Methods for determining and inhibiting rheumatoid arthritis associated with the BRAF oncogene in a subject
Inventors
Assignees
US Department of Veterans Affairs • Government of the United States of America
Publication Number
US-8680066-B2
Publication Date
2014-03-25
Expiration Date
2032-04-05
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Abstract
The invention provides methods for determining whether a subject is suffering from a rheumatoid arthritis associated with the BRAF oncogene comprising contacting isolated fibroblasts from the subject with a molecule or pool of molecules directed to the BRAF oncogene; and culturing the sample in the presence of the agent and determining whether BRAF oncogene expression by the cell is decreased and/or whether cells in the sample return to a less transformed phenotype, exhibit decreased cell proliferation and/or exhibit increased contact inhibition, any of which is indicative that the subject is suffering from a rheumatoid arthritis associated with the BRAF oncogene.
Core Innovation
The invention provides methods for determining whether a subject is suffering from rheumatoid arthritis (RA) associated with the BRAF oncogene. This involves contacting isolated fibroblasts from the subject with a molecule or pool of molecules directed to the BRAF oncogene, culturing the sample in the presence of the agent, and determining whether BRAF oncogene expression is decreased and/or whether cells in the sample return to a less transformed phenotype, exhibit decreased cell proliferation, and/or exhibit increased contact inhibition. Any of these outcomes is indicative that the subject is suffering from a rheumatoid arthritis associated with the BRAF oncogene.
The background of the invention identifies synovial fibroblasts as responsible for destroying articular cartilage and bone in rheumatoid arthritis but the mechanism for fibroblast transformation remained elusive. The invention addresses this problem by discovering that gain-of-function mutations of the BRAF oncogene, such as the strong V600R mutation found in melanomas and other cancers, are present in synovial fibroblasts from some RA patients. BRAF-specific siRNA inhibited proliferation of synovial fibroblasts with these mutations, suggesting that BRAF mutations underlie fibroblast transformation in RA and establishing a new target for therapeutic intervention.
Additionally, aberrant splice variants of BRAF that retain an intact kinase domain and partially lose the N-terminal autoinhibitory domain were identified in fibroblasts from RA patients. These variants also contribute to fibroblast proliferation and transformation, further implicating BRAF alterations in RA pathogenesis. The invention thus solves the problem of unknown molecular mechanisms of synovial fibroblast transformation by identifying mutant and aberrant BRAF oncogenes as causative factors and providing methods to detect and inhibit them.
Claims Coverage
The patent includes multiple independent claims covering methods for determining RA associated with BRAF oncogene, methods for detecting mutated or variant BRAF oncogenes, and methods for inhibiting synovial cell growth associated with BRAF mutations.
Method for determining RA associated with BRAF oncogene by agent contact and cell phenotype assessment
A method comprising contacting synovial tissue sample with an agent directed to the BRAF oncogene, culturing the sample in presence of the agent, and determining whether BRAF expression is decreased and/or whether synovial cells show less transformed phenotype, decreased proliferation, or increased contact inhibition, any of which indicates RA associated with BRAF oncogene.
Use of nucleic acid molecules as agents targeting BRAF oncogene
The agent directed to the BRAF oncogene can be a nucleic acid molecule, specifically an RNA molecule such as siRNA or antisense RNA, targeting the BRAF oncogene sequence to decrease expression or activity.
Detection of mutant or alternatively spliced BRAF oncogenes
Methods include detecting mutant BRAF oncogene sequences with mutation at codon 600 changing valine to arginine or other amino acids, or detecting alternatively spliced BRAF transcripts missing specific nucleotide sequences within SEQ ID NO:1 encoding truncated mutant BRAF proteins missing amino acid sequences corresponding to defined exons.
Method for determining RA associated with BRAF oncogene by agent association
A method involving contacting synovial tissue sample with agent directed to BRAF oncogene or transcripts, detecting association of the agent with BRAF oncogene or its transcripts from synovial cells, where such association indicates RA associated with BRAF oncogene, including mutants with codon 600 valine changes.
Method for inhibiting synovial cell growth associated with BRAF oncogene
A method for inhibiting synovial cell growth in a subject with RA associated with BRAF oncogene by contacting synovial cells with an agent that binds the BRAF oncogene, thereby inhibiting growth.
Use of nucleic acid molecules for inhibiting BRAF oncogene in synovial cells
The molecule that targets the BRAF oncogene for inhibition can be a nucleic acid molecule such as RNA.
The claims cover inventive methods for diagnosing rheumatoid arthritis associated with mutant or aberrant BRAF oncogene by contacting synovial samples with agents targeting BRAF and assessing changes in gene expression or cell phenotype, as well as methods of inhibiting synovial cell growth by agents, including nucleic acid molecules, that bind the BRAF oncogene or protein. These methods encompass detection of specific point mutations and alternatively spliced BRAF transcripts encoding mutant proteins.
Stated Advantages
Identifies BRAF mutation as a mechanism for synovial fibroblast transformation responsible for articular cartilage and bone destruction in rheumatoid arthritis.
Establishes BRAF as a novel therapeutic target for treating rheumatoid arthritis.
Provides methods to determine whether a subject's rheumatoid arthritis is associated with BRAF oncogene alterations by assessing changes in fibroblast phenotype and proliferation after treatment with BRAF-targeting agents.
Demonstrates that inhibiting BRAF expression decreases proliferation of mutated synovial fibroblasts, confirming the functional importance of BRAF mutations in RA.
Documented Applications
Diagnosis and determination of rheumatoid arthritis in subjects by detecting mutant or aberrant expression of BRAF oncogene in isolated synovial fibroblasts.
Treatment of rheumatoid arthritis by administering agents that bind or inhibit the BRAF oncogene or mutant BRAF proteins.
Screening assays for identifying molecules that bind or block BRAF protein in rheumatoid arthritis cells by assessing alteration of BRAF activity after molecule exposure.
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