Patent Application
20210057040
The present disclosure relates to a gene panel for personalized medicine, a method of constructing the same, and a personalized treatment method using the same.
Genomic data analysis has been developed based on the advancement of next-generation sequencing (NGS) equipment and IT technology that deals with large-scale information, and through human whole genome sequencing, has led to the prediction of individuals' diseases and the provision of personalized disease prevention and treatment. In addition, the development of genome analysis technology has made it possible to secure a genome analysis technology platform and sufficient data needed for clinical application. Even though patients have the same cancer, the molecular and biological characteristics of cancer vary depending on each individual, and patients also exhibit different cancer progression and response to treatment. Therefore, it is important to consider individuals' personal characteristics for prescription. For this reason, the need for personalized medicine has emerged, and there are various studies related to the development of technology for characterizing molecular and biological alterations in patients.
Since differences in disease susceptibility or treatment response between individuals are attributed to differences in genetic variations, it is possible to prevent diseases or to provide personalized medicine optimized for each individual by analyzing genomic data. Targeted therapies have enabled personalized treatment of protein and gene abnormalities of an individual patient, but there are limitations in that mutations of target proteins are limited and drugs corresponding thereto must be used in combination. Accordingly, there is a need for personalized therapies that target gene mutations in individual patients.
The present inventors found cancer cell-specific mutations in essential genes and constitutive genes which are needed for cell survival, and designed siRNAs targeting the mutations to remove only cancer cells, thereby completing the present disclosure.
An aspect provides a gene panel for personalized medicine, the gene panel including 10 or more consecutive polynucleotides including mutations of polynucleotides of genes listed in Table 3 below, or complementary polynucleotides thereof:
Another aspect provides a method of constructing a gene panel for personalized medicine, the method including constructing a target gene set by screening the intersection of essential genes and constitutive genes.
Still another aspect provides a method of providing information for personalized treatment, the method including detecting mutations of the gene panel defined as above in a biological sample isolated from an individual; and from the detection results, determining, as a treatment target for the individual, genes in which mutations have occurred.
An aspect provides a gene panel for personalized medicine, the gene panel including 10 or more consecutive polynucleotides including mutations of polynucleotides of genes listed in Table 3 below, or complementary polynucleotides thereof:
The “polynucleotide” may be DNA or RNA. Further, the polynucleotide may be single-stranded or double-stranded. Further, the polynucleotide may include those consisting of natural nucleotides as well as a nucleotide selected from the group consisting of natural nucleotides, analogues of natural nucleotides, nucleotides having modifications in a sugar base, or phosphate moiety of natural nucleotides, and combinations thereof, as long as it is able to hybridize with a complementary nucleotide by hydrogen bonds (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
The polynucleotide shows a single nucleotide polymorphism on a mutation site. Therefore, when one single-stranded polynucleotide is associated with the risk of developing intractable diseases including cancer, it may be determined that a polynucleotide complementary to the single-stranded polynucleotide is naturally associated with the risk of developing intractable diseases including cancer. For example, in a polynucleotide of SEQ ID NO: 1, a nucleotide at position of hg19. 54656673 is “C or T”. In this case, the gene panel includes the “C or T” nucleotide at position of hg19. 54656673, and 10 or more consecutive nucleotides selected from the polynucleotide of SEQ ID NO: 1, as well as a complementary single-stranded polynucleotide having a “C or T” nucleotide at position corresponding to the position of hg19. 54656673.
The polynucleotides may be primers, probes, or antisense nucleic acids. The “primer” refers to a single-stranded polynucleotide that may act as a starting point in nucleotide polymerization by a polymerase. For example, the primer may be a single-stranded polynucleotide capable of acting as a starting point of template-directed DNA synthesis under appropriate conditions of an appropriate temperature and an appropriate buffer (i.e., in the presence of four different nucleoside triphosphates and a polymerase). The appropriate length of the primer may vary depending on various factors, for example, temperature and use of the primer. The primer may have a length of 15 nt to 30 nt. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with a template.
The sequence of the primer does not need to be completely complementary to some sequences of the template, but the primer should have sufficient complementarity within a range where it may perform its inherent actions through hybridization with the template. Accordingly, the primer includes not only the polynucleotide itself, but also a sequence that specifically hybridizes with the polynucleotide, which may serve as a starting point for polymerization. For example, the primer may be a sequence perfectly complementary to the polynucleotides of SEQ ID NOS: 1 to 10 as well as a sequence having complementarity within a range where it may act as a primer through hybridization with the sequences. The design of the primer may be easily carried out by a person skilled in the art by referring to a sequence of a given target nucleotide sequence to be amplified. For example, the primer may be designed using commercially available primer design programs. Examples of the commercially available primer design programs may include program PRIMER 3.
When the polynucleotide is used as a PCR primer, in addition to the polynucleotide, a primer specifically binding to a complementary strand thereof may be included.
The “probe” refers to a polynucleotide that specifically binds to a specific target sequence. The polynucleotide may be DNA or RNA. The polynucleotide may be single-stranded. Further, the polynucleotide may include those consisting of natural nucleotides as well as a nucleotide selected from the group consisting of natural nucleotides, analogues of natural nucleotides, nucleotides having modifications in a sugar base, or phosphate moiety of natural nucleotides, and combinations thereof, as long as it is able to hybridize with a complementary nucleotide by hydrogen bonds. The probe may have a length of 5 nt to 100 nt, 10 nt to 90 nt, 15 nt to 80 nt, 20 nt to 70 nt, or 30 nt to 50 nt. The polynucleotide includes PNA. In addition, the polynucleotide may have a detectable label (e.g., Cy3, Cy5 fluorescent substance) attached, for example, to the 3′-end or 5′-end, in order to facilitate detection of the polynucleotide or a complex, to which the polynucleotide is bound, in the analysis reaction.
The probe may be a nucleotide sequence perfectly complementary to a target sequence including a mutation site. In addition, the probe may have a substantially complementary nucleotide sequence within a range that does not interfere with specific hybridization to the target sequence including the mutation site. Further, the probe may have a modified nucleotide within a range that does not impair specific hybridization to the target sequence including the mutation site. Examples of the probe may be selected from the group consisting of a perfect match probe consisting of a sequence perfectly complementary to the polynucleotide including the mutation site and a probe having a sequence perfectly complementary to all sequence excluding the mutation site with regard to the polynucleotide including the mutation site.
The “antisense nucleic acid” refers to a nucleic acid-based molecule that has a complementary nucleotide sequence with respect to a target sequence and is able to form a dimer therewith. The antisense nucleic acid may be the polynucleotide or a fragment thereof, or those complementary thereto. The antisense nucleic acid may have a length of 10 nt or more, more specifically, 10 nt to 200 nt, 10 nt to 150 nt, or 10 nt to 100 nt, but an appropriate length may be selected to increase detection specificity.
By using the above primer, probe, or antisense nucleic acid, a nucleotide sequence having a specific allele at the mutation site may be amplified or the presence thereof may be identified.
The polynucleotides may be polynucleotides labeled with a detectable label. The detectable label, which is a labeling material capable of generating a detectable signal, may be a labeling material capable of generating a detectable signal, including a fluorescent material, for example, a material such as Cy3 and Cy5. The detectable label may identify results of nucleic acid hybridization.
In addition, the mutations may be silent mutations. Specifically, amino acid substitution means that an amino acid sequence is changed by change of one or more nucleotides. The silent mutation means that even though nucleotides are changed, an amino acid encoded thereby is not changed. The mutations of the polynucleotides may include T at position of hg19. 54656673 in SEQ ID NO: 1, A at position of hg19. 54649456 in SEQ ID NO: 2, G at position of hg19. 134109517 in SEQ ID NO: 3, G at position of hg19. 19030598 in SEQ ID NO: 4, A at position of hg19. 86585178 in SEQ ID NO: 5, T at position of hg19. 62564024 in SEQ ID NO: 6, A at position of hg19. 96950285 in SEQ ID NO: 7, A at position of hg19. 46543189 in SEQ ID NO: 8, C at position of hg19. 75663394 in SEQ ID NO: 9, and T at position of hg19. 36912832 in SEQ ID NO: 10. Further, the gene panel according to one embodiment may be for targeted treatment of a cancer or an intractable disease, and the cancer may be, for example, brain cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate cancer, colorectal cancer, colon cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, ureter cancer, or cervical cancer. The intractable disease may be, for example, albinism, alcaptonuria, lactose intolerance, hereditary hemorrhagic telangiectasia, thalassemia, congenital dyserythropoietic anaemia, Evans syndrome, pituitary hypofunction, Huntington's disease, hereditary motor sensory neuropathy, or other autonomic nervous system disorders.
As described above, the gene panel according to one specific embodiment is based on an individual patient's tumor genes, and thus tumor treatment effects may be improved through a personalized gene therapy. In addition, since the gene panel targets not only the existing missense mutations, but also silent mutations in which protein mutations do not occur, it is possible to effectively perform a targeted therapy for various diseases caused by gene mutations.
Another aspect provides a method of constructing a gene panel for personalized medicine, the method including constructing a target gene set by screening the intersection of essential genes and constitutive genes. Specifically, the essential genes, which are genes essential for cell survival, encode proteins for maintaining functions such as cell metabolism, DNA replication, protein translation, etc. The essential genes may be those shown in Table 1 below. In addition, the constitutive genes, which are genes originally possessed by living organisms and always fulfill their functions regardless of environmental conditions, may be selected from housekeeping genes which are always expressed in cells and indispensable for cell survival. The constitutive genes may be those shown in Table 2 below. Further, the genes forming the intersection of the essential genes and the constitutive genes are essential for cell survival, and may transcribe genes regardless of cellular environmental conditions. The genes may be those shown in Table 3 below.
Further, the method may include extracting mutant genes common to the target gene set from a plurality of public cell line databases, and collecting base sequence information of the mutant genes; and verifying each mutant gene from the base sequence information of the mutant genes. The method of constructing the gene panel for personalized medicine according to one specific embodiment includes extracting mutant genes common to the target gene set from a plurality of public cell line databases, and collecting base sequence information of the mutant genes. Specifically, the plurality of public cell line databases may include cancer databases of Cancer Cell Line Encyclopedia (CCLE), National Cancer Institute (NCI), or Catalogue of Somatic Mutations in Cancer (COSMIC). The extracting of the mutant genes refers to identifying and/or discriminating information about nucleotide substitution, addition, or deletion in a nucleotide sequence that constitutes an exon of a subject gene. Such nucleotide substitution, addition, or deletion may occur due to various causes, and may be caused by structural differences including, for example, mutations, cleavage, deletions, duplications, inversions and/or translocations of chromosomes. The extracting of the mutant gene may be performed by mapping the genome sequencing data to a standard base sequence. In addition, the collecting of the base sequence information of the mutant genes may be performed by obtaining genome sequencing data of a subject. In this regard, the genome sequencing data may be exome sequencing data, whole genome sequencing data, or sequence data of a gene known to be related to a disease.
Further, the method of constructing the gene panel for personalized medicine according to one specific embodiment includes verifying each mutant gene from the base sequence information of the mutant genes. Specifically, the verifying of the mutant gene may be performed by comparing expression levels between a normal gene and the gene in which the mutation has occurred.
Still another aspect provides a method of providing information for personalized treatment, the method including detecting mutations of the gene panel defined as above in a biological sample isolated from an individual; and from the detection results, determining, as a treatment target for the individual, genes in which mutations have occurred.
The method of providing information according to one specific embodiment includes detecting mutations of the gene panel defined as above in a biological sample isolated from an individual. Specifically, the individual refers to a subject for predicting the risk of developing a cancer or an intractable disease caused by gene mutations. The individual may include vertebrates, mammals, or humans (Homo sapiens). For example, the human may be Korean. Further, the biological sample may be a tissue, a cell, whole blood, serum, plasma, saliva, sputum, a cerebrospinal fluid, or urine. The detecting of mutations of the gene panel defined as above may be performed by isolating a nucleic acid from the biological sample and then determining the mutation site. The methods of isolating the nucleic acid and determining the mutation site are known in the art. The method of isolating the nucleic acid may be performed by, for example, directly isolating DNA from the biological sample or amplifying a specific region by a nucleic acid amplification method such as PCR. The isolated nucleic acid sample includes not only purely isolated nucleic acids, but also crudely isolated nucleic acids, for example, cell lysates containing nucleic acids. The nucleic acid amplification method includes PCR, ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, and nucleic acid-based sequence amplification (NASBA). The isolated nucleic acid may be DNA or RNA. The DNA may be genomic DNA, cDNA, or recombinant DNA. The RNA may be mRNA. Further, the method of determining the mutation site may directly determine nucleotides at the mutation site by, for example, a known nucleotide sequencing method. The method of determining nucleotide sequences may include the Sanger (or dideoxy) sequencing method or the Maxam-Gilbert (chemical cleavage) method. In addition, the nucleotide at the mutation site may be determined by hybridizing a probe including the sequence of the mutation site with a target polynucleotide and analyzing the hybridization result. The degree of hybridization may be confirmed by, for example, labeling a detectable label on a target nucleic acid and detecting the hybridized target nucleic acid, or may be confirmed by an electrical method. A single base primer extension (SBE) method may also be used.
The method of providing information of one specific embodiment includes determining a gene, in which mutations have occurred, as a treatment target of the individual, based on the detection result. Specifically, when the mutations of the gene panel include Tat position of hg19. 54656673 in SEQ ID NO: 1, A at position of hg19. 54649456 in SEQ ID NO: 2, G at position of hg19. 134109517 in SEQ ID NO: 3, G at position of hg19. 19030598 in SEQ ID NO: 4, A at position of hg19. 86585178 in SEQ ID NO: 5, T at position of hg19. 62564024 in SEQ ID NO: 6, A at position of hg19. 96950285 in SEQ ID NO: 7, A at position of hg19. 46543189 in SEQ ID NO: 8, C at position of hg19. 75663394 in SEQ ID NO: 9, and T at position of hg19. 36912832 in SEQ ID NO: 10, it is determined that the individual belongs to a group with a high risk of developing cancer caused by gene mutations. In addition, by identifying the gene in which the mutation has occurred, it is possible not only to predict the risk of developing a specific disease, but also to determine the gene as a treatment target for a specific disease of an individual, thereby performing an efficient targeted therapy.
A gene panel of one aspect, based on an individual's genomic sequence mutation information, may detect gene mutations related to intractable diseases including cancer, and therefore, personalized treatments considering cancer progression or change in patients and treatment models of the diseases may be constructed. In addition, by broadening understanding of the molecular mechanisms of tumor evolution and carcinogenesis through identification of an individual's genome mutations, it is possible to efficiently discover new drugs personalized for patients, and it may also be a starting point for the development of a personalized therapy.
Hereinafter, exemplary embodiments are provided for better understanding of the present disclosure. However, the following exemplary embodiments are provided for easier understanding of the present disclosure, and the contents of the present disclosure are not limited by the following exemplary embodiments.
1-1. Selection of Constitutive Genes
Through literature search, 556 essential genes were selected based on siRNA screening, and 3804 genes (constitutive genes) present in all cells above a predetermined level were selected from housekeeping genes through RNA Seq expression. The related literatures are as follows.
Measuring error rates in genomic perturbation screens: gold standards for human functional genomics (Mol Syst Biol. 2014 July; 10(7): 733.)
Highly parallel identification of essential genes in cancer cells (Proc Natl Acad Sci USA. 2008 Dec. 23; 105(51): 20380-20385.)
Essential Gene Profiles in Breast, Pancreatic, and Ovarian Cancer Cells (Cancer Discov. 2012 February; 2(2):172-189. doi: 10.1158/2159-8290.)
Thereafter, the intersection (220 genes) of the essential genes and the housekeeping genes were selected as screening gene sets. The essential genes, constitutive genes, and gene sets are shown in Tables 1 to 3 below.
1-2. Selection of Cancer Cell Line and Target Mutation
Gene mutations common to the selected gene sets were extracted using WES/WGS public data of various cancer cell lines from cancer databases of Cancer Cell Line Encyclopedia (CCLE), National Cancer Institute (NCI), and Catalogue of Somatic Mutations in Cancer (COSMIC).
1-3. siRNA Synthesis
To inhibit protein expression of the cancer cell line mutant genes selected in Example 1-2, siRNAs were designed. Real time RT-PCR primers and probes for examining expression inhibitory efficiency of siRNAs were also designed such that they specifically react with only the mutant genes, and the results are shown in Table 4 below.
1-4. Examination of Inhibition of Protein Expression of Cancer Cell Line Mutant Genes by siRNA
(1) RD Cell Line
To examine whether protein expression of target genes in cancer cell lines was inhibited by siRNAs designed in Example 1-3, Western blotting was performed. In detail, each 25 nM of Cont siRNA and CNOT3 siRNA designed in Example 3 were added to RD cells, respectively, followed by incubation for 24 hr. Thereafter, the incubated cells were harvested and proteins were extracted therefrom. Western blotting was performed to examine changes in CNOT3 gene expression.
As a result, as shown in
(2) NCI-H69 Cell Line
Experiments were performed in the same manner as in (1), except that NCI-H69 cell line and ZC3H13 siRNA were used.
As a result, as shown in
(3) MDAMB-231 Cell Line
Experiments were performed in the same manner as in (1), except that MDAMB-231 cell line and KARS siRNA were used.
As a result, as shown in
(4) SK-OV-3 Cell Line
Experiments were performed in the same manner as in (1), except that SK-OV-3 cell line and EIF3D siRNA were used.
As a result, as shown in
1-5. Examination of cytotoxicity of siRNA
(1) RD Cell Line
To examine effects of the siRNAs designed in Example 1-3 on cancer cell death, RD cells were treated with CNOT siRNA, and then changes in the increase rate of cytotoxicity were examined. In detail, RD cells were seeded at a density of 1×105 cells/ml in a 6-well plate, and then each 25 nM of Cont siRNA and CNOT siRNA were added thereto, followed by incubation for 24 hr. Thereafter, annexin V staining was performed, and the increase rate of cytotoxicity was examined using a FACSCalibur flow cytometer (Becton Dickinson). Then, the increase rate of cytotoxicity was examined by measuring absorbance at 490 nm using a Pierce LDH Cytotoxicity Assay Kit (thermo scientific) which is a product to measure cell damage using lactate dehydrogenase (LDH) released from cells.
As a result, as shown in
(2) NCI-H69 Cell Line
Experiments were performed in the same manner as in (1), except that NCI-H69 cell line and ZC3H13 siRNA were used.
As a result, as shown in
(3) MDAMB-231 Cell Line
Experiments were performed in the same manner as in (1), except that MDAMB-231 cell line and KARS siRNA were used.
As a result, as shown in
(4) SK-OV-3 Cell Line
Experiments were performed in the same manner as in (1), except that SK-OV-3 cell line and EIF3D siRNA were used.
As a result, as shown in
In other words, it was confirmed that cytotoxicity was increased in cells treated with siRNA against the target, indicating that the gene panel according to one specific embodiment may be usefully applied to a targeted therapy for an individual.
2-1. Target Capture and Library Construction
For NGS experiment, genomic DNAs were isolated from cancer tissue specimens (tissue, blood, FFPE, FNA, etc.) derived from various cancer patients using a QiAmp DNA Mini kit (Qiagen, Valencia, Calif., USA). Thereafter, concentrations, purities, and degradation of the isolated genomic DNAs were determined using a Nanodrop 8000 UV-Vis spectrometer (Thermo Scientific Inc., DE, USA), a Qubit 2.0 Fluorometer (Life technologies Inc., Grand Island, N.Y., USA), and a 2200 TapeStation Instrument (Aglient Technologies, Santa Clara, Calif., USA). Specimens meeting the QC criteria were used in subsequent experiments.
The genomic DNA (˜250 ng) obtained from each tissue was subjected to shearing using Covaris S220 (Covaris, MA, USA), and then subjected to end-repair, A-tailing, paired-end adaptor ligation and amplification to construct sequencing libraries. To capture 220 genomic regions selected in Example 1, reaction was allowed using a composition containing all the prepared polynucleotides for a hybridization time of 24 hr at 65° C., and the genomic DNA library fragments captured by the hybridization were purified. Binding property of biotin and streptavidin attached to polynucleotides was used in the purification. In detail, after binding the streptavidin coated with magnetic beads and biotin attached to the captured library fragments, the captured library fragments were separated from the mixture using a magnetic force. Thereafter, the purified gene DNA library fragments were amplified in a PCR equipment with an index barcode tag, and the conditions are shown in Table 5 below.
2-2. Sequencing
The gene fragments captured in Example 2-1 were injected into an NGS instrument (Miseq, illumina, USA), and sequence information of each DNA fragment was obtained and aligned, thereby obtaining sequence information about each gene in the cancer sample. Sequencing reaction was performed using a TruSeq Rapid PE Cluster kit and a TruSeq Rapid SBS kit (Illumina, USA) under 100 bp paired-end conditions.
2-3. Variant Calling
Data of sequencing reads obtained in Example 2-2 were aligned to UCSC hg19 reference genome (http://genome.ucsc.edu) using Burrows-Wheeler Aligner (BWA) algorithm. PCR duplication was removed using a Picard-tools-1.8 (http://picard.sourceforge.net/), and single nucleotide variation (SNV) was identified using GATK-2.2.9 algorithm.
The above descriptions of the present disclosure are for illustrative purposes only. It will be understood by those skilled in the art that the present disclosure may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above embodiments are not limitative, but illustrative in all aspects.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0013081 | Feb 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/001247 | 1/30/2019 | WO | 00 |
