG5 siRNA. This resulted in a marked downregulation of autophagy via a decrease in the number and intensity of LC3-GFP positive autophagosomes ( figure 1 A) and the decreased ratio of LC3II to LC3I in Western blot assay ( figure...

Embodiment 2

[0305] Example 2. Secondary high-throughput characterization of candidate genes

[0306] To elucidate the molecular pathways by which newly identified genes are involved in the regulation of autophagy, additional high-throughput assays were used to characterize these hit genes ( Figure 4 ). In one experiment, the function of mTORC1, an essential mediator of starvation-induced autophagy, was investigated. To identify candidate genes that regulate autophagy by altering mTORC1 activity, in situ immunoblotting assays were used to evaluate phosphorylation levels of ribosomal S6 protein (rpS6), a downstream target of mTORC1 signaling. To confirm this system, H4 cells were transfected with mTOR siRNA. Compared with the non-targeting siRNA control, the phosphorylation level of rpS6 was significantly decreased in mTOR siRNA-transfected cells ( Figure 5 ). Knockdown of only 14 (6%) of 219 validated genes induced autophagy by in situ immunoblotting assays in cells, and was strong...

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Abstract

The present disclosure relates to methods for the modulation of autophagy and the treatment of autophgy-related diseases, including cancer, neurodegenerative diseases and pancreatitis.

Description

[0001] related application [0002] This application claims priority to US Provisional Application 61 / 247,251, filed September 30, 2009, and US Provisional Application 61 / 247,309, filed September 30, 2009. The entire contents of which are hereby incorporated by reference. [0003] Government funding [0004] The present invention is accomplished under the support of the U.S. government, and the research grant numbers granted by the U.S. National Institutes of Health are AG012859 and AG027916. The government has certain rights in this invention. Background technique [0005] Autophagy is a catabolic process that mediates the renewal of intracellular components in a lysosome-dependent manner (Levine and Klionsky, (2004) Dev Cell 6, 463-377). Autophagy is initiated by the formation of an isolation membrane, which expands to engulf part of the cytoplasm to form double-layered vesicles called autophagosomes. Autophagosomes then fuse with lysosomes to form autolysosomes, where t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C07K16/18A61K31/00A61K31/713
CPCA61P1/18A61P5/00A61P7/00A61P7/06A61P13/12A61P21/00A61P21/04A61P25/00A61P25/14A61P25/16A61P25/20A61P25/28A61P25/32A61P35/00A61P43/00C12N15/113C12N2310/14A61K31/713C07K16/18C07K16/22C07K16/24C12N5/0618C12N2320/10C12N2320/30G01N33/502
Inventor 袁君英M·M·利平斯基
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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