Claims for Patent: 10,446,261

Summary for Patent: 10,446,261

Title:	System and method for analyzing splicing codes of spliceosomal introns
Abstract:	A system and method for analyzing splicing codes of spliceosomal introns is disclosed. One embodiment comprises methods of identifying introns and exons in genomic DNA or pre-mRNA sequences by locating characteristic markers in splicing junctions by computation and/or manually. Exon sequences predicted by computation can be verified and characterized by employing standard amplification methods, such as comparative genomic, RNA-seq, next-generation sequencing, RT-PCR. DNA/RNA/oligo, electrophoretic or protein chip technologies. If a given sample is verified, its polypeptide can be translated based on genetic codons. Its functions can be deduced based on its characteristics, computation predictions and related knowledge databases. These data can be used to compare databases which correlate the characterized intron or exon or gene to characterized diseases or genetic mutations. Isoforms can be detected and analyzed at mRNA and protein levels alone and with other isoforms predicted by computation, characterized by experiments and stored in existing databases.
Inventor(s):	Zhuo; Degen (Miami, FL)
Assignee:	BIOTAILOR, INC. (Miami, FL)
Application Number:	13/372,180
Patent Claims:	1. A method of identifying at least one potentially alternatively spliced transcript in at least one pre-mRNA sequence obtained from biochemical analysis of a biological sample from a species, the method comprising: (a) generating a splicing code table of the species, comprising the substeps of: determining substantially all of the 5' splice sites and 3' splice sites from relevant existing databases, wherein the relevant existing databases comprise genome sequences of the species, and wherein the genome sequences comprise at least an intron dataset; dividing the 5' splice sites into 5' exonic (E5) and 5' intronic (I5) splicing sequences; dividing the 3' splice sites into 3' intronic (I3) and 3' exonic (E3) splicing sequences; constructing the splicing code table by parsing out E5 sequences, I5 sequences, I3 sequences, and E3 sequences from the intron dataset, each splicing code comprising E5 junction sequence-the first intronic dinucleotide-I3 junction sequence, wherein an E5 junction sequence comprises eight twelve nucleotides of an E5 sequence upstream of the 5' splice site and an I3 junction sequence comprises six to ten nucleotides of an I3 sequence upstream of the 3' splice site, and storing the splicing code table in a programmable, searchable computer database; generating by a computer processor putative markers, wherein each of the putative markers comprises an E5-the first intronic dinucleotide-I3 sequence of all genes of interest of the species; (b) aligning the at least one pre-mRNA sequence with each of the putative markers in the splicing code table; (c) determining that the at least one RNA sequence is an alternatively spliced transcript candidate if the at least one pre-mRNA sequence is found to have a substantially identical match with at least one of the putative markers in the splicing code table and it is not identical to putative markers; or determining that the at least one pre-mRNA sequence is not an alternatively spliced transcript candidate if no substantially identical matches are found between the at least one pre-mRNA sequence and any of the putative markers in the splicing code table; (d) verifying, if the at least one RNA sequence is determined to be an alternatively spliced transcript candidate in step (c), that the at least one RNA sequence is a real alternatively spliced transcript by a biochemical assay, wherein the biochemical assay in step (d) comprises at least one of the RT-PCR, RNA sequencing, DNA sequencing, RNA-seq sequencing and array hybridization, and the biochemical assay comprises use of at least one primer and/or probe, wherein the at least one primer and/or probe is designed based on the E5 sequences and the E3 sequences of the at least one of the putative markers with which the at least one pre-mRNA sequence is found to be substantially identical and wherein the at least one primer and/or probe is designed to assay an alternatively spliced transcript. 2. The method according to claim 1, wherein the biochemical assay comprises RT-PCR, and the biochemical assay comprises use of a first primer and a second primer, wherein the first primer is designed based on the E5 sequence and the second primer is designed based on the E3 sequence. 3. The method according to claim 1, wherein the biochemical assay comprises array hybridization and the biochemical assay comprises use of a probe, and wherein the probe is designed based on a splice junction sequence connecting the E5 sequence and the E3 sequence. 4. The method according to claim 1, wherein the biochemical assay comprises RNA-seq and the biochemical assay comprises use of a probe, and wherein the probe is designed based on a junction sequence connecting the E5 sequence and the E3 sequence. 5. The method according to claim 1, wherein size of the putative markers has a range of 150 bp to 50,000 bp. 6. The method according to claim 1, wherein the species is a eukaryotic organism. 7. The method according to claim 6, wherein the species is a mammal. 8. The method according to claim 7, wherein the species is a mouse. 9. The method according to claim 7, wherein the species is a human. 10. The method according to claim 6, wherein the species is a vertebrate, selected from the group consisting of chicken and zebrafish, or an invertebrate, selected from a fungus, a protest, C. elegans and D. melanogaster, or a eukaryotic virus. 11. The method according to claim 1, wherein in step (c) the E5 sequence plus the first intronic dinucleotide and the I3 sequence of each of the putative markers in the splicing code table with which the at least one RNA sequence is aligned are mapped to a gene encoding a receptor, an ion-channel or a neurotransmitter, selected from a group consisting of insulin receptor, G protein-coupled receptors, 5-Hydroxytryptamine receptors, Acetylcholine receptors (muscarinic), Adenosine receptors, Adrenoceptors, Anaphylatoxin receptors, Angiotensin receptors, Apelin receptor, Bile acid receptor, Bombesin receptors, Bradykinin receptors, Calcitonin receptors, Calcium-sensing receptors, Cannabinoid receptors, Chemokine receptors, Cholecystokinin receptors, Corticotropin-releasing factor receptors, Dopamine receptors, Endothelin receptors, Estrogen (G protein coupled) receptor, Formylpeptide receptors, Free fatty acid receptors, Frizzled receptors, GABAB receptors, Galanin receptors, Ghrelin receptor, Glucagon receptor family, Glycoprotein hormone receptors, Gonadotrophin-releasing hormone receptors, Histamine receptors, Hydroxycarboxylic acid receptors, Kisspeptin receptor, Leukotriene receptors, Lysophospholipid receptors, Melanin-concentrating hormone receptors, Melanocortin receptors, Melatonin receptors, Metabotropic glutamate receptors, Motilin receptor, Neuromedin U receptors, Neuropeptide FF/neuropeptide AF receptors, Neuropeptide S receptor, Neuropeptide W/neuropeptide B receptors, Neuropeptide Y receptors, Neurotensin receptors, Opioid receptors, Orexin receptors, P2Y receptors, Parathyroid hormone receptors, Peptide P518 receptor, Platelet-activating factor receptor, Prokineticin receptors, Prolactin-releasing peptide receptor, Prostanoid receptor, Protease-activated receptors, Relaxin family peptide receptors, Somatostatin receptors, Tachykinin receptors, Thyrotropin-releasing hormone receptor, Trace amine receptor, Urotensin receptor, VIP and PACAP receptors, Vasopressin and oxytocin receptors, Class A Orphans, Class B Orphans, Class C Orphans Non-signaling 7TM chemokine-binding proteins, BLT1, BLT2, CysLT1, CysLT2, OXE, CCRL2, CMKLR1, GPR1, GPR3, GPR4, GPR6, GPR12, GPR15, GPR17, GPR18, GPR19, GPR20, GPR21, GPR22, GPR25, GPR26, GPR27, GPR31, GPR32, GPR33, GPR34, GPR35, GPR37, GPR37L1, GPR39, GPR42, GPR45, GPR50, GPR52, GPR55, GPR61, GPR62, GPR63, GPR65, GPR68, GPR75, GPR78, GPR79, GPR82, GPR83, GPR84, GPR85, GPR87, GPR88, GPR101, GPR119, GPR120, GPR132, GPR135, GPR139, GPR141, GPR142, GPR146, GPR148, GPR 149, GPR150, GPR151, GPR152, GPR153, GPR160, GPR161, GPR162, GPR171, GPR173, GPR174, GPR176, GPR182, GPR183, LGR4, LGR5, LGR6, LPAR6, MAS1, MAS1L, MRGPR, MRGPRE, MRGPRF, MRGPRG, MRGPRX1, MRGPRX2, MRGPRX3, MRGPRX4, OPN3, OPN5, OXGR1, P2RY8, P2RY10, SUCNR1, TAAR2, TAAR3, TAAR4, TAAR5, TAAR6, TAAR8, TAAR9, Calcium-Activated Potassium Channels, CatSper and Two-Pore Channels, Cyclic Nucleotide-Regulated Channels, Inwardly Rectifying Potassium Channels, Transient Receptor Potential Channels, Two-P Potassium Channels, Voltage-Gated Calcium Channels, Voltage-Gated Potassium Channels, Voltage-Gated Sodium Channels, 5-HT3 receptors, GABAA receptors, Glycine receptors, Ionotropic glutamate receptors, Nicotinic acetylcholine receptors, P2X receptors, ZAC, Thyroid Hormone Receptors, Retinoic acid receptors, Peroxisome proliferator-activated receptors, Rev-Erb receptors, RAR-related orphan receptors, Liver X receptor-like receptors, Vitamin D receptor-like receptors, Hepatocyte nuclear factor-4 receptors, Retinoid X receptors, Testicular receptors, Tailess-like receptors, COUP-TF-like receptors, Estrogen receptors, Estrogen-related receptors, 3-Ketosteroid receptors, Nerve growth factor IB-like receptors, Fushi taruzu F1-like receptors, Germ cell nuclear factor receptors, DAX-like receptors, Human Epidermal growth factor Receptor 1, Human Epidermal growth factor Receptor 2, Estrogen receptor-.alpha. (ER.alpha.; NR3A1, ESR1), Estrogen receptor-.beta. (ER.beta.; NR3A2, ESR2), Estrogen-related receptor-.alpha. (ERR.alpha.; NR3B1, ESRRA), Estrogen-related receptor-.beta. (ERR.beta.; NR3B2, ESRRB), Estrogen-related receptor-.gamma. (ERR.gamma.; NR3B3, ESRRG), Glucocorticoid receptor (GR; NR3C1), Mineralocorticoid receptor (MR; NR3C2), Progesterone receptor (PR; NR3C3, PGR), and Androgen receptor (AR; NR3C4). 12. The method according to claim 11, wherein the gene encodes an insulin receptor. 13. The method of claim 1, wherein each splicing code comprises an E5 junction sequence comprising nine nucleotides of an E5 sequence upstream of the 5' splice site and an I3 junction sequence comprising nine nucleotides of an I3 sequence upstream of the 3' splice site. 14. A method of identifying at least one potentially alternatively spliced transcript of a gene from a biological tissue from a species, the method comprising: (a) obtaining at least one pre-mRNA sequence from the biological tissue; (b) generating a splicing code table of the species, comprising the substeps of: (i) determining substantially all of the 5' splice sites and 3' splice sites from relevant existing databases, wherein the relevant existing databases comprise genome sequences of the species, and wherein the genome sequences comprise at least an intron dataset; (ii) dividing the 5' splice sites into 5' exonic (E5) and 5' intronic (I5) splicing sequences; (iii) dividing the 3' splice sites into 3' intronic (I3) and 3' exonic (E3) splicing sequences; (iv) constructing the splicing code table by parsing out E5 sequences, I5 sequences, I3 sequences, and E3 sequences from the intron dataset, each splicing code comprising E5 junction sequence-the first intronic dinucleotide-I3 junction sequence, wherein an E5 junction sequence comprises eight to twelve nucleotides of an E5 sequence upstream of the 5' splice site and an I3 junction sequence comprises six to ten nucleotides of an I3 sequence upstream of the 3' splice site, and storing the splicing code table in a programmable, searchable computer database; and (v) based on the splicing code table, generating by a computer processor a set of putative markers, wherein each of the set of putative markers comprises an E5-the first intronic dinucleotide-I3 sequence of the gene; (d) identifying the at least one potentially alternatively spliced transcript in the at least one pre-mRNA sequence by a biochemical assay, wherein: the biochemical assay comprises at least one of RT-PCR, RNA sequencing, DNA sequencing, DNA-seq sequencing and array hybridization; and the biochemical assay comprises use of at least one primer and/or probe, and wherein the at least one primer and/or probe is designed based on the E5 sequences and the E3 sequences of the at least one of the putative markers and wherein the at least one primer and/or probe is designed to assay an alternatively spliced transcript. 15. The method according to claim 14, wherein the at least one potentially alternatively spliced transcript is a truncated isoform of the gene; and step (b) further comprising, after substep (iv), a substep of removing putative markers to which the E5 sequences and the E3 sequences correspond are mapped to be a membrane-binding domain. 16. The method according to claim 15, wherein the species is a mammal. 17. The method according to claim 16, wherein the gene encodes a receptor, an ion-channel or a neurotransmitter, selected from a group consisting of insulin receptor, G protein-coupled receptors, 5-Hydroxytryptamine receptors, Acetylcholine receptors (muscarinic), Adenosine receptors, Adrenoceptors, Anaphylatoxin receptors, Angiotensin receptors, Apelin receptor, Bile acid receptor, Bombesin receptors, Bradykinin receptors, Calcitonin receptors, Calcium-sensing receptors, Cannabinoid receptors, Chemokine receptors Cholecystokinin receptors, Corticotropin-releasing factor receptors, Dopamine receptors, Endothelin receptors, Estrogen (G protein coupled) receptor, Formylpeptide receptors, Free fatty acid receptors, Frizzled receptors, GABAB receptors, Galanin receptors, Ghrelin receptor, Glucagon receptor family, Glycoprotein hormone receptors, Gonadotrophin-releasing hormone receptors, Histamine receptors, Hydroxycarboxylic acid receptors, Kisspeptin receptor, Leukotriene receptors, Lysophospholipid receptors, Melanin-concentrating hormone receptors, Melanocortin receptors, Melatonin receptors, Metabotropic glutamate receptors, Motilin receptor, Neuromedin U receptors, Neuropeptide FF/neuropeptide AF receptors, Neuropeptide S receptor, Neuropeptide W/neuropeptide B receptors, Neuropeptide Y receptors, Neurotensin receptors, Opioid receptors, Orexin receptors, P2Y receptors, Parathyroid hormone receptors, Peptide P518 receptor, Platelet-activating factor receptor, Prokineticin receptors, Prolactin-releasing peptide receptor, Prostanoid receptor, Protease-activated receptors, Relaxin family peptide receptors, Somatostatin receptors, Tachykinin receptors, Thyrotropin-releasing hormone receptor, Trace amine receptor, Urotensin receptor, VIP and PACAP receptors, Vasopressin and oxytocin receptors, Class A Orphans, Class B Orphans, Class C Orphans Non-signaling 7TM chemokine-binding proteins, BLT1, BLT2, CysLT1, CysLT2, OXE, CCRL2, CMKLR1, GPR1, GPR3, GPR4, GPR6, GPR12, GPR15, GPR17, GPR18, GPR19, GPR20, GPR21, GPR22, GPR25, GPR26, GPR27, GPR31, GPR32, GPR33, GPR34, GPR35, GPR37, GPR37L1, GPR39, GPR42, GPR45, GPR50, GPR52, GPR55, GPR61, GPR62, GPR63, GPR65, GPR68, GPR75, GPR78, GPR79, GPR82, GPR83, GPR84, GPR85, GPR87, GPR88, GPR101, GPR119, GPR120, GPR132, GPR135, GPR139, GPR141, GPR142, GPR146, GPR148, GPR149, GPR150, GPR151, GPR152, GPR153, GPR160, GPR161, GPR162, GPR171, GPR173, GPR174, GPR176, GPR182, GPR183, LGR4, LGR5, LGR6, LPAR6, MAS1, MAS1L, MRGPR, MRGPRE, MRGPRF, MRGPRG, MRGPRX1, MRGPRX2, MRGPRX3, MRGPRX4, OPN3, OPN5, OXGR1, P2RY8, P2RY10, SUCNR1, TAAR2, TAAR3, TAAR4, TAAR5, TAAR6, TAAR8, TAAR9, Calcium-Activated Potassium Channels, CatSper and Two-Pore Channels, Cyclic Nucleotide-Regulated Channels, Inwardly Rectifying Potassium Channels, Transient Receptor Potential Channels, Two-P Potassium Channels, Voltage-Gated Calcium Channels, Voltage-Gated Potassium Channels, Voltage-Gated Sodium Channels, 5-HT3 receptors, GABAA receptors, Glycine receptors, Ionotropic glutamate receptors, Nicotinic acetylcholine receptors, P2X receptors, ZAC, Thyroid Hormone Receptors, Retinoic acid receptors, Peroxisome proliferator-activated receptors, Rev-Erb receptors, RAR-related orphan receptors, Liver X receptor-like receptors, Vitamin D receptor-like receptors, Hepatocyte nuclear factor-4 receptors, Retinoid X receptors, Testicular receptors, Tailess-like receptors, COUP-TF-like receptors, Estrogen receptors, Estrogen-related receptors, 3-Ketosteroid receptors, Nerve growth factor IB-like receptors, Fushi taruzu F1-like receptors, Germ cell nuclear factor receptors, DAX-like receptors, Human Epidermal growth factor Receptor 1, Human Epidermal growth factor Receptor 2, Estrogen receptor-.alpha. (ER.alpha.; NR3A1, ESR1), Estrogen receptor-.beta. (ER.beta.; NR3A2, ESR2), Estrogen-related receptor-.alpha. (ERR.alpha.; NR3B1, ESRRA), Estrogen-related receptor-.beta. (ERR.beta.; NR3B2, ESRRB), Estrogen-related receptor-.gamma. (ERR.gamma.; NR3B3, ESRRG), Glucocorticoid receptor (GR; NR3C1), Mineralocorticoid receptor (MR; NR3C2), Progesterone receptor (PR; NR3C3, PGR), and Androgen receptor (AR; NR3C4). 18. The method according to claim 17, wherein the gene encodes an insulin receptor. 19. The method of claim 14, wherein each splicing code comprises an E5 junction sequence comprising nine nucleotides of an E5 sequence upstream of the 5' splice site and an I3 junction sequence comprising nine nucleotides of an I3 sequence upstream of the 3' splice site. 20. A method for identifying at least one potentially alternatively spliced transcript in at least one pre-mRNA sequence obtained from biochemical analysis of a biological sample from a species, the method comprising: (a) generating a splicing code table of the species, comprising the substeps of: determining substantially all of the 5' splice sites and 3' splice sites from relevant existing databases, wherein the relevant existing databases comprise genome sequences of the species, and wherein the genome sequences comprises at least an intron dataset; dividing the 5' splice sites into 5' exonic (E5) and 5' intronic (I5) splicing sequences; dividing the 3' splice sites into 3' intronic (I3) and 3' exonic (E3) splicing sequences; constructing the splicing code table by parsing E5 sequences, I5 sequences, I3 sequences, and E3 sequences from the intron dataset, each splicing code comprising E5 junction sequence-the first intronic dinucleotide-I3 junction sequence, wherein an E5 junction sequence comprises eight to twelve nucleotides of an E5 sequence upstream of the 5' splice site and an I3 junction sequence comprises six to ten nucleotides of an I3 sequence upstream of the 3' splice site, and storing the splicing code table in a programmable, searchable computer database; generating by a computer processor putative markers, wherein each of the putative markers comprises an E5-the first intronic dinucleotide-I3 sequence of all genes of interest of the species; (b) aligning the at least one pre-mRNA sequence with each of the putative markers in the splicing code table; (c) determining that the at least one RNA sequence is an alternatively spliced transcript candidate if the at least one pre-mRNA sequence is found to have a substantially identical match with at least one of the putative markers in the splicing code table and it is not identical to putative markers; or determining that the at least one pre-mRNA sequence is not an alternatively spliced transcript candidate if no substantially identical matches are found between the at least one pre-mRNA sequence and any of the putative markers in the splicing code table; and (d) verifying, if the at least one RNA sequence is determined to be an alternatively spliced transcript candidate in step (c), that the at least one RNA sequence is a real alternatively spliced transcript by a biochemical assay, wherein the biochemical assay in step (d) comprises contacting RNA isolated from the biological sample with at least one of primer and/or probe, wherein the at least one primer and/or probe is designed based on the E5 sequences and the E3 sequences of the at least one of the putative markers with which the at least one pre-mRNA sequence is found to be substantially identical and wherein the at least one primer and/or probe is designed to bind the alternatively spliced transcript, and detecting resultant binding between the RNA isolated from the biological sample and the at least one primer and/or probe. 21. The method of claim 20, wherein each splicing code comprises an E5 junction sequence comprising nine nucleotides of an E5 sequence upstream of the 5' splice site and an I3 junction sequence comprising nine nucleotides of an I3 sequence upstream of the 3' splice site.

Details for Patent 10,446,261

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Jubilant Hollisterstier Llc	N/A	positive skin test control-histamine	Injection	103891	03/13/1924		2028-01-06
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2028-01-06