Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases

A technology of therapeutic composition, protein fragment, applied in the field of polynucleotide and its complement

Active Publication Date: 2013-05-08
ATYR PHARM INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Likewise, no systematic experimental analyzes have been performed to examine the use of such resectins as biotherapeutics, diagnostics, or drug targets in the treatment of various medical conditions, or their potential association with human disease

Method used

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  • Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases
  • Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases
  • Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of leucyl-tRNA synthetases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0731] Use protein topology and migration analysis platform to identify proteolytic fragments and alternative splicing products from AARS

[0732] In order to identify AARS fragments from cell lines, conditioned media, and tissues, samples were prepared as follows:

[0733] Mouse macrophages (RAW 264.7), cytosol and conditioned medium: 15x10 with serum-free DMEM medium 6 The density of cells / flask handles the cells. After 48 hours, the conditioned medium and cell pellet were collected and processed. 200 μg of protein was separated from the secretory and cytosolic proteome fraction by SDS-PAGE, and gel slices were prepared for analysis by mass spectrometry.

[0734] Mouse pancreatic tissue: The pancreas was excised from three mice, dounced homogenized, and sonicated in PBS containing protease inhibitors. The cytosolic protein group was separated by centrifugation, and 200 μg of protein was separated by SDS-PAGE, and gel slices were prepared for analysis by mass spectrometry.

[0735]...

Embodiment 2

[0740] Use deep sequencing to identify splice variants

[0741] High-throughput sequencing of a cDNA library enriched with aminoacyl tRNA synthetase transcripts was used to identify splice variants of aminoacyl tRNA synthetase. Prepare cDNA templates from total RNA extracts from tissues such as adult brain and fetal brain, and use primer sequences specific for all annotated exons of human aminoacyl tRNA synthetase and related proteins to enrich aminoacyl tRNA synthesis Enzyme transcript.

[0742] Human total RNA was obtained from Clontech. For cell lines and mouse tissue samples, RNA extraction II kit (MN) was used to extract total RNA. The genomic DNA in the total RNA sample is digested by DNase I. In order to obtain mature messenger RNA (mRNA), the RNA sample was enriched twice by combining poly A+ RNA and digesting the 5'-cap-free RNA by 5'-phosphate-dependent exonuclease. The complementary DNA (cDNA) is synthesized from mature RNA using primers that anneal to the exon seque...

Embodiment 3

[0746] Use bioinformatics to identify AARS peptides

[0747] Use bioinformatics to identify AARS protein fragments (excision protein or appendacrine peptide). Use a program such as FASTA (available from the website http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_www.cgi) or NCBI's BLASTP program (available from the website http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthom) to compare the amino acid sequence of the full-length human aminoacyl tRNA synthetase with the full-length amino acid sequence of its ortholog from the bacterial E. coli. When there are gaps in the aligned bacterial sequences, or regions with low homology between the two species, the excision protein sequence from the human protein is identified as a sequence coverage area. The peptides and corresponding DNA sequences in Tables 3, 6, and 9 include examples identified in this way.

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Abstract

Provided are compositions comprising newly identified protein fragments of aminoacyl-tRNA synthetases, polynucleotides that encode them and complements thereof, related agents, and methods of use thereof in diagnostic, drug discovery, research, and therapeutic applications.

Description

[0001] Cross references to related applications [0002] This application requires U.S. Provisional Patent Application No. 61 / 345,533 filed on May 17, 2010, U.S. Provisional Patent Application No. 61 / 345,531 filed on May 17, 2010 and U.S. Provisional Patent Application No. 61 / 345,532 filed on May 17, 2010, the entire content of each provisional application is incorporated herein by reference. [0003] Statement on Sequence Listing [0004] The sequence listing related to this application is provided in text format instead of the paper copy, and is hereby incorporated into this specification by reference. The name of the text file containing the sequence listing is 120161_456PC_SEQUENCE_LISTING_txt. The text file is approximately 205KB, created on May 12, 2011, and submitted electronically via EFS-Web. Technical field [0005] The present invention generally relates to compositions containing newly identified protein fragments of aminoacyl-tRNA synthetase and other proteins, polynucl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16A61K38/17A61K39/395A61P35/00A61P29/00A61P3/00
CPCA61K39/395G01N33/573C12N9/93C12Q1/527A61K38/00A61K2039/505G01N33/50A61K38/53A61P3/00A61P3/10A61P7/00A61P25/00A61P29/00A61P31/00A61P35/00A61P37/02A61P43/00C12Y601/01C07K16/40
Inventor 莱斯利·安·格林尼凯尔·P·基昂格洪非阿兰·P·瓦瑟洛特罗咏诗杰弗里·D·沃特金斯谢丽尔·L·奎恩约翰·D·门德莱恩
Owner ATYR PHARM INC
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