Methods and systems for identifying patient specific driver mutations
Inventors
Altschuler, Yoram • Tarcic, Gabi
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10047356-B2
Publication Date
2018-08-14
Expiration Date
Abstract
Methods for identifying patient specific driver mutations are provided. The methods provided identify specific patient derived markers associated with aberrant signal transduction pathways, in biological samples of a cancer patient.
Core Innovation
The invention provides a diagnostic platform for identifying one or more patient specific driver mutations in a biological sample of a cancer patient. The method obtains a plurality of mRNAs from the biological sample, generates a cDNA library, and amplifies specific cDNAs using primers complementary to polynucleotides encoding for known signal transduction proteins. The amplified cDNAs are used to form individual expression constructs operably linked to a promoter.
An addressable array is formed including a first set of expression constructs harboring the amplified cDNAs from the biological sample and a second set of expression constructs harboring the corresponding wild type cDNAs. Viable assay cells are added to each locus, and an expression vector encoding a Fluorescence Translocation Reporter (FTR) gene is introduced for each locus, with a target gene portion linked to a specific reporter gene portion.
At least one attribute of the expressed FTR is compared between assay cells expressing the cDNAs from the patient sample and assay cells expressing the corresponding wild type cDNAs. The platform identifies that the cDNA from the biological sample comprises a candidate patient specific driver mutation when there is a disparate result in the at least one attribute of the FTR compared to the corresponding wild type expressed cDNAs, including fluorescence protein localization or fluorescence protein translocation.
Claims Coverage
The document provides two independent claims. Each claim includes patient mRNA-to-cDNA generation, primer-based amplification of polynucleotides encoding known signal transduction proteins, construction and arraying of patient and corresponding wild-type expression constructs, and an FTR-based comparison of a fluorescence attribute to identify candidate patient specific driver mutations.
Patient mRNA to cDNA amplification for known signal transduction proteins
Obtain a plurality of mRNAs from the biological sample, generate a cDNA library from the plurality of mRNAs, and amplify specific cDNAs using primers complementary to polynucleotides encoding for known signal transduction proteins.
Addressable array comparing patient cDNA constructs versus wild-type cDNA constructs
Form individual expression constructs of the amplified cDNAs operably linked to a promoter, and form an addressable array of a first set of expression constructs harboring the amplified cDNAs from the biological sample and a second set of expression constructs of the corresponding wild type cDNAs.
FTR expression and comparison of fluorescence attribute between patient and wild type
Add an expression vector encoding for a Fluorescence Translocation Reporter (FTR) gene comprising a target gene portion linked to a specific reporter gene portion for each locus in the array, add viable assay cells to each locus, and compare at least one attribute of the expressed FTR in the assay cells expressing the cDNAs from the biological sample with its corresponding wild type expressed cDNAs, wherein a disparate result identifies that the cDNA from the biological sample comprises a candidate patient specific driver mutation.
Across the independent claims, the method identifies candidate patient specific driver mutations by generating and amplifying patient-derived cDNA corresponding to polynucleotides encoding for known signal transduction proteins, assembling patient versus corresponding wild-type expression constructs in an addressable array, and using an FTR gene to compare a fluorescence attribute and call a candidate mutation based on a disparate result.
Stated Advantages
Provides identification of one or more patient specific driver mutations in a cancer patient biological sample.
Enables candidate mutation identification by comparing an FTR attribute between patient-derived cDNA constructs and corresponding wild-type cDNA constructs.
Supports distinguishing changes in fluorescence protein localization or fluorescence protein translocation attributes between patient-derived and wild-type expressed FTR.
Documented Applications
Diagnostic identification of patient specific driver mutations in a biological sample of a cancer patient using an FTR-based comparison with corresponding wild type cDNAs.
Use of signal-pathway activity emulation and prediction of therapy sensitivity/resistance by detecting altered reporter localization associated with candidate mutations.
Detection of autocrine/paracrine effects by using altered reporter localization as evidence of pathway changes.
Interested in licensing this patent?