Patent Application
20230374608
The contents of the electronic sequence listing (J022770014US03-SEQ-HJD.xml; Size: 235,625 bytes; and Date of Creation: Apr. 18, 2023) is herein incorporated by reference in its entirety.
Breast cancer survival rates indicate what portion of people with the same type and stage of breast cancer are still alive a certain amount of time (e.g., 5 years) after they are diagnosed. The extensive heterogeneity of breast cancer, however, complicates a precise assessment of prognosis, making therapeutic decisions difficult and treatments inappropriate in some cases.
Provided herein, in some aspects, is a molecular profiling platform that may be used, for example, to identify exon splicing events (e.g., exon inclusion or exon exclusion) that are specific to breast cancer and can be used for survival prognosis. Alternative splicing is a biological phenomenon that increases protein diversity. In one type of alternative splicing, referred to as “exon skipping,” exons are either spliced out of the transcript based on cellular conditions or are not spliced out but instead remain in the transcript and are “skipped” over. Exon skipping events are regulated by RNA-binding proteins (RPBs) and the spliceosome complex. A common metric for evaluating the extent of exon skipping is percent spliced in (PSI or Ψ), which represents the percentage of transcripts that include a specific exon or splice site.
Prior approaches for analyzing cancer tissue samples separately analyzed a group of normal samples (non-cancerous samples) and a group of cancer samples (samples known to be cancerous) to generate two distributions. Data in the non-overlapping parts of the two distributions would be analyzed to assess the differences between the two groups of samples. Due to the heterogeneity of the biological data, where alternative splicing can occur for reasons other than having cancer (e.g., exon skipping can occur naturally for non-cancerous (normal) healthy patients), the conventional “two-distribution” approach is not well suited to identifying exon skipping events that are predictive of cancer.
The present disclosure provides, in some aspects, methods that combine the analysis (e.g., PSI values) determined for normal and cancer tissue samples and analyze the combined input using a probabilistic model (GMM) to identify subpopulations (clusters) within the overall population that can be further analyzed to assess whether they are cancer-specific. Some of the data described herein is based on an analysis of ˜9300 normal and tumor samples from The Cancer Genome Atlas (TCGA), which identified ˜67,000 exon skipping events. From this data, a subset of exon splicing events (e.g., exon inclusion or exon exclusion) specific to breast cancer was identified.
In some aspects, the present disclosure provides a method comprising assaying nucleic acids of a sample for the presence or absence of a target exon comprising a nucleotide sequence of any one of SEQ ID NOS: 22-24, 26-36, 38-40, 73-75, 77-79, 82-100, 102-104. In some embodiments, the target exon comprises a nucleotide sequence of any one of SEQ ID NOS: 27, 98, 102, or 104.
In other aspects, the present disclosure provides a method comprising assaying nucleic acids of a sample for the presence or absence of at least 2 target exons, wherein each target exon comprises a nucleotide sequence of any one of SEQ ID NOS: 23, 27, 35, 85, 88, 89, 98, 101, 102, or 104. In some embodiments, each target exon comprises a nucleotide sequence of any one of SEQ ID NOS: 27, 98, 101, 102, or 104.
In yet other aspects, the present disclosure provides a method comprising assaying nucleic acids of a sample for the presence or absence of at least 3 target exons, wherein each target exon comprises a nucleotide sequence of any one of SEQ ID NOS: 21, 23, 27, 30, 31, 32, 35, 36, 39, 85, 87-89, 91, 94, 98, or 101-104.
In still further aspects, the present disclosure provide a method comprising assaying nucleic acids of a sample for the presence or absence of at least 8 different target exons, wherein each target exon comprises a nucleotide sequence of any one of SEQ ID NOs: 21-40 or 73-104.
In some embodiments, the sample is a breast tissue sample. For example, the sample may be obtained from a subject suspect of having, at risk of, or diagnosed with breast cancer. In some embodiments, the subject is a female subject.
In some embodiments, the nucleic acids comprise messenger ribonucleic acid (mRNA), or complementary deoxyribonucleic acid (cDNA) synthesized from mRNA obtained from the sample.
In some embodiments, the methods further comprise detecting the presence of a target exon comprising a nucleotide sequence of any one of SEQ ID NOs: 24, 28, 31, 33, and/or 38 or the absence of a target exon comprising a nucleotide sequence of any one of SEQ ID NOs: 82, 87 and/or 91, and assigning a favorable survival prognosis to the sample. In some embodiments, the methods further comprise detecting the presence of a target exon comprising a nucleotide sequence of any one of SEQ ID NOs: 21-23, 25-27, 29, 30, 32, and/or 34-40 or the absence of a target exon comprising a nucleotide sequence of any one of SEQ ID NOs: 73-81, 83-86, 88-90, and/or 92-104, and assigning an unfavorable survival prognosis to the sample.
Also provided herein are complementary deoxyribonucleic acids (cDNAs) comprising a nucleotide sequence of any one of SEQ ID NOs: 1-20 or 105-136. In some embodiments, the cDNAs comprise a nucleotide sequence of any one of SEQ ID NOs: 22-24, 27-34, 36, 38, or 40. Compositions comprising the cDNAs are also contemplated herein. In some embodiments, the compositions further comprise a probe or pair of primers that binds the cDNA. Some compositions of the present disclosure comprise (a) a messenger ribonucleic acid (mRNA) comprising a nucleotide sequence of any one of SEQ ID NOs: 1-20 or 105-136 and (b) a probe or a pair of primers that binds a nucleotide sequence of any one of SEQ ID NOs: 1-20 or 105-136. In some embodiments, the probe or primer comprises a detectable label.
Further provided herein are kits comprising a molecule that can detect the presence or absence of a target exon comprising a nucleotide sequence of any one of SEQ ID NOS: 22-24, 26-36, 38-40, 73-75, 77-79, 82-100, 102-104, and a detection reagent selected from buffers, salts, polymerases, and deoxyribonucleotide triphosphates (dNTPs). In some embodiments, the molecule comprise a probe or primer that bind a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 22-24, 26-36, 38-40, 73-75, 77-79, 82-100, 102-104.
Also provided herein are kits comprising: (a) molecules that can detect the presence or absence of at least 2 target exons, wherein each target exon comprises a nucleotide sequence of any one of SEQ ID NOS: 23, 27, 35, 85, 88, 89, 98, 101, 102, or 104, (b) molecules that can detect the presence or absence of at least 3 target exons, wherein each target exon comprises a nucleotide sequence of any one of SEQ ID NOS: 21, 23, 27, 30, 31, 32, 35, 36, 39, 85, 87-89, 91, 94, 98, or 101-104, or (c) molecules that can detect the presence or absence of at least 8 different target exons, wherein each target exon comprises a nucleotide sequence of any one of SEQ ID NOs: 21-40 or 73-104, and a detection reagent selected from buffers, salts, polymerases, and deoxyribonucleotide triphosphates (dNTPs). In some embodiments, at least one of the probes and/or primers comprises a detectable label.
Alternative splicing is a key mechanism of biological diversity in eukaryotes because it allows multiple mRNA isoforms to be transcribed and translated from a single gene. The human genome includes more than 20,000 genes; however, more than 95% of multi-exonic pre-mRNAs are alternatively spliced to generate nearly 200,000 isoforms. The alternative splicing isoforms translated into proteins can have distinct or even opposing functions. Alternative splicing is involved in a wide range of biological processes, including immune cell maturation and processing.
Studies examining the cancer transcriptome have enabled unprecedented insight into cancer cell heterogeneity and generated novel classifications. This progress has not yet fully translated into clinical benefit. Isoforms as well as alterations in alternative splicing are associated with numerous diseases and can contribute to cancer malignancy by regulating the expression of oncogenes and tumor suppressors. Aberrant alternative splicing profiles can arise in cancer due to mutations at the splice sites or splicing-regulatory elements, but can also reflect changes in splicing regulators. Recurrent mutations in core splicing machinery are found in myeloid leukemia, as well as in sporadic mutations in lung and breast cancer, suggesting that alternative alterations play a key role in tumorigenesis. Alterations in alternative splicing result in the generation of a repertoire of novel isoforms in tumors that, together with fusion molecules, can be viewed as another class of neoantigens.
Provided herein, in some aspects, are methods that comprise assaying a sample for a particular cancer isoform including or excluding a particular exon. In some embodiments, a sample is assayed for multiple exon inclusion or exon exclusion isoforms as provided herein. The data provided by the present disclosure demonstrates that at least one of fifty-two different exon inclusion or exon exclusion isoforms can be detected in ˜91% of all breast cancer samples tested.
Some aspects of the present disclosure comprise assaying a sample for (the presence or absence of) a nucleic acid (e.g., an exon inclusion event or an exon exclusion event) comprising a nucleotide sequence (e.g., an exon) of any one of SEQ ID NOS: 21-40 and 105-136. It should be understood that the phrase “assaying a sample for a nucleic acid comprising a nucleotide sequence of SEQ ID NO: X” encompasses assaying a sample for the presence or absence of a nucleic acid that includes the full length nucleotide sequence identified by SEQ ID NO: X (all nucleotides of SEQ ID NO: X); and the phrase also includes assaying a sample for the presence or absence of a nucleic acid that includes a fragment of the nucleotide sequence identified by SEQ ID NO: X. The length of the fragment is not limited and may be, for example, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides.
In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 23. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 24. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 25. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 26. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 28. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 31. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 36. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 37. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 38. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 39. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 40. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 105. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 106. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 107. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 108. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 109. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 110. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 111. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 112. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 113. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 114. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 115. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 117. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 118. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 119. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 120. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 121. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 122. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 123. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 124. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 125. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 126. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 127. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 128. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 129. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 130. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 131. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 132. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 133. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 134. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 135. In some embodiments, the methods comprise assaying a sample for a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 136.
In some embodiments, methods of the present disclosure comprise assaying a sample for a (at least one) nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 22-24, 27-34, 36, 38, or 40. In some embodiments, the methods further comprise assaying the sample for a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 21, 25, 26, 35, 37, or 39.
In some embodiments, methods of the present disclosure comprise assaying the sample for a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 21, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 22, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 23, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 24, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 25, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 26, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 27, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 28, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 29, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 30, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 31, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 32, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 33, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 34, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 35, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 36, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 37, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 38, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 39, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 40, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 105, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 106, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 107, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 108, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 109, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 110, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 111, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 112, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 113, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 114, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 115, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 116, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 117, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 118, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 119, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 120, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 121, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 122, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 123, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 124, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 125, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 126, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 127, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 128, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 129, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 130, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 131, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 132, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 133, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 134, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 135, a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 136.
In some embodiments, the methods of the present disclosure comprise assaying the sample for 2 (or at least 2) of the 52 exons (selected from exons comprising a nucleotide sequence of any one of SEQ ID NOS: 21-40 and 105-136). In some embodiments, the methods of the present disclosure comprise assaying the sample for 3 (or at least 3) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 4 (or at least 4) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 5 (or at least 5) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 6 (or at least 7) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 7 (or at least 7) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 8 (or at least 8) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 9 (or at least 9) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 10 (or at least 10) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 11 (or at least 11) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 12 (or at least 12) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 13 (or at least 13) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 14 (or at least 14) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 15 (or at least 15) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 16 (or at least 16) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 17 (or at least 17) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 18 (or at least 18) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 19 (or at least 19) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 20 (or at least 20) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 21 (or at least 21) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 22 (or at least 22) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 23 (or at least 23) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 24 (or at least 24) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 25 (or at least 25) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 26 (or at least 26) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 27 (or at least 27) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 28 (or at least 28) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 29 (or at least 29) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for (or at least 30) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 31 (or at least 31) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 32 (or at least 32) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 33 (or at least 33) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 34 (or at least 34) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 35 (or at least 35) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 36 (or at least 36) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 37 (or at least 37) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 38 (or at least 38) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 39 (or at least 39) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 40 (or at least 40) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 41 (or at least 41) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 42 (or at least 42) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 43 (or at least 43) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 44 (or at least 44) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 45 (or at least 45) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 46 (or at least 46) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 47 (or at least 47) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 48 (or at least 48) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 49 (or at least 49) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 50 (or at least 50) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 51 (or at least 51) of the 52 exons. In some embodiments, the methods of the present disclosure comprise assaying the sample for 52 exons.
It should be understood that a method “comprising assaying the sample for fifty-two (52) exon splicing isoforms (e.g., exon inclusion or exon exclusion, each comprising a different nucleotide sequence of SEQ ID NOS: 21-40 and 105-136” is a method that comprises assaying for all 52 isoforms provided in Table 1,Table 2 and Table 3.
Not every sample will have more than one exon splicing isoform (e.g., exon inclusion or exon exclusion) of the present disclosure. In many embodiments, only one of the exon splicing isoforms of the present disclosure will be detected in a sample. Nonetheless, a sample may be assayed for one or more (e.g., 1 to 52) of the 52 exon splicing isoforms. For example, a single sample may include only the exon splicing isoform comprising the sequence of SEQ ID NO:1 or SEQ ID NO: 21. All 52 or a subset of the 52 (less than 52) of the exon splicing isoforms of Table 1, Table 2, and Table 3 may be assayed in order to detect that exon splicing isoform comprising the sequence of SEQ ID NO:1 or SEQ ID NO: 21
It should also be understood that the step of “assaying for an exon splicing isoform(s) (e.g., exon inclusion or exon exclusion)” or “assaying for a nucleic acid” encompasses assaying for mRNA comprising the exon splicing isoform(s) or assaying for complementary DNA (cDNA) comprising the exon splicing isoform(s) (e.g., comprising the sequence of any one of SEQ ID NOS: 21-40 and 105-136). As is known in the art, cDNA is synthesized from mRNA.
There are many different known methods for assaying a sample for the presence or absence of a particular nucleotide sequence, any of which may be used in accordance with the present disclosure. For example, standard polymerase chain reaction (PCR) methods (e.g., reverse transcription PCR (RT-PCR)) may be performed using mRNA obtained from a sample. In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase. The cDNA is then used as a template for exponential amplification using PCR. Thus, kits provided herein may include any one or more reagents used in a PCR such as, for example, primers or probes that bind to a particular nucleic acid comprising an exon splicing event (e.g., exon inclusion or exon exclusion), polymerases, buffers, deoxyribonucleotide triphosphates (dNTPs), and salts.
In some embodiments, an Archer® FusionPlex® assay is used to assay for a nucleotide sequence (e.g., exon). This assay may include using custom designed probes with and an Anchored Multiplexed PCR (AMP™) following by next generation sequencing (NGS) (e.g., with an Illumina® platform). Thus, kits provided herein may include any one or more reagents used in a Archer® FusionPlex® assay.
In other embodiments, targeted sequencing using long-read sequencing technology (e.g., PacBio®, built on Single Molecule, Real-Time (SMRT) Sequencing technology,) is used to assay for a nucleotide sequence (e.g., exon). Thus, kits provided herein may include any one or more reagents used in a long-read sequencing technology.
In other embodiments, Droplet Digital™ PCR (ddPCR™) (BioRad®) is used to assay for a nucleotide sequence (e.g., exon). For example, combinations of primers and probes may be designed to detect selected exon splicing isoforms in single cell suspension or in cells isolated from frozen tumor tissues, e.g., using Laser Capture Microdissection. More than one isoform may be detected in the single cell, for example. Thus, kits provided herein may include any one or more reagents used in a Droplet Digital™ PCR (ddPCR™) assay.
In yet other embodiments, ViewRNA™ In Situ Hybridization (ISH) (Thermo Fisher Scientific) may be used to assay for a nucleotide sequence (e.g., exon). For example, splice junction probes may be designed to enable specific detection of the exon splicing isoforms of the present disclosure in tissue sections (e.g., breast cancer tissue sections) through Fluorescent In Situ Hybridization (FISH). More than one isoform may be detected in the same cell, for example. Thus, kits provided herein may include any one or more reagents used in an ISH assay.
In still other embodiments, nCounter® technology (nanoString™) is used to assay for a nucleotide sequence (e.g., exon). For example, the nCounter® Analysis System utilizes a novel digital barcode technology for direct multiplexed measurement of analytes and offers high levels of precision and sensitivity (<1 copy per cell). The technology uses molecular “barcodes” and single molecule imaging for the direct hybridization and detection of hundreds of unique transcripts in a single reaction. Each color-coded barcode is attached to a single target-specific probe corresponding to an analyte (e.g., exon) of interest. Combined together with invariant controls, the probes form a multiplexed CodeSet. Thus, kits provided herein may include any one or more reagents used in a nCounter® assay or other nanoString™ nucleic acid detection assay.
Other nucleic acid detection methods may be used.
Some aspects of the present disclosure comprise assaying a sample for the presence or absence of a nucleic acid (e.g., an exon inclusion event) comprising a nucleotide sequence of any one of SEQ ID NOS: 1-20, each of which include an exon inclusion event as well as a sequence directly upstream from and a sequence directly downstream from the exon inclusion event (any one of SEQ ID NOS: 21-40). Some aspects of the present disclosure comprise assaying a sample for the presence or absence of a nucleic acid (e.g., an exon exclusion event) comprising a nucleotide sequence of any one of SEQ ID NOS: 105-136, each of which include an exon exclusion event as well as a sequence directly upstream from and a sequence directly downstream from the exon exclusion event (any one of SEQ ID NOS: 41-72).
A probe is a synthetic (non-naturally-occurring) nucleic acid that is wholly or partially complementary to and thus binds to a nucleic acid of interest (e.g., a nucleic acid comprising or comprised within a nucleotide sequence of any one of SEQ ID NOS: 1-20,21-40, 41-72, or 105-136). In some embodiments, a probe comprises DNA. In some embodiments, a probe comprises RNA. In some embodiments, a probe comprise DNA and RNA. It should be understood that the term “probe” encompasses “primer,” which, as is known in the art, is a synthetic nucleic acid (e.g., DNA) used as a starting point for nucleic acid (e.g., DNA) synthesis. The length of a probe may vary, depending on the nucleic acid detection assay being used. For example, a probe may have a length of at least 15, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some embodiments, a probe has a length of 15 to 30 nucleotides, 15 to 50 nucleotides, or 15 to 100 nucleotides. Depending on the application, a probe may be longer than 100 nucleotides.
In some embodiments, one or more probe is designed to bind directly to an exon (e.g., exon inclusion event or exon exclusion event) of any one of SEQ ID NOS: 21-40 and 105-136. The probe may bind, for example, to a 5′ region, a central region, or a 3′ region of an exon.
In some embodiments, one or more probe is designed to bind to a nucleotide sequence directly upstream (5′) from an exon of any one of SEQ ID NOS: 21-40 and 105-136. In other embodiments, one or more probe is designed to bind to nucleotide sequence directly downstream (3′) from an exon of any one of SEQ ID NOS: 21-40 and 105-136. In some embodiments, a first probe (e.g., primer) of a pair of probes is designed to bind to nucleotide sequence directly upstream (5′) from an exon of any one of SEQ ID NOS: 21-40 and 105-136, and a second probe (e.g., primer) of the pair of probes is designed to bind to nucleotide sequence directly downstream (3′) from an exon of any one of SEQ ID NOS: 21-40 and 105-136 such that the pair of probes flank the exon.
In some embodiments, one or more probe is designed to bind to an exon junction. An exon junction comprises (a) nucleotide sequence that includes a 5′ region of an exon (e.g., of any one of SEQ ID NOS: 21-40 and 105-136) and nucleotide sequence directly upstream from the 5′ region of the exon, or (b) nucleotide sequence that includes a 3′ region of an exon (e.g., of any one of SEQ ID NOS: 21-40 and 105-136) and nucleotide sequence directly downstream from the 3′ region of the exon. Table 6 provides examples of cDNA sequences that include exon inclusion events (underlined) as well as sequences directly upstream from and downstream from the exon inclusion event. Any one or more probe may be designed to bind to any region of a nucleotide sequence of Table 6 (SEQ ID NOS: 1-20), e.g., for the purpose of detecting (e.g., amplifying or labeling) the nucleotide sequence in a sample. Table 7 provides examples of cDNA sequences that include exon exclusion events (underlined) as well as sequences directly upstream from and downstream from the exon exclusion event. Any one or more probe may be designed to bind to any region of a nucleotide sequence of Table 7 (SEQ ID NOS: 41-72), e.g., for the purpose of detecting (e.g., amplifying or labeling) the nucleotide sequence in a sample.
In some embodiments, the mRNA is obtained from a biological sample. Biological samples include tissue samples or fluid samples. Non-limiting examples of tissue samples include blood samples and breast tissue samples. Non-limiting examples of fluid samples include cerebrospinal fluid (CSF) samples and urine samples.
In some embodiments, the mRNA is obtained from a breast tissue sample. The breast tissue sample, in some embodiments, is obtained from a female subject (e.g., human female subject), although it may alternatively be obtained from a male subject (e.g., human male subject).
In some embodiments, the sample is obtained from a subject diagnosed with a cancer, such as breast cancer. For example, the subject may have, may be at risk of having, or may be suspected of having a cancer of a breast duct, breast lobule, or breast tissue in between the duct and lobule. Non-limiting examples of breast cancer that may be sampled include ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma of the breast, medullary carcinoma of the breast, mucinous carcinoma of the breast, papillary carcinoma of the breast, cribriform carcinoma of the breast, invasive lobular carcinoma, inflammatory breast cancer, Paget's disease of the nipple, Phyllodes tumors of the breast, metastatic breast cancer, and triple negative breast cancer (TNBC).
Methods of the present disclosure, in some embodiments, comprise assigning a favorable prognosis or unfavorable prognosis to a cancer patient, based on the presence of a nucleic acid in the sample (e.g., an exon inclusion event or an exon exclusion) comprising a nucleotide sequence (e.g., an exon) of any one of SEQ ID NOS: 21-40 and 105-136. Thus, in some embodiments, methods herein comprise obtaining a sample from a subject, assaying the sample for a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 21-40 and 105-136, and assigning a favorable prognosis or unfavorable prognosis to the sample/patient (e.g., breast tissue sample) (see, e.g., Table 4 or Table 5). In some embodiments, a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 21-40 or 105-136 is detected in the sample obtained from the patient.
In some embodiments, a favorable prognosis is assigned to the sample when a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 24, 28, 31, 33, 38, 114, 119, or 123 is detected. In some embodiments, a favorable prognosis is an at least 70% probability of surviving at least 2000 days. In some embodiments, a favorable prognosis is an at least 75% probability of surviving at least 2000 days. In some embodiments, a favorable prognosis is an at least 70% probability of surviving at least 4000 days. In some embodiments, a favorable prognosis is an at least 75% probability of surviving at least 4000 days.
In other embodiments, an unfavorable prognosis is assigned to the sample when a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOS: 21-27, 29, 30, 32, 34-37, 39, 40, 105-113, 115-118, 120-122, or 124-136 is detected. In some embodiments, an unfavorable prognosis is an at least 75% probability of surviving less than 2000 days.
Alternative splicing is a biological phenomenon that increases transcript and protein diversity. In one type of alternative splicing, referred to as “exon skipping,” exons are either spliced “in” or spliced “out” of the transcript based on cellular conditions (
Due to alternative splicing, different transcript isoforms (exon configurations) of the same gene might be expressed in tumor and normal samples. Therefore, even though a gene is expressed in both tumor and normal tissues, transcripts might harbor an exon configuration that is distinctive to cancer.
A conventional approach for identification of cancer biomarkers is based on gene expression. Researchers aim to detect whether a gene is specifically expressed in tumors using microarrays or RNA sequencing. We took a splicing-based approach rather than a gene-based approach to identify cancer biomarkers.
To identify splicing biomarkers in cancer, we took the steps outlined below, i.e., (i) Transcript sequencing, (ii) TCGA analysis, and (iii) Clustering analysis using a novel methodology to identify splicing-based biomarkers.
Sequencing: Long read sequencing using PacBio® Single Molecule Real Time Sequencing (SMRT) technology. This technology is capable of sequencing full-length cDNA transcripts without the need of cDNA fragmentation, and therefore can be used to directly infer the connectivity of exons in transcripts without the need of computational reconstruction. We used this technology to sequence transcripts in 81 cancer and tumor samples. We obtained 298K transcripts corresponding to ˜14K genes, yielding a median of 8 30 isoforms per gene. This represents a ˜2-fold increase over the public human reference transcriptome (Gencode version 25) for those set of genes. This set of transcripts is called PacBio® Transcriptome.
Data Analysis Step 1, TCGA analysis: Quantification of exon skipping events in a large cohort of breast cancer patients available from TCGA using the PacBio®
Transcriptome as background. The aim of the step is to compute percent spliced-in (PSI) for exons undergoing alternative splicing. This step was performed using the rMATS software. rMATS identified 67,255 skipping events in the PacBio® transcriptome, and computed the PSI levels for each of those exons across all samples (n=1,748, including 1,111 breast cancer tumors and 637 normal). Given the size of the TCGA sequencing data, this step was performed using the ISB Cancer Genomics Cloud (Google Cloud) platform.
Data Analysis Step 2, Clustering: Apply a methodology of the present disclosure called ts3 (Tumor Specific Splice Site Detection) to find exons that are included (e.g., spliced in) and excluded (spliced out) only in cancer (
We applied our methodology based on Gaussian mixture modeling to identify exon splicing events specific to breast cancer patients from the TCGA cohort. As a result, we identified 20 exon inclusion events (spliced “in” exons) that are specifically expressed in cancer and have prognosis power. These exon inclusion events have the following properties:
We also identified 32 exon exclusion events (spliced “out” exons) that are specific to breast cancer and have prognosis power. These exon exclusion events have the following properties:
Because they are specific to cancer, these exon events are referred to as “exon inclusion biomarkers or exon exclusion biomarkers.”
The exon splicing sequences were identified using long read SMRT PacBio® sequencing (see, e.g., Rhoads A et al. Genomics Proteomics Bioinformatics 2015; 13: 278-289, and Huddleston J et al. Genome Research 2014; 24: 688-696).
We found 2 types of exon splicing biomarkers, with favorable and unfavorable prognosis. Table 1 indicates that 15 exon inclusion events have unfavorable prognosis (worse outcome, lower survival time), and 5 exon inclusion events have favorable prognosis (better outcome, increased survival time). Table 2 indicates that 29 exon exclusion events have unfavorable prognosis, and 3 exon exclusion events have favorable prognosis.
In terms of exon biomarkers detection, breast cancer TCGA patients can be divided in four groups, (i) unfavorable biomarkers only (60.9% or 502 patients), (ii) favorable biomarkers only (2.9% or 24 patients), and (iii) mixed unfavorable and favorable biomarkers (27.7% or 228 patients), and (iv) no detected biomarkers (8.5% or 70 patients) (
Therefore, while it is common to detect more than one biomarker in the patient, we observed that patients tend to have the same type of exon splicing biomarker (all unfavorable or all favorable). Additional work is ongoing to devise a strategy to utilize these exon biomarkers in the clinical
We classified patients into different groups based on the outcome (unfavorable, favorable, mixed, no prediction) and number of exon splicing biomarkers (exactly one event, more than one event). The classification is available in the Table 3. For instant, unfavorable prognosis was ascertained to 11.9% of patient (exactly one event).
¶Human genome build hg38
¶Human genome build hg38
In this example, we analyzed the splicing events listed in Table 4 and Table 5 (see
Visual inspection of data suggests the existence of a subpopulation of samples in which the target exon is included, or “spliced-in”. This subpopulation (classification “4” samples in
Nonetheless, only one of the clusters (e.g., C4 of
The cluster C4 contains 97 breast cancer patients out of 824 analyzed, which means that the exon inclusion event was detected in ˜12% of TCGA breast cancer patients. Moreover, survival analysis of breast cancer patients in cluster C4 versus the remaining breast cancer patients in TCGA indicates that patients in C4 (expressing the targeting exon) have a worse overall survival (
Furthermore, the expression (expressed as PSI) of a different target exon varies substantially across cancer and normal samples (see, e.g.,
Visual inspection of data suggests the existence of a subpopulation of samples in which the target exon is excluded, or “spliced-out”. This subpopulation (classification “4” samples in
Nonetheless, only two of the clusters (e.g., C1 and C3 of
The cluster C1 contains 37 breast cancer patients out of 824 analyzed, which means that the exon exclusion event was detected in ˜4% of TCGA breast cancer patients. Moreover, survival analysis of breast cancer patients in cluster C1 versus the remaining breast cancer patients in TCGA indicates that patients in C1 (the targeting exon is spliced out) have a worse overall survival (
AGACGAGAGCTGCCCGGAGGACTGGGCAGCA
GCTGTTCCAGCAGAGACATCAGCAAAAGCCA
TCTAGAGGTGGATCCAGAGTGTGGACTAACA
GAGAAAAGAAGTGGAGGGAGAGCAG
GTCTGC
ACAGAGTCTCGCTCTGTTGCCCAGGCTAGAG
TGCAATGGCGTAATCTCAGCTCACTGCAACCT
CCGCCTCCCGTGTTCAAGCGATTCTCCTGCCT
CAGCCTCCTGAGTAGCTGGGATCACAG
ACAG
CCTCTTTTAGTGTAAGCATAAAGAACATTTTT
GGTTCACTTGCTGCTACCCTCTTGTGCCCACT
TTGGCTTAATAAATCCCAATCCAGCCTAGCTG
ATTTACTGAAGAACAAAGGGATGACTAGTTTT
TGCTACGCCAAG
GGGTTCAACAGAAATATTTGG
TTTGCCACTGTGGCACAAAGGCAGCCAGGGA
CGACATGGAAACACATGAAA
GTGCAGATGGGG
AACTCAGCTCGCCTTGAAGGAGATAACATTAT
GGTAAATTTCTCTTACATGCTCAACTTCCTGC
ATGTAAAATGGCTGAAG
TCTGAACAGGAATTA
TCTCCCCCTAAAAAAAGATAAAACTCCTTCCT
GGTTATTAACTGAAATGCTGATCGAGCTTTAT
CCTAAAGAAGATCAGTCGTGGACAAGAACCT
TGTGAAATGTTCCCCATTTGAGACCCTAAAAC
TAATGAAAATCACAGCTTTTGG
ATAAACATTG
GACGGGGATCTCACTATGTGGCCCAGGCTGG
TCTCGAACTCCAAGCTCAAGCGATCCTCCCAC
CTCAGCCTCCCAAAGTACTGGGATTACAGGC
AGGAGCCACCATGCCAAGCCAACACTCTTGTT
CTTAAAGGGCCAGACAGTCAGCATTTTAGCTT
TGCAGGCCTGTTGCTCTATTGCAACAACTCTG
CTGGACTGTGTTCCAGTAAAACATTATGGACG
CTGAAATGTGAATTTCATGTCATTTTCACGTG
TCATGAAATATTCTTCTGTTTTTTTTTTTCAAC
CACTTAAAAACATAAAAAGCCATTTTTAGCTT
GCAGCCTGTACCAAAGCAGGAAGCAGGCTAG
GTTCATCCTGCCTGCCCATTCTCCCACCCCTG
GTCCAGTGAATTACTGGCAAAGAAACAACTG
CATGACCGTTTCTTCACTAAAGCCTCTTCTTG
CTTTCACAGCCCTTTACAGTCTGCAAGGGGCA
TTCTGATGCCTCTTGTTGGTGAGATGGCAGCC
TCATTTTACAGATGAGGACATAGGCCCCAGG
GAGCAAGTGACTTACCCGTGGTCACTCAGCTT
GTGTGTGGTAGGGCAGGATCCCACCCCAGGC
CCCCGCCTCCCTCTCCCACCCAACGCTACTCA
CCGCTTGGCCATGGCCTGGAGCCGGCAGACT
TTTCCTGAGGGACGTCCGGCCTAATAATCAAC
TTGGCAATATATCTGGCTCGTAGACTGCGGC
GATGGGCGTTGATGTGGATATCCTAGATTCCT
CTGGGTTTTCCTTCTTCAAAGTCCTTTCAAAC
CTGTAACAGAAATCTGCTTCACAGATATCTGA
GTCAGTGGGACAGTGGAAGGCAGTGCCTGAA
TGTCCCAGAAGTCCTCCCTCCAGTTGCCTTTT
GGGTCCTGCTGTCATTATCAATAGGACCTTCG
GAGGGACTTCTTGGTTCCCCATCCTATGTCTT
AGGGAAAGAATTGTTGCTGTATTTTGCAGTCA
TTTACTGGGCACCTGTATAAGCTGGAGATGG
CCTAGCCCCAGCGCATGTCCTCCTCCAGGAA
GGCTTCCTGGGTTGTCCTGGGAGAATCAATA
GCCCCTTCCCTGCAGCCTCACTGTGCCTAAGC
AGACACCAATCCTAGCTAGCACTTAGGGGTTT
GTGAACAGGTCTGCCTCCTGCACTAGGCTGT
GATCCCGGACCTGTCTCTGCATCCCTTGCAGG
TGGGAAAGGATCTGCATATGGCAGCCTTTTTT
TTTTTTTTTTTTTTTTTGAGACAGAGTCTCATT
CTATTGCCTGGGCTGGAGCACAGTGGCGAGA
TCTCGGCTCACCACAACCTCCACCTCCCAGGT
TCAAGTGATTCTCCTGCCTCAGCCTCCTGAGT
ACCTGGGACTACAGGCGTGAGCCACCATGCC
CGGCTAATTTTTGTATTTTTAGTAGAGACGGG
GTTTCACTATGTTGGCCAGGCTGGTCTTGAAC
TCCTGACCTCGTGATCCGCCTGCCTTGGCCTC
CCAAAGTGCCGGGATTACAGGCGTGAGCCAC
TGTGCCCAGCCGGCAGGCTTTTATTAAGCGTT
AGATGGGAGGATAGAGGAGTGAAGTGGTACT
GGCAGGAAGTACCAAGGTTCCAGCTGGCGTA
ATCAGGAAGGCTGCATGGAGGAAGCAGCCTT
TGAGCTGCCTGTGGAGTGGTGGGCAGGGTGT
TGTGAAGTGGCAATCACTGGATTTTGCTTCTG
GTACGAGGTGTGGCCAGATGCAAGAAAGAGC
AGGGTGGACTTTGGTGCAATTGGTGGGGGTC
TGGTCTGTAGGGTTCCCGTGGGGAGCCGTGG
AGGGAGGCAGCAAAGGAGGGAGGGGCACAG
AGGATGCTGGACTGTGTTTAAGAGGCAGCAG
GGAGCCATGGCAGGTGCTTGAGGAGAAGCGA
GTGATGTGTTTAAAGCAGCCCTTTCAGGAGG
CTCAGGCTCACAGCAGGATGTGCACAGTAGC
CCTGTCTTGAGCTAAAGCAGATGAAGGTTTTG
CCCTCTGCACTTCCCCACGTGAGAAACGAAG
ATGCACCCGCAGATTCCTTGAGGCAGCTCCC
CCACTTCTCAGTTGCCAGAAATCAGCCCAGAG
AAACAAACCCGTAATCAGCCCAGGGTGCTTTC
CCTTCCCTTTCTCGAGGGGGCTGCTGGTTCGC
ACATAAGGAGTGGGTCACTCCCGCTTGGGAG
AAAGCAGCAGAATTCCTTCACAGCCAGGTAA
GATGTGCCAGTGGTCGATGGATGAAATCTAG
CCGGGGAGTTGGAATCTGTGTTGCCAGCAGT
GACCTGTGAGCAGTGACAAAGCCAAAG
GTAC
TGTTTGATGATGTCTCTCTGAACCTCAGAGAC
GTCTCTTAGGCTGACCTTCACCCAGGCGAGA
AGCACTCCCTCAGCAGAGCCAGCCCACGTGC
ACTCGCCGAGCTCCAGGCCTGGCGCTGGCTA
CCTGCCTCCAGAGCTTTTTCTTCAGGAACACT
CCTTTTCTGTGTG
TAATGATCTGGGATGACCTG
CAGAGTAAGTTTCTTCACTTAGCTCCTACTAA
CAGTGGTGGTTGGGTGGCTGTTTACTGACTG
GATTTCTTACCCTTTTAAGGTCTGTTGAAAGG
AAGTAACCGAATTCCCATGCTTTGATTGGGTT
GGCTCTTTATTTTAATTTAATAAGACTGCCAT
TTCCAGGATCTTTTGCTTTCTTAAAGGACTCT
ATCATCTATGTCTATCCCGATTTGTCAAAGTG
TGGAATTTGGGCGGGAACATGTTTCAAAGTAT
GACACGTGTTATGTAACACTATTTCCCCATAA
CTTTGTCATCAGCAGGAAACCAGAGGATTCTG
TCCTAGTAAGGATCCCTACTAATTTGAAATGA
TTGTGTGGTCATTCATACAGTTATATCTTTAG
ACTGCTAATAGTCTTGAGTCTTGGAGATAATC
CACAGTACTTTATAGAATTAGGTCATCAATCA
TTATAAAGTACCATGTCTTACTAATGTTCTTT
CTGGTACATTCAGATTGAACAGCTCATTCATT
ATTAGTACCAAACATTTCAACCTGTTGTAGAC
ATATACCCTTTTATGAGTTTGGGGTGGTGGTT
GTTGTTGTTGTTCTTCTTCTTCTTTTAAATATA
GAAATCTATTATTTTTACCTTTTTCTCAAAGCA
AGATTCCCATACTAACTATGTACTTCAATCCA
TATCAGAAGGAATCCCCCTCTAAAATGAAGAT
TGTTCTATATCCAG
GAGCCTGAGGAAGAGGGC
CCCTCATGGCGAGGAGTCCCTTTAGAGGTTG
CTGGGCCTGCTTGTGGCCTTGTCTGGTGTGA
AATGGGCTGG
ATGAGGACGATGTGCACAGATG
AGTATAGTGGTGTGATCATAGCTCACTGCAGC
TTCAACCTCCTGTGGTGGTGATCCTCCTGCCT
CAGCCTCCTAAGTTGCTGGGACTACAG
GAGCC
ACCTTGCTGGCTCTGGTGATAAAGAGATGAA
AAAGACAAAAGTTCCTCCCTGCAAAGAGCTCA
TGGTGCAATGGAAGAGATAGAAAGCTGCATT
GTGACAG
ATCGACCTTGGACATGTCCAATAAAA
GGGGAGTCAGACAAGAGCATCATGCTTCTTA
AAAGCCCAGACCCCTGGCTATAACACATCGA
AGATTCTCAGAAGAGAATTGAGGAGCGGACA
GGCGCCACACTCCGTTGTGGTCACTGCCTCTT
CCTGGCCCACCACACTCCTGTCCTCTGCATGT
ACTGAGAGCTCTGTCCAGGATGCCAGGGTCC
TGCCTCGGCAGAGAGGCGGTGCCAGATGCCC
CACAGCAGCTGGTGGGAGTGCCCACAGCTGG
AGGGCAGGGGAGGAGCCTGGCCTCTGGCTGG
TGTTTCCTTCCCAGCTCTCAAGAACTGGAGAC
TTTGGTTACAGAAGTGAAGGCTGCTCCCTCAC
AGACTTCCTAGTGTCCGATGGTACCACATGGA
AGGATCAGAGTTTTGAAGGACTGGGCCAGAA
CCCAGATAGGGCACAAGGCTGCCAGCGCCTG
CATTGAGGGAGCTATGATGTGACGGGGGCTC
CTGCAGAAGATGGCCTTCCTTGTACAG
AGTCG
TTGATAAATTCAAAATATATGTAAAACATATG
CAAGCTGTATAGCAGAACAATAAAATGAACAC
CTATGAATTCACCACTCAATCCAATAATCAAA
ATGACCAGTATTGAATGTGCTTACTTCCAGAG
GGTTGTTATGAACTAGACTGGTCCAACAGGA
AAGTATGATAGATGTGAACTGGGGCTTCTTTT
CAACCTTTTCCGGAAGCTCTCAAGCTGTTCTT
GTGGATAAGACAGAGAATATGTACTCCAATG
CAAAGACTTTTGGTTGAATTATAACTGGCTGA
AG
GTGACTGTAGGACGAGGATTTGGTGTCACAT
GACTTGAGCCCTGGAAATAAGCATCAGTGCA
GACGAGTGCTCTATGAGAAGCTATCTAGTTAA
AGCTCAAGGAGCCACAAAGGGATTTCCTGGC
AGCACAGTCACCAGAAACACTGAGGGAGAAC
TCTCTGAACAGAGGAATTGTGACCCCAAGAC
AGTAGTTTTTAGACGTGACACCAAAAGCACAA
TCCATAAAAGAACAAATTGATAAATTGGACTT
TTTTAAAATTTAAAACTTCTGCTCTATGAAAC
AGACTTTTAAGAGATGGGAAG
TGCAGGAATCA
CACCCTGAACGCGCCCCATCTCTTCTGATCTC
GGAAGCTAACCAAGGTCAGACCTGGTTAGTG
CTTGGATGGGAGATCACCTATTACTTTTTCT
T
GCAGTGACTTAAAATTTGGAGGCAAATTTTCC
TTAAGAGGATATCAAGTTCCAGTATCTTCAGA
TGTTGATAAGCCGTTAG
AATCTCCCTGGAAAA
TCTAGAGGAGGAAGCGGGAAGATGTACACCA
GGGGAGGGGAAAGCTGCAGTCTTCCTTGCCC
ACAGTCTGCTTTGATTGATTCAGTCATTGATG
TTAAAGCAGAATTTGGGTTCTAGCTTCCTACA
GAGAAAACTCCTGTTTCCTGAAGTGATCAAAT
AACGGTGGTTGGATGAACAGCAACAGATAAT
GGAATCTCTTAATGTACTACACAGTGAATTG
AAAAATAAGGTTGAAACATTTTCTGAATCAA
G
TTCCAAAAGCTGAGACAAGATCTTGAAATGGT
ATCAAGATAGATATTATAGCAG
GGACAGATAT
TTTGGCCAGACCACCAGTGTGTGGATAGGTTT
ACAAAATGATGATTATGAAACATGGCTAAATG
GAAAGCCTGTGGTATATTCTAACTGGTCTCCA
TTTGATATAATAAAT
TGCCTTCTGCTGAATATCC
CTGTTGTATTTTCAGTTCTTCCAGTGTGAATC
AGTTAATATTCTCGGGAACGAGGGAGAGGTT
GATCCTATGAGGAAATCAACCACAGTGAAAA
GGCTTGGGCCGCTTTTGTTTTCACCTGCTTTT
GTTGAACAAATTTGATTTCCGGAGTCAGTCAT
TTTACTGTCAAGACATTTCTTCGGCATTCTGC
AACAG
TTTCCAACATGGCTAGATCCATCAGAAA
TCGGGAACGAGGGAGAGGTTGATCCTATGAG
GAAATCAACCACAGTGAAAAGGCTTGGGCCG
CTTTTGTTTTCACCTGCTTTTGTTGAACAAATT
TGATTTCCGGAGTCAGTCATTTTACTGTCAAG
ACATTTCTTCGGCATTCTGCAACAG
TTTCCAAC
TGTGGTGGCCGCCGGTGGCGTGGAGAAG
GCG
GACCACATAATGAAATATAAAAAAAAGTGTGT
CAAGGCCG
GAAGCCTGTGGGATCCGAACATCAC
AACTGAACAAAGACAACATTGTTCCTGGAACG
CCCTCTTTTTAAAAAAG
GGGCTGCGGGCGCCTG
GACAACGGCAACACGACAAGAAGTCCTTGGC
CTCTACCGCAGCATTTTCAGGCTTGCGAGGAA
ATGGCAGGCGACATCAGGGCAGATGGAAGAC
ACCATCAAAGAAAAACAGTACATACTAAATGA
AGCCAGAACGCTGTTCCGGAAAAACAAAAAT
C
CCATTCGTTATCTATGACATGAATTCCTTAAT
GATGGGAGAAGATAAAATCAAGTTCAAACAC
ATCACCCCCCTGCAGGAGCAGAGCAAAGAGG
TGGCCATCCGCATCTTTCAGGGCTGCCAGTTT
CGCTCCGTGGAGGCTGTGCAGGAGATCACAG
AGTATGCCAAAAGCATTCCTGGTTTTGTAAAT
CTTGACTTGAACGACCAAGTAACTCTCCTCAA
ATATGGAGTCCACGAGATCATTTACACAATGC
TGGCCTCCTTGATGAATAAAGATGGGGTTCTC
ATATCCGAGGGCCAAGGCTTCATGACAAGGG
AGTTTCTAAAGAGCCTGCGAAAGCCTTTTGGT
GACTTTATGGAGCCCAAGTTTGAGTTTGCTGT
GAAGTTCAATGCACTGGAATTAGATGACAGC
GACTTGGCAATATTTATTGCTGTCATTATTCT
CAGTGGAG
ACCGCCCAGGTTTGCTGAATGTGAA
CCCGCCCTCCTCAGCCTGATCCTGGAAGAGA
CTCGGGGCCCCCCAGCCTCCGCCAACCCAG
C
GTATCCTGCATAGATTTCAGGTACATCACTGA
TGTCCTGACTGAGGAGGATGCCCTAG
CCTGTC
AGACTTTGGCAGCGTGTAAGGTCTGAGGACA
GGGGCACCGGAGGCCGAGGATGAGAGGCCA
GTGCCTGTTTCCAGGCAGCCAGGGCCTCAGA
AACTCCGGCCGGAGCACTCACCCGTCGGTGG
AGGCCGTTACCAGGGCCACCTTATTTGCGAG
CGGGTCCCGGCGGGTCATCCCGGAGCTGGCC
ATCCGCACCGAATTCCAAGCCCGGGCACAGA
GGCCTAGCAGCCCCGCCTTGTGCATGGATCA
GACCAGCAA
ACATGGGCCCCGTCCTGGGCCAAA
AAATTAAAAAATGGACACTTGGGGCCAGGCG
CAGTGGCTTACGCCTGTAATTCCACCACTTTG
GGGAGCTGAGGCGGGCAGATCACTTGACATC
GAGAGTTCAAGACCAGCCTGACCAACATGGA
GAAACCCCGTCTCTACTAAAAATACAAAAAAT
TATCCAGACGTAGTGGCACATGCCTGTAATCT
CAGCTACTTGGGAGGCTGAGGTAGGAGAATC
GCTTGAACCCGGGAGGCAGAGGTTGTGGTGA
GCCGAGATCATGCCATTGAACTCCAGCCTGG
GCAACAAGAGCGAAACTCCGTCTCCAAAAAA
AAAAAAAGACACTTATTTAGGCTTTCCATATA
TCATG
GGAAGACATGTAAGGAATTTGCATAAGA
GGGTTGGAGCATGACAAGCAGGAGACAGACG
TTCACTTCAACTCCCTGACTGGGGAGGGGAA
CTTCAATTGGCGCTTTGTGTTCCGCTTTGACT
ACCTGCCCACGGAGCGGGAGGTGAGCGTCCG
GCGCAGGTCTGGACCCTTTGCCCTGGAGGAG
GCGGAGTTCCGGCAGCCTGCAGTGCTGGTCC
TGCAG
CTATGAGCTCAGAGTTGTCATCTGGAAC
GTTCCTGAGGAAGAAAAACATGAGGTTGGAA
TAAG
GAAAGAGTTCCTAGAGAGAGAAGACAAA
GTTCCTGAGGAAGAAAAACATGAGGTTGGAA
TAA
GAAAGAGTTCCTAGAGAGAGAAGACAAAG
GAATGTACACTGGAGACCACAGTTCATGCCA
GCGGCTGGAATAAG
GGCACTGTGTTTAGTCTTG
GCTGCCCATGCCCAACCTGAAGGACGAGCTG
CATCACTCAGGATGGAACACCTGCAGCAGCT
GCTTCGGTGATAGCACCAAGTCGCGCACCAA
GCTGGTGCTGCCCAGTCTCATCTCCTCTCGCA
TCTATGTGGTGGACGTGGGCTCTGAGCCCCG
GGCCCCAAAGCTGCACAAG
CTACGAAATGTGG
CATTGCTCTCTGTCCCTGAGATCTTGCTGGAG
ACAGGGCTACTCAGTCCCTCTTTGCCAGGTAA
ACAAAGGATTCAGATTGAAGGACTGCTCAGA
CACCCTCCGAAGAGGTGGCCCTGCCTGCGCT
CCTCCTGGCTGCAGAGTACCCCACCAGCGC
G
TACTTGGGTGTCCCCGCCTCTGATACCTCCCT
GCTGGAGGAAACAGCAGGAAAAGAGAACCAG
GCAGGCAGGCAGACATCCCCACGGAGCAGCG
TTGGGCCCCCAAGGTGCCTGACCCACTTCCTA
GAGTACTGAACAGTCCCAGAGTGTCACAGCT
GATGTGCAGGACAGCCTGGAGCTCTCACCTT
CAACACGGGGTGTACCTGAGACTTCCAGTGG
ATGAGGGTCAGCCTCTGGAGCTGTGAAAACC
TGGGCCGACAGCGGAGGCAGAGCTGCACTAA
TGTTCCCACACGAGTCCTTCCCACCCAACACC
TTGGTGCAGGGAGACGGAAGGAGCCTGGAGC
CAGGG
CTAGAAGAAGTCTTCACTTCCCAGGAGA
GTGAAGTTCAAGGGCATCTTGAACGTGGTGC
ACTTGGAGACAGTGAGGGAAGCAGGGGTGAA
GTGGCTGCTACCTGAGTCCCTTCTGGAGCTCC
ATTTTGCTTGGTCTTGGAGAAGGCTTCTCAGC
TGCCCTCCCAGCTAGT
GAGTTACATCTGCTAAC
GCTTCCTGACCTTCTGGGACCAGTGCACAGA
GAGACTCAGCTGCTTCCTCTGCCCGGTGGAG
CGGGTGCTTCTCACCTTCTGCAACCAGTATGG
TGCCCGCCTCTCCCTGCGCCAGCCAGGCTTG
GCTGAGGCTG
TGTGTGTGAAGTTCCTGGAGGAT
TAAAGATGATCTTCAGAGAGCAATTGCCTTGA
GTTTGGCCGAATCAAACAGGGCATTCAGGGA
GACTGGAATAACTGATGAGGAACAAGCCATT
AGCAG
AGTTCTTGAAGCCAGCATAGCAGAGAAT
TGGAGTGCAATGGCGCTATCTCGGCTCACTG
CAACCTCCGCTTCCCGGATTCAAGCGATTCTC
CTGCCTCAGCCTCCCGAGTAGGTGGGACTAC
AG
GACCAGAGGAGCGAGAGCAGCAAGAACCAC
GTATGATGGAAGGTCTGATCTTCATGTTGGAA
TAACTAACACAAATG
GTATAATGAGGAAAAGG
TATGCCTCCTTTCACTCTGGGAGTCTTCCAGA
GGTCTTGCACTCGGGAGAGCATGCTCAGGTT
TCCCCAGCTCTACAAAATCACCCAGAATGCCA
AAGACTTCAACACAAGGGTAAATAAGGTTGAT
CTCAGAATTGTCACCTCAAAAAGGCCCTGCCT
TCCACTGTTCAGTTCTGGTCATCTGCCTATGA
GATATCTGAAGCTTGAAAGAGAACACTTGAAA
ATCACTGAGACCGTGACTCCCATCCCAGCACA
CACAGCAAGCCAA
ATACTGTGTTGACCAGTGGT
TGACTTTCATGAAGGCGTTAGAGCTG
GTAATT
GACCAAATCCTCAGCTGTCCTCTTCATCTTGA
TCTTCTCTCTGATCTTCAAGCTGGAGGAGCTG
ACACATCTACTTCCCAGTAATATTTCCCCGAA
GAGAAATATTGGCAGCCGAAGACACCAAAAG
CAGAAAAATCACATGGATTTGAATTCTTAAAT
GTGCAGCAG
GTCTAAGGCCCGCCTGTTCTGTGC
AAGGATGACACTGGTGAGAACCCTATGAGGG
AGTGAAGCAGCCTGGACTCTTACCACAAGAG
GGAGGTGTTATAAGAGCAATGCAGAGGTTGG
AGTGGGCAGCAGTTGGGGCAGGAGGAAGCCG
ACTGCTGCCTGGTCTGCAAAGAAGTCCTTTCA
AGTCTCTAGGACTGGACTCTTCCTAAGCAAGT
CCG
AGAAGGAAGCACCCTCACTATGTGGCTCTA
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.
Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein.
This application is a divisional of U.S. application Ser. No. 17/253,974, filed Dec. 18, 2020, which is a national stage filing under 35 U.S.C. § 371 of international application number PCT/US2019/039794, filed Jun. 28, 2019, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/692,121, filed Jun. 29, 2018, and U.S. provisional application No. 62/818,582, filed Mar. 14, 2019, each of which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62692121 | Jun 2018 | US | |
| 62818582 | Mar 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17253974 | Dec 2020 | US |
| Child | 18302737 | US |
