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Osteoarthritis biomarkers and uses thereof

a biomarker and osteoarthritis technology, applied in the field of identification and selection of novel biomarkers, can solve the problems of chondrocytes having a very limited ability to up-regulate their anabolic activity, affecting the efficiency of treatment regimens, and affecting the quality of life of chondrocytes, so as to improve the effect of treatment regimens, prevent, treat, manage and/or ameliorate osteoarthritis

Inactive Publication Date: 2005-10-06
GENENEWS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The patent is about identifying and selecting new biomarkers that can be used to diagnose and treat osteoarthritis. The invention provides methods for measuring the expression of these biomarkers and using them to diagnose and stage the disease. The biomarkers can also be used to monitor disease progression and the effectiveness of therapies. The invention also includes the use of these biomarkers in identifying new therapeutic targets and developing new treatments for osteoarthritis."

Problems solved by technology

In most cases, due to the essential weight-bearing function of the knees and hips, osteoarthritis in these joints causes much more disability than osteoarthritis of the hands.
These symptoms frequently progress to the point that they have a significant impact in terms of lost productivity and or quality of life consequences for the patient.
Unfortunately, chondrocytes have very limited ability to up-regulate their anabolic activity and increase the synthesis of proteoglycan and type II collagen in response to damage or loss of cartilage matrix.
Currently there is no known medical treatment to reverse the effects of this cartilage damage.
“Early stage osteoarthritis” is currently very difficult to diagnose.
Currently there are no recognized biochemical markers used to confirm the diagnosis of early stage osteoarthritis.
These symptoms, however, often do not correlate well with the pathological stages of damage to the cartilage.
A more reliable measure of “early stage” osteoarthritis can be obtained by determining the extent of cartilage damage, however there is currently no method for measuring cartilage deterioration which is relatively non-invasive.
Again, there are no non-invasive methods which can be used to accurately confirm the diagnosis of “late stage osteoarthritis”.

Method used

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  • Osteoarthritis biomarkers and uses thereof
  • Osteoarthritis biomarkers and uses thereof
  • Osteoarthritis biomarkers and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Microarray Construction

[0566] An array according to one aspect of the invention was constructed as follows.

[0567] PCR products (˜40 ul) of cDNA clones from OA cartilage cDNA libraries, in the same 96-well tubes used for amplification, are precipitated with 4 ul (1 / 10 volume) of 3M sodium acetate (pH 5.2) and 100 ul (2.5 volumes) of ethanol and stored overnight at −20° C. They are then centrifuged at 3,300 rpm at 4° C. for 1 hour. The obtained pellets were washed with 50 ul ice-cold 70% ethanol and centrifuged again for 30 minutes. The pellets are then air-dried and resuspended well in 50% dimethylsulfoxide (DMSO) or 20 ul 3×SSC overnight. The samples are then deposited either singly or in duplicate onto Gamma Amino Propyl Silane (Corning CMT-GAPS or CMT-GAP2, Catalog No. 40003, 40004) or polylysine-coated slides (Sigma Cat. No. P0425) using a robotic GMS 417 or 427 arrayer (Affymetrix, CA). The boundaries of the DNA spots on the microarray are marked with a diamond scriber. The i...

example 2

RNA Isolation

from Whole Blood

[0569] 100 ul whole blood is obtained in a microcentrifuge tube and spun at 2,000 rpm (800 g) for 5 min at 4° C. and the supernatant removed. Pelleted cells are homogenized using TRIzol® (GIBCO / BRL) in a ratio of approximately 6 μl of TRIzol® for every 10 μl of the original blood sample and vortexed well. Samples are left for 5 minutes at room temperature. RNA is extracted using 12 μl of chloroform per 10 μl of TRIzol®. Sample is centrifuged at 12,000×g for 5 minutes at 4° C. and upper layer is collected. To upper layer, isopropanol is added in ratio of 5 μl per 10 μl of TRIzol®. Sample is left overnight at −20° C. or for one hour at −20° C. RNA is pelleted in accordance with known methods, RNA pellet air dried, and pellet resuspended in DEPC treated ddH2O. RNA samples can also be stored in 75% ethanol where the samples are stable at room temperature for transportation.

from Centrifuged Lysed Blood

[0570] 10 ml whole blood is obtained in a Vacutaine...

example 3

Target Nucleic Acid Preparation and Hybridization

Preparation of Fluorescent DNA Probe from mRNA

[0572] Fluorescently labeled target nucleic acid samples of RNA are prepared for analysis with an array of the invention.

[0573] 1 μg Oligo-dT primers are annealed to 10 ug of total RNA isolated from blood from patient diagnosed with osteoarthritis or suspected of having osteoarthritis in a total volume of 10 ul, by heating to 70° C. for 10 min, and cooled on ice. The mRNA is reverse transcribed by incubating the sample at 42° C. for 40 min in a 25 μl volume containing a final concentration of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 25 mM DTT, 25 mM unlabeled dNTPs, 400 units of Superscript II (200 U / uL, Gibco BRL), and 15 mM of Cy3 or Cy5 (Amersham). The reaction is stopped by the addition of 2.5 μl of 55500 mM EDTA and5 μl of 1M NaOH, and incubation at 65° C. for 10 min. The reaction mixture is neutralized by addition of 12.5 μl of 1M TrisHCl (pH7.6).

[0574] The labeled targe...

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PUM

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Abstract

The invention relates to the identification and selection of novel biomarkers and the identification and selection of novel biomarker combinations which are differentially expressed in osteoarthritis and / or in a particular stage of osteoarthritis, as well as a means of selecting the novel biomarker combinations. The biomarkers of the invention may be utilized to diagnose arthritis, differentiate between two stages of osteoarthritis and diagnose a particular stage of osteoarthritis. The invention provides kits comprising polynucleotides and / or proteins which specifically and / or selectively hybridize to the products of the biomarkers of the invention. Such kits are useful in diagnosing arthritis, differentiating between two stages of osteoarthritis, diagnosing a particular stage of osteoarthritis monitoring disease progression, and monitoring the efficacy of therapeutic regimens. The invention also provides for methods of using the products of the biomarkers of the invention in the identification of novel therapeutic targets for osteoarthritis.

Description

[0001] This application is entitled to and claims priority benefit under 35 U.S.C. § 119 to U.S. provisional application Ser. No. 60 / 493,607, filed Aug. 8, 2003 and U.S. provisional application Ser. No. 60 / 516,823, filed Nov. 3, 2003, each of which is incorporated herein by reference in its entirety.1. FIELD OF THE INVENTION [0002] The invention relates to the identification and selection of novel biomarkers and the identification and selection of novel biomarker combinations which are differentially expressed in osteoarthritis and / or in a particular stage of osteoarthritis, as well as a means of selecting the novel biomarker combinations. The measurement of expression of the products of the biomarkers and combinations of biomarkers of the invention demonstrates particular advantage in one or more of the following: (a) diagnosing individuals as having arthritis, (b) differentiating between two stages of osteoarthritis (OA) and (c) diagnosing individuals as having a particular stage ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28C12NC12N15/113C12Q1/68G01N33/53G01N33/564
CPCC12N15/113C12N2310/11C12N2310/12C12N2310/14C12N2310/15C12Q1/6883C12Q2600/158G01N33/564G01N2800/105G01N2800/52
Inventor LIEW, CHOONG-CHINYAGER, THOMASDEMPSEY, ADAMCHAO, SAMUELZHANG, HONGWEIMARSHALL, WAYNE
Owner GENENEWS
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