EP3390635A1 - Oligomères antisens pour le traitement de la sclérose tubéreuse de bourneville - Google Patents
Oligomères antisens pour le traitement de la sclérose tubéreuse de bournevilleInfo
- Publication number
- EP3390635A1 EP3390635A1 EP16876615.2A EP16876615A EP3390635A1 EP 3390635 A1 EP3390635 A1 EP 3390635A1 EP 16876615 A EP16876615 A EP 16876615A EP 3390635 A1 EP3390635 A1 EP 3390635A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- mrna
- nucleobases
- tsc2
- intron
- ric pre
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 208000026911 Tuberous sclerosis complex Diseases 0.000 title claims abstract description 33
- 230000000692 anti-sense effect Effects 0.000 title claims description 138
- 238000011282 treatment Methods 0.000 title description 7
- 102100031638 Tuberin Human genes 0.000 claims abstract description 525
- 101000795659 Homo sapiens Tuberin Proteins 0.000 claims abstract description 439
- 238000000034 method Methods 0.000 claims abstract description 153
- 108050009309 Tuberin Proteins 0.000 claims abstract description 134
- 239000000203 mixture Substances 0.000 claims abstract description 57
- 230000001965 increasing effect Effects 0.000 claims abstract description 54
- 230000002950 deficient Effects 0.000 claims abstract description 51
- 230000014509 gene expression Effects 0.000 claims abstract description 45
- 108020004999 messenger RNA Proteins 0.000 claims description 679
- 230000000717 retained effect Effects 0.000 claims description 502
- 108020005067 RNA Splice Sites Proteins 0.000 claims description 313
- 108090000623 proteins and genes Proteins 0.000 claims description 248
- 102000004169 proteins and genes Human genes 0.000 claims description 203
- 239000002773 nucleotide Substances 0.000 claims description 200
- 125000003729 nucleotide group Chemical group 0.000 claims description 200
- 238000011144 upstream manufacturing Methods 0.000 claims description 114
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 98
- 108700028369 Alleles Proteins 0.000 claims description 62
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 59
- 230000000295 complement effect Effects 0.000 claims description 50
- 235000000346 sugar Nutrition 0.000 claims description 40
- 108091092195 Intron Proteins 0.000 claims description 39
- 201000010099 disease Diseases 0.000 claims description 39
- 239000008194 pharmaceutical composition Substances 0.000 claims description 39
- 108091034117 Oligonucleotide Proteins 0.000 claims description 35
- 230000000694 effects Effects 0.000 claims description 34
- 150000007523 nucleic acids Chemical class 0.000 claims description 32
- 238000012986 modification Methods 0.000 claims description 29
- 230000004048 modification Effects 0.000 claims description 29
- 230000035772 mutation Effects 0.000 claims description 27
- 108020004707 nucleic acids Proteins 0.000 claims description 27
- 102000039446 nucleic acids Human genes 0.000 claims description 27
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 25
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 25
- 108091027974 Mature messenger RNA Proteins 0.000 claims description 24
- 238000004519 manufacturing process Methods 0.000 claims description 22
- 238000002347 injection Methods 0.000 claims description 20
- 239000007924 injection Substances 0.000 claims description 20
- 208000035475 disorder Diseases 0.000 claims description 19
- 241000282414 Homo sapiens Species 0.000 claims description 18
- 230000002829 reductive effect Effects 0.000 claims description 17
- 230000001594 aberrant effect Effects 0.000 claims description 16
- SZCZSKMCTGEJKI-UHFFFAOYSA-N tuberin Natural products COC1=CC=C(C=CNC=O)C=C1 SZCZSKMCTGEJKI-UHFFFAOYSA-N 0.000 claims description 14
- 108091093037 Peptide nucleic acid Proteins 0.000 claims description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 11
- 210000004072 lung Anatomy 0.000 claims description 11
- 238000000185 intracerebroventricular administration Methods 0.000 claims description 10
- 238000010255 intramuscular injection Methods 0.000 claims description 10
- 239000007927 intramuscular injection Substances 0.000 claims description 10
- 239000007928 intraperitoneal injection Substances 0.000 claims description 10
- 238000007913 intrathecal administration Methods 0.000 claims description 10
- 238000010253 intravenous injection Methods 0.000 claims description 10
- 238000010254 subcutaneous injection Methods 0.000 claims description 10
- 239000007929 subcutaneous injection Substances 0.000 claims description 10
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 10
- 230000027455 binding Effects 0.000 claims description 9
- 230000036961 partial effect Effects 0.000 claims description 9
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 8
- 230000002685 pulmonary effect Effects 0.000 claims description 8
- 230000007812 deficiency Effects 0.000 claims description 7
- ANCLJVISBRWUTR-UHFFFAOYSA-N diaminophosphinic acid Chemical compound NP(N)(O)=O ANCLJVISBRWUTR-UHFFFAOYSA-N 0.000 claims description 7
- 230000001939 inductive effect Effects 0.000 claims description 7
- 230000000699 topical effect Effects 0.000 claims description 7
- 210000003754 fetus Anatomy 0.000 claims description 5
- 230000000670 limiting effect Effects 0.000 claims description 3
- 210000001161 mammalian embryo Anatomy 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 2
- 102000002027 Tuberin Human genes 0.000 claims 21
- 210000004027 cell Anatomy 0.000 description 98
- 235000018102 proteins Nutrition 0.000 description 54
- 230000014759 maintenance of location Effects 0.000 description 23
- 230000006870 function Effects 0.000 description 17
- 230000014616 translation Effects 0.000 description 15
- 230000008685 targeting Effects 0.000 description 14
- -1 ethylene nucleic acid Chemical class 0.000 description 12
- 238000003559 RNA-seq method Methods 0.000 description 11
- 210000004940 nucleus Anatomy 0.000 description 11
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 10
- 210000000805 cytoplasm Anatomy 0.000 description 10
- 230000001086 cytosolic effect Effects 0.000 description 10
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 210000003491 skin Anatomy 0.000 description 8
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 7
- 101710175981 Hamartin Proteins 0.000 description 7
- 102000044632 Tuberous Sclerosis Complex 1 Human genes 0.000 description 7
- 230000008499 blood brain barrier function Effects 0.000 description 7
- 210000001218 blood-brain barrier Anatomy 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 125000005647 linker group Chemical group 0.000 description 7
- 239000002502 liposome Substances 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 108700024394 Exon Proteins 0.000 description 6
- 102100034343 Integrase Human genes 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical group C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 150000002632 lipids Chemical group 0.000 description 6
- 108091033319 polynucleotide Proteins 0.000 description 6
- 239000002157 polynucleotide Substances 0.000 description 6
- 102000040430 polynucleotide Human genes 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 210000004556 brain Anatomy 0.000 description 5
- 210000003169 central nervous system Anatomy 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 238000013519 translation Methods 0.000 description 5
- 102100040768 60S ribosomal protein L32 Human genes 0.000 description 4
- 108091035707 Consensus sequence Proteins 0.000 description 4
- 101000672453 Homo sapiens 60S ribosomal protein L32 Proteins 0.000 description 4
- 101000795643 Homo sapiens Hamartin Proteins 0.000 description 4
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 4
- 108020004485 Nonsense Codon Proteins 0.000 description 4
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 description 4
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 230000037433 frameshift Effects 0.000 description 4
- 230000037434 nonsense mutation Effects 0.000 description 4
- 230000035515 penetration Effects 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 229920000768 polyamine Polymers 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000004094 surface-active agent Substances 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 208000026350 Inborn Genetic disease Diseases 0.000 description 3
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical group C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 230000005856 abnormality Effects 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000003416 augmentation Effects 0.000 description 3
- 238000007622 bioinformatic analysis Methods 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 210000003618 cortical neuron Anatomy 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 208000016361 genetic disease Diseases 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 238000010820 immunofluorescence microscopy Methods 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 210000004789 organ system Anatomy 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- OIVLITBTBDPEFK-UHFFFAOYSA-N 5,6-dihydrouracil Chemical compound O=C1CCNC(=O)N1 OIVLITBTBDPEFK-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 208000035657 Abasia Diseases 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 238000012935 Averaging Methods 0.000 description 2
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 2
- 206010010904 Convulsion Diseases 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- NBSCHQHZLSJFNQ-QTVWNMPRSA-N D-Mannose-6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-QTVWNMPRSA-N 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 206010012559 Developmental delay Diseases 0.000 description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 2
- 102100022466 Eukaryotic translation initiation factor 4E-binding protein 1 Human genes 0.000 description 2
- 108050000946 Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 2
- 102100031561 Hamartin Human genes 0.000 description 2
- 238000009015 Human TaqMan MicroRNA Assay kit Methods 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 201000006347 Intellectual Disability Diseases 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 2
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000004952 Polyamide Substances 0.000 description 2
- 239000004721 Polyphenylene oxide Substances 0.000 description 2
- 102000046951 Ras Homolog Enriched in Brain Human genes 0.000 description 2
- 102000009738 Ribosomal Protein S6 Kinases Human genes 0.000 description 2
- 108010034782 Ribosomal Protein S6 Kinases Proteins 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- XVIYCJDWYLJQBG-UHFFFAOYSA-N acetic acid;adamantane Chemical compound CC(O)=O.C1C(C2)CC3CC1CC2C3 XVIYCJDWYLJQBG-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 235000009697 arginine Nutrition 0.000 description 2
- 208000021018 autosomal dominant inheritance Diseases 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000004700 cellular uptake Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229940124447 delivery agent Drugs 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 2
- 229940043264 dodecyl sulfate Drugs 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 231100000221 frame shift mutation induction Toxicity 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000012224 gene deletion Methods 0.000 description 2
- 229960002442 glucosamine Drugs 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 235000004554 glutamine Nutrition 0.000 description 2
- 125000003827 glycol group Chemical group 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000002161 motor neuron Anatomy 0.000 description 2
- 238000007481 next generation sequencing Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 238000012259 partial gene deletion Methods 0.000 description 2
- 239000003961 penetration enhancing agent Substances 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 230000003285 pharmacodynamic effect Effects 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 229920002647 polyamide Polymers 0.000 description 2
- 229920000570 polyether Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 125000002948 undecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- LSPHULWDVZXLIL-UHFFFAOYSA-N (+/-)-Camphoric acid Chemical compound CC1(C)C(C(O)=O)CCC1(C)C(O)=O LSPHULWDVZXLIL-UHFFFAOYSA-N 0.000 description 1
- OAWXYINGQXLWOE-UHFFFAOYSA-N (2-acetyloxybenzoyl) 2-acetyloxybenzoate Chemical compound CC(=O)OC1=CC=CC=C1C(=O)OC(=O)C1=CC=CC=C1OC(C)=O OAWXYINGQXLWOE-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- AYRXSINWFIIFAE-UHFFFAOYSA-N 2,3,4,5-tetrahydroxy-6-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhexanal Chemical compound OCC1OC(OCC(O)C(O)C(O)C(O)C=O)C(O)C(O)C1O AYRXSINWFIIFAE-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- 229940080296 2-naphthalenesulfonate Drugs 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- ZRPLANDPDWYOMZ-UHFFFAOYSA-N 3-cyclopentylpropionic acid Chemical compound OC(=O)CCC1CCCC1 ZRPLANDPDWYOMZ-UHFFFAOYSA-N 0.000 description 1
- XMIIGOLPHOKFCH-UHFFFAOYSA-M 3-phenylpropionate Chemical compound [O-]C(=O)CCC1=CC=CC=C1 XMIIGOLPHOKFCH-UHFFFAOYSA-M 0.000 description 1
- BOPYMSJWZABKAZ-UHFFFAOYSA-N 4-amino-1-ethylpyrimidin-2-one Chemical compound CCN1C=CC(N)=NC1=O BOPYMSJWZABKAZ-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 208000003120 Angiofibroma Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-DCSYEGIMSA-N Beta-Lactose Chemical compound OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-DCSYEGIMSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 206010051290 Central nervous system lesion Diseases 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N D-Maltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- UNXHWFMMPAWVPI-QWWZWVQMSA-N D-Threitol Natural products OC[C@@H](O)[C@H](O)CO UNXHWFMMPAWVPI-QWWZWVQMSA-N 0.000 description 1
- HEBKCHPVOIAQTA-QWWZWVQMSA-N D-arabinitol Chemical compound OC[C@@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-QWWZWVQMSA-N 0.000 description 1
- SHZGCJCMOBCMKK-SVZMEOIVSA-N D-fucopyranose Chemical compound C[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O SHZGCJCMOBCMKK-SVZMEOIVSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- SRBFZHDQGSBBOR-AGQMPKSLSA-N D-lyxopyranose Chemical compound O[C@@H]1COC(O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-AGQMPKSLSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000792859 Enema Species 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- 108050000948 GTP-binding protein Rheb Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 108010042653 IgA receptor Proteins 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- HEBKCHPVOIAQTA-IMJSIDKUSA-N L-arabinitol Chemical compound OC[C@H](O)C(O)[C@@H](O)CO HEBKCHPVOIAQTA-IMJSIDKUSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000012098 Lipofectamine RNAiMAX Substances 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 208000035752 Live birth Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000038030 PI3Ks Human genes 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 1
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 208000034189 Sclerosis Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108010033576 Transferrin Receptors Proteins 0.000 description 1
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- WNLRTRBMVRJNCN-UHFFFAOYSA-L adipate(2-) Chemical compound [O-]C(=O)CCCCC([O-])=O WNLRTRBMVRJNCN-UHFFFAOYSA-L 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- PNNNRSAQSRJVSB-KCDKBNATSA-N aldehydo-L-fucose Chemical compound C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-KCDKBNATSA-N 0.000 description 1
- PYMYPHUHKUWMLA-YUPRTTJUSA-N aldehydo-L-lyxose Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-YUPRTTJUSA-N 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000008055 alkyl aryl sulfonates Chemical class 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- SRBFZHDQGSBBOR-STGXQOJASA-N alpha-D-lyxopyranose Chemical compound O[C@@H]1CO[C@H](O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-STGXQOJASA-N 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 229940072107 ascorbate Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000002869 basic local alignment search tool Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 229940077388 benzenesulfonate Drugs 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-M benzenesulfonate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-M 0.000 description 1
- 229940050390 benzoate Drugs 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- XMIIGOLPHOKFCH-UHFFFAOYSA-N beta-phenylpropanoic acid Natural products OC(=O)CCC1=CC=CC=C1 XMIIGOLPHOKFCH-UHFFFAOYSA-N 0.000 description 1
- 125000002619 bicyclic group Chemical class 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- FATUQANACHZLRT-KMRXSBRUSA-L calcium glucoheptonate Chemical compound [Ca+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)C([O-])=O FATUQANACHZLRT-KMRXSBRUSA-L 0.000 description 1
- MIOPJNTWMNEORI-UHFFFAOYSA-N camphorsulfonic acid Chemical compound C1CC2(CS(O)(=O)=O)C(=O)CC1C2(C)C MIOPJNTWMNEORI-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 208000020291 cardiac rhabdomyoma Diseases 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000017455 cell-cell adhesion Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000012350 deep sequencing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- GPRLSGONYQIRFK-DYCDLGHISA-N deuteron Chemical compound [2H+] GPRLSGONYQIRFK-DYCDLGHISA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- PGUYAANYCROBRT-UHFFFAOYSA-N dihydroxy-selanyl-selanylidene-lambda5-phosphane Chemical compound OP(O)([SeH])=[Se] PGUYAANYCROBRT-UHFFFAOYSA-N 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- POULHZVOKOAJMA-UHFFFAOYSA-M dodecanoate Chemical compound CCCCCCCCCCCC([O-])=O POULHZVOKOAJMA-UHFFFAOYSA-M 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 239000013583 drug formulation Substances 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- 239000007920 enema Substances 0.000 description 1
- 229940079360 enema for constipation Drugs 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 206010016629 fibroma Diseases 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- FBPFZTCFMRRESA-GUCUJZIJSA-N galactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-GUCUJZIJSA-N 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- MNWFXJYAOYHMED-UHFFFAOYSA-N heptanoic acid Chemical compound CCCCCCC(O)=O MNWFXJYAOYHMED-UHFFFAOYSA-N 0.000 description 1
- IPCSVZSSVZVIGE-UHFFFAOYSA-M hexadecanoate Chemical compound CCCCCCCCCCCCCCCC([O-])=O IPCSVZSSVZVIGE-UHFFFAOYSA-M 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000011577 humanized mouse model Methods 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-M hydrogensulfate Chemical compound OS([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-M 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000005414 inactive ingredient Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- SUMDYPCJJOFFON-UHFFFAOYSA-N isethionic acid Chemical compound OCCS(O)(=O)=O SUMDYPCJJOFFON-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 201000007785 kidney angiomyolipoma Diseases 0.000 description 1
- 229940001447 lactate Drugs 0.000 description 1
- 229940099584 lactobionate Drugs 0.000 description 1
- JYTUSYBCFIZPBE-AMTLMPIISA-N lactobionic acid Chemical compound OC(=O)[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O JYTUSYBCFIZPBE-AMTLMPIISA-N 0.000 description 1
- 229940070765 laurate Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229940049920 malate Drugs 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- KVBGVZZKJNLNJU-UHFFFAOYSA-M naphthalene-2-sulfonate Chemical compound C1=CC=CC2=CC(S(=O)(=O)[O-])=CC=C21 KVBGVZZKJNLNJU-UHFFFAOYSA-M 0.000 description 1
- 210000001577 neostriatum Anatomy 0.000 description 1
- 230000003188 neurobehavioral effect Effects 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 230000004942 nuclear accumulation Effects 0.000 description 1
- 210000004492 nuclear pore Anatomy 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- 229940049964 oleate Drugs 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 125000004437 phosphorous atom Chemical group 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229940075930 picrate Drugs 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-M picrate anion Chemical compound [O-]C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-M 0.000 description 1
- 229950010765 pivalate Drugs 0.000 description 1
- IUGYQRQAERSCNH-UHFFFAOYSA-N pivalic acid Chemical compound CC(C)(C)C(O)=O IUGYQRQAERSCNH-UHFFFAOYSA-N 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000024155 regulation of cell adhesion Effects 0.000 description 1
- 230000021014 regulation of cell growth Effects 0.000 description 1
- 230000034563 regulation of cell size Effects 0.000 description 1
- 230000009712 regulation of translation Effects 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- HEBKCHPVOIAQTA-ZXFHETKHSA-N ribitol Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CO HEBKCHPVOIAQTA-ZXFHETKHSA-N 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- JRPHGDYSKGJTKZ-UHFFFAOYSA-K selenophosphate Chemical compound [O-]P([O-])([O-])=[Se] JRPHGDYSKGJTKZ-UHFFFAOYSA-K 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000003584 silencer Effects 0.000 description 1
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 210000003523 substantia nigra Anatomy 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 210000001103 thalamus Anatomy 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 208000009999 tuberous sclerosis Diseases 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- ZDPHROOEEOARMN-UHFFFAOYSA-N undecanoic acid Chemical compound CCCCCCCCCCC(O)=O ZDPHROOEEOARMN-UHFFFAOYSA-N 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical class CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Definitions
- Tuberous sclerosis complex is a disorder characterized by growth of benign tumors in multiple organ systems (Au, K., et al., J. Child Neurol., 2004, 19: 699-709).
- Tumors of the central nervous system (CNS) are the leading cause of morbidity and mortality, followed by renal disease.
- Patients can suffer from abnormalities of the brain that may include seizures, intellectual disability, and developmental delay, as well as abnormalities of the skin, lung, kidneys, and heart.
- the disorder affects as many as 25,000 to 40,000 individuals in the United States and about 1 to 2 million individuals worldwide, with an estimated prevalence of one in 6,000 newborns.
- TSC is a genetic disorder with an autosomal dominant inheritance pattern, caused by inherited defects or de novo mutations that occur on two genes, TSC1 and TSC2. Only one of the genes needs to be affected for TSC to be present.
- the TSC1 gene on chromosome 9, produces a protein called hamartin.
- the TSC2 gene discovered in 1993, is on chromosome 16 and produces the protein tuberin.
- mTOR evolutionarily conserved kinase
- the invention provides compositions and methods for treating tuberous sclerosis complex, including antisense oligomers (ASOs) that promote constitutive splicing at intron splice sites of a TSC 2 retained-intron-containing pre-mRNA (RIC pre-mRNA).
- ASOs antisense oligomers
- RIC pre-mRNA TSC 2 retained-intron-containing pre-mRNA
- the invention further provides compositions and methods for increasing production of mature TSC2 mRNA and, in turn, TSC2 protein, in cells of a subject in need thereof, for example, a subject that can benefit from increased production of TSC2 protein.
- the described methods may be used to treat subjects having tuberous sclerosis complex caused by mutations in the TSC2 gene, including missense, splicing, frameshift and nonsense mutations, as well as whole gene deletions, that result in deficient tuberin protein production.
- a method of increasing expression of a target protein wherein the target protein is tuberin, by cells having a retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes tuberin protein
- the method comprising contacting the cells with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding tuberin protein, whereby the retained intron is constitutively spliced from the RIC pre- mRNA encoding tuberin protein, thereby increasing the level of mRNA encoding tuberin protein, and increasing the expression of tuberin protein in the cells.
- ASO antisense oligomer
- the target protein is tuberin.
- the target protein or the functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces a target protein or functional RNA that is deficient in amount or activity in the subject.
- the cells are in or from a subject having a condition caused by a deficient amount or activity of tuberin protein.
- the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele.
- the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has (a) a first mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild- type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and (b) a second mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild-type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and wherein when the subject has a first mutant allele a.iii., the second mutant allele is b.i.
- the target protein is produced in a form having reduced function compared to the equivalent wild-type protein. In some embodiments, the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.
- the targeted portion of the RIC pre- mRNA is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron. In some embodiments, the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
- the targeted portion of the RIC pre-mRNA is in the retained intron within: (a) the region +6 to +500, +6 to +495, or +6 to +100 relative to the 5' splice site of the retained intron; or (b) the region -16 to -500, -16 to -400, or -16 to -100 relative to the 3' splice site of the retained intron.
- the targeted portion of the RIC pre-mRNA is within: (a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.
- the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.
- the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 2-8.
- the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating alternative splicing of pre-mRNA transcribed from a gene encoding the functional RNA or target protein.
- the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or the functional RNA.
- the RIC pre-mRNA was produced by partial splicing of a full-length pre- mRNA or partial splicing of a wild-type pre-mRNA.
- the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
- the target protein produced is full-length protein, or wild- type protein.
- the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10- fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about
- the total amount of target protein produced by the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5- fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3 -fold, at least about 3.5-fold, at least about 4-fold, at least about 5-
- the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35
- nucleobases 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
- nucleobases 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
- the antisense oligomer is at least 80%, at least 85%>, at least 90%, at least 95%, at least 98%), at least 99%, or 100%>, complementary to the targeted portion of the RIC pre-mRNA encoding the protein.
- the targeted portion of the RIC pre-mRNA is within a sequence selected from SEQ ID NOs: 5097-5105.
- the antisense oligomer comprises a nucleotide sequence that is at least about 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-5096.
- the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.
- the cell comprises a population of RIC pre- mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the most abundant retained intron in the population of RIC pre- mRNAs.
- the binding of the antisense oligomer to the most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
- the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNAs.
- the binding of the antisense oligomer to the second most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
- the condition is a disease or disorder.
- the disease or disorder is tuberous sclerosis complex.
- the target protein and the RIC pre-mRNA are encoded by the TSC2 gene.
- the method further comprises assessing TSC2 protein expression.
- the antisense oligomer binds to a targeted portion of a tuberin RIC pre- mRNA, wherein the targeted portion is within a sequence selected from SEQ ID NOS: 49, 50, 51, 52, 53, 54, 55, 56, and 57.
- the subject is a human.
- the subject is a non-human animal. In some embodiments, the subject is a fetus, an embryo, or a child. In some embodiments, the cells are ex vivo. In some embodiments, the antisense oligomer is administered by topical application to the skin, pulmonary delivery to the lung, intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the 9 nucleotides at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron, are identical to the corresponding wild-type sequence. In some
- the 16 nucleotides at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site are identical to the corresponding wild-type sequence.
- an antisense oligomer as used in a method described above.
- an antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 9-5096.
- a pharmaceutical composition comprising the antisense oligomer described above, and an excipient.
- a method of treating a subject in need thereof by administering the pharmaceutical composition by topical application to the skin, pulmonary delivery to the lung, intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
- compositions comprising an antisense oligomer for use in a method of increasing expression of a target protein or a functional RNA by cells to treat tuberous sclerosis complex in a subject in need thereof, associated with a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron-containing pre-mRNA (RIC pre-mRNA) encoding the target protein or the functional RNA, wherein the target protein is: (a) the deficient protein; or (b) a compensating protein which functionally augments or replaces the deficient protein or in the subject; and wherein the functional RNA is: (a) the deficient RNA; or (b) a compensating functional RNA which functionally augments or replaces the deficient functional RNA in the subject; wherein the RIC pre-mRNA comprises a retained in
- compositions comprising an antisense oligomer for use in a method of treating a condition associated with tuberin protein in a subject in need thereof, the method comprising the step of increasing expression of tuberin protein by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes the tuberin protein, the method comprising contacting the cells with the antisense oligomer, whereby the retained intron is constitutively spliced from the RIC pre-mRNA transcripts encoding tuberin protein, thereby increasing the level of mRNA encoding the tuberin protein, and increasing the expression of tuberin protein, in the cells of the subject.
- RIC pre-mRNA retained-intron-containing pre-mRNA
- the condition is a disease or disorder.
- the disease or disorder is tuberous sclerosis complex.
- the target protein and RIC pre-mRNA are encoded by the TSC2 gene.
- the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron.
- the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within: (a) the region +6 to +100 relative to the 5' splice site of the retained intron; or (b) the region -16 to -100 relative to the 3' splice site of the retained intron. In some embodiments, the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 500, 400, 300, 200, or 100 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 100, 200, 300, 400, or 500 nucleotides upstream of the 3' splice site of the at least one retained intron.
- the targeted portion of the RIC pre-mRNA is within: (a) the region +2e to - 4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the ex on flanking the 3' splice site of the retained intron.
- the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. sequence identity to SEQ ID NO: 1.
- the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 2-8.
- the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the pre-mRNA transcribed from a gene encoding the target protein or functional RNA.
- the antisense oligomer does not increase the amount of the functional RNA or functional protein by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or functional RNA.
- the RIC pre- mRNA was produced by partial splicing from a full-length pre-mRNA or a wild-type pre- mRNA.
- the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
- the target protein produced is full-length protein, or wild-type protein.
- the retained intron is a rate-limiting intron. In some embodiments, said retained intron is the most abundant retained intron in said RIC pre-mRNA. In some embodiments, the retained intron is the second most abundant retained intron in said RIC pre-mRNA.
- the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, said antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0- methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety.
- the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
- nucleobases 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
- the antisense oligomer is at least 80%, at least 85%, at least 90%), at least 95%, at least 98%>, at least 99%, or is 100% complementary to the targeted portion of the RIC pre-mRNA encoding the protein. In some embodiments, the antisense oligomer binds to a targeted portion of a tuberin RIC pre-mRNA, wherein the targeted portion is within a sequence selected from SEQ ID NOs: 5097-5105.
- the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-5096. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.
- a pharmaceutical composition comprising the antisense oligomer described above, and an excipient.
- a method of treating a subject in need thereof by administering the pharmaceutical composition by topical application to the skin, pulmonary delivery to the lung, intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
- a pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a deficient TSC2 mRNA transcript, wherein the deficient TSC2 mRNA transcript comprises a retained intron, wherein the antisense oligomer induces splicing out of the retained intron from the deficient TSC2 mRNA transcript; and a pharmaceutical acceptable excipient.
- the deficient TSC2 mRNA transcript is a TSC2 RIC pre-mRNA transcript.
- the targeted portion of the TSC2 RIC pre-mRNA transcript is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' spliced site of the retained intron.
- the TSC2 RIC pre-mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.
- the TSC2 RIC pre-mRNA transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 2-8.
- the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.
- the antisense oligomer comprises from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
- nucleobases 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
- the antisense oligomer is at least 80%>, at least 85%>, at least 90%), at least 95%, at least 98%>, at least 99%, or is 100%> complementary to a targeted portion of the TSC2 RIC pre-mRNA transcript.
- the targeted portion of the TSC2 RIC pre-mRNA transcript is within a sequence selected from SEQ ID NOs: 5097-5105.
- the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-5096.
- the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.
- the second 85%>, 90% at least 95%, at least 98%>, at least 99%, or is 100%> complementary to a targeted portion of the TSC2 RIC pre-mRNA transcript.
- composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
- a method of inducing processing of a deficient TSC2 mRNA transcript to facilitate removal of a retained intron to produce a fully processed TSC2 mRNA transcript that encodes a functional form of a tuberin protein comprising: (a) contacting an antisense oligomer to a target cell of a subject; (b) hybridizing the antisense oligomer to the deficient TSC2 mRNA transcript, wherein the deficient TSC2 mRNA transcript is capable of encoding the functional form of tuberin protein and comprises at least one retained intron; (c) removing the at least one retained intron from the deficient TSC2 mRNA transcript to produce the fully processed TSC2 mRNA transcript that encodes the functional form of tuberin protein; and (d) translating the functional form of tuberin protein from the fully processed TSC2 mRNA transcript.
- the retained intron is an entire retained intron.
- the deficient TSC2 mRNA transcript is an entire retained intron.
- a method of treating a subject having a condition caused by a deficient amount or activity of tuberin protein comprising: administering to the subject an antisense oligomer comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-5096.
- FIG. 1 shows a schematic representation of an exemplary retained-intron-containing (RIC) pre-mRNA transcript.
- the 5' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -3e to -le and +1 to +6 (numbers labeled "e” are exonic and unlabeled numbers are intronic).
- the 3' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -15 to -1 and +le (numbers labeled "e” are exonic and unlabeled numbers are intronic).
- Intronic target regions for ASO screening comprise nucleotides +6 relative to the 5' splice site of the retained intron (arrow at left) to -16 relative to the 3' splice site of the retained intron (arrow at right).
- intronic target regions for ASO screening comprise nucleotides +6 to +100 relative to the 5' splice site of the retained intron and -16 to -100 relative to the 3' splice site of the retained intron.
- Exonic target regions comprise nucleotides +2e to -4e in the ex on flanking the 5' splice site of the retained intron and +2e to -4e in the ex on flanking the 3' splice site of the retained intron.
- n or N denote any nucleotide
- y denotes pyrimidine.
- the sequences shown represent consensus sequences for mammalian splice sites and individual introns and exons need not match the consensus sequences at every position.
- FIG. 2A-FIG. 2B illustrate schematic representations of the Targeted Augmentation of Nuclear Gene Output (TANGO) approach.
- FIG. 2A shows a cell divided into nuclear and cytoplasmic compartments.
- a pre-mRNA transcript of a target gene consisting of exons (rectangles) and introns (connecting lines) undergoes splicing to generate an mRNA, and this mRNA is exported to the cytoplasm and translated into target protein.
- the splicing of intron 1 is inefficient and a retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus, and if exported to the cytoplasm, is degraded, leading to no target protein production.
- FIG. 2B shows an example of the same cell divided into nuclear and cytoplasmic compartments.
- Treatment with an antisense oligomer (ASO) promotes the splicing of intron 1 and results in an increase in mRNA, which is in turn translated into higher levels of target protein
- FIG. 3 shows intron-retention in the TSC2 gene with intron 4 detail.
- the identification of intron-retention events in the TSC2 gene using RNA sequencing (RNAseq) is shown, visualized in the UCSC genome browser.
- the upper panel shows the read density
- the TSC2 transcript expressed in HCN human cortical neurons
- HCN human cortical neurons
- nuclear fraction nuclear fraction
- a graphic representation of the TSC2 gene is shown to scale.
- the read density is shown as peaks.
- the highest read density corresponds to exons (black boxes), while no reads are observed for the majority of the introns (lines with arrow heads) in either cellular fraction.
- Higher read density is detected for introns 4, 25/26, and 31/32 (indicated by the arrows) in the nuclear fraction compared to the cytoplasmic fraction indicating that splicing efficiency of introns 4, 25/26, and 31/32 is low, resulting in intron retention.
- the retained-intron containing pre-mRNA transcripts are retained in the nucleus and are not exported out to the cytoplasm.
- the read density for intron 4 in HCN is shown in detail in the lower panel.
- FIG. 4 shows TSC2 gene IVS 4 ASO walk.
- FIG. 5 shows intron-retention in the TSC2 gene with introns 25 and 26 detail.
- Intron retention in the TSC2 gene was identified by RNA sequencing (RNAseq), visualized in the UCSC genome browser, as described herein in the Examples.
- RNAseq RNA sequencing
- the read density for introns 25 and 26 in HCN is shown in detail in the lower panel.
- Introns 25 and 26 flank exon 26, an alternatively spliced exon.
- FIG. 6 shows TSC2 gene IVS 25 and 26 ASO walk.
- the splice site intronic regions flanking alternative exon 26 are not targeted to avoid affecting the inclusion level of exon 26.
- ASOs were designed to cover these regions by shifting 5 nucleotides at a time.
- the TSC2 exon-intron structure is drawn to scale.
- FIG. 7 shows intron-retention in the TSC2 gene with introns 31 and 32 detail.
- Intron retention in the TSC2 gene was identified by RNA sequencing (RNAseq), visualized in the UCSC genome browser, as described herein in the Examples.
- the read density for introns 31 and 32 is shown in detail in the lower panel. Introns 31 and 32 flank exon 32, an alternatively spliced exon. This is evidenced in the graphic representations of the TSC2 transcripts and the RNAseq data, such that the rectangle depicting exon 32 is present in some transcripts while absent in others, and the read density corresponding to exon 32 in the cytoplasmic fraction is significantly lower than that of the constitutively spliced exons in TSC2.
- the read density for intron 31 is shown in detail in the lower panel indicating 43% intron retention as calculated by bioinformatic analysis.
- FIG. 8 shows TSC2 gene IVS 31 and 32 ASO walk.
- the splice site intronic regions flanking alternative exon 32 are not targeted to avoid affecting the inclusion level of exon 32.
- ASOs were designed to cover these regions by shifting 5 nucleotides at a time with the exception of TSC2-IVS32-33 and TSC2- IVS32-51.
- the TSC2 exon-intron structure is drawn to scale.
- FIG. 9 depicts a schematic of the ReSeq Genes for TSC2 intron 4 corresponding to NM 000548.
- the Percent Intron Retention (PIR) of the circled intron is shown.
- FIG. 11 depicts a schematic of the ReSeq Genes for TSC2 intron 25 corresponding to NM 000548.
- the Percent Intron Retention (PIR) of the circled intron is shown.
- FIG. 13 depicts a schematic of the ReSeq Genes for TSC2 intron 26 corresponding to NM 000548.
- the Percent Intron Retention (PIR) of the circled intron is shown.
- FIG. 14 depicts a schematic of the ReSeq Genes for TSC2 intron 31 corresponding to NM 000548.
- the Percent Intron Retention (PIR) of the circled intron is shown.
- FIG. 16 depicts a schematic of the ReSeq Genes for TSC2 intron 32 corresponding to NM 000548.
- the Percent Intron Retention (PIR) of the circled intron is shown.
- introns in primary transcripts of protein-coding genes having more than one intron are spliced from the primary transcript with different efficiencies. In most cases only the fully spliced mRNA is exported through nuclear pores for subsequent translation in the cytoplasm. Unspliced and partially spliced transcripts are detectable in the nucleus. It is generally thought that nuclear accumulation of transcripts that are not fully spliced is a mechanism to prevent the accumulation of potentially deleterious mRNAs in the cytoplasm that may be translated to protein. For some genes, splicing of the least efficient intron is a rate- limiting post-transcriptional step in gene expression, prior to translation in the cytoplasm.
- the present invention provides compositions and methods for upregulating splicing of one or more retained TSC2 introns that are rate-limiting for the nuclear stages of gene expression to increase steady-state production of fully-spliced, mature mRNA, and thus, translated tuberin protein levels.
- These compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at an intron splice sites of a retained-intron-containing TSC2 pre- mRNA that accumulates in the nucleus.
- ASOs antisense oligomers
- TSC2 protein is increased using the methods of the invention to treat a condition caused by TSC2 deficiency.
- the methods of the invention are used to increase TSC2 production to treat a condition in a subject in need thereof.
- the subject has condition in which TSC2 is not necessarily deficient relative to wild-type, but where an increase in TSC2 mitigates the condition nonetheless.
- the condition is a caused by a TSC2 haploinsufficiency.
- the condition is an autosomal dominant disorder.
- the condition is an autosomal recessive disorder.
- Tuberous sclerosis complex is a disease characterized by tumor growth in multiple organ systems (Au et al., J. Child Neurol. 2004, 19, 699-709). Tumors are usually benign but are occasionally malignant. Approximately 90% of tuberous sclerosis complex cases display cortical tuber; facial angiofibroma and renal angiomyolipoma occur in more than 80% of cases. In addition, approximately 80% of cases display subependymal nodule, approximately 50% of cases display cardiac rhabdomyoma, and 51% to approximately 88% of cases display ungual/ subungual fibroma. Tumors of the central nervous system (CNS) are the leading cause of morbidity and mortality, followed by renal disease (Au et al., J. Child Neurol. 2004, 19, 699- 709).
- CNS central nervous system
- Tuberous sclerosis complex is a genetic disorder with an autosomal dominant inheritance pattern and a high mutation rate (Au et al., J. Child Neurol. 2004, 19, 699-709).
- Linkage of tuberous sclerosis complex to chromosomal regions 9q34.3 and 16pl3.3 led to the identification of the TSC1 and TSC2 genes, respectively.
- More than 69% of tuberous sclerosis complex cases result from haploinsufficency of TSC2, and disease in approximately two thirds of patients results from de novo mutation or deletion (Au et al., J. Child Neurol. 2004, 19, 699-709).
- Severe disease is thought to require a "second hit" reduction of the other allele.
- the prevalence of tuberous sclerosis complex is as high as one in 5,800 live births, or approximately 300 births per year in the United States, with 200 cases per year resulting from mutation of TSC2.
- the disease is described, e.g., by OMTM #613254 (Online Mendelian Inheritance in Man, Johns Hopkins University, 1966-2015), incorporated by reference herein.
- the TSC2 gene codes for a protein named tuberin.
- the TSC2 gene contains 41 exons, two of which are alternatively spliced, and spans approximately 40 kb on chromosome 16 (Au et al., J. Child Neurol. 2004, 19, 699-709).
- Tuberin is a 200 kDa protein that forms a complex with the TSC1 gene product hamartin.
- the tuberin-hamartin complex plays a role in the regulation of cell growth, translation, cell size, and cell adhesion and migration through a variety of signal cascades.
- the tuberin-hamartin complex functions in the phosphatidyl-inositol-3- kinase and protein B pathway (PI3K/PKB) that regulates translation.
- PI3K/PKB phosphatidyl-inositol-3- kinase and protein B pathway
- the tuberin- hamartin complex suppresses cell growth and translation by suppressing mTOR kinase activity, resulting in suppression of the p70 ribosomal protein S6 kinase (S6K) 1 and activation of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1).
- S6K ribosomal protein S6 kinase
- 4E-BP1 eukaryotic translation initiation factor 4E binding protein 1
- the GAP domain of tuberin hydrolyzes guanosine triphosphate (GTP) bound to the small G protein Rheb, a Ras homolog enriched in brain, thus preventing mTOR activation.
- tuberin and hamartin regulate cell growth and cell adhesion through the MAP kinase (MAPK) and E-cadherin-beta-catenin pathways, respectively.
- MAPK MAP kinase
- E-cadherin-beta-catenin pathways respectively.
- Mutations include frameshift and protein truncations, nonsense mutations, mutations that affect splicing, in-frame mutations, deletions, insertions, duplications, and large deletions and rearrangement. Mutations of TSC2 that yield truncated protein products fail to form the tuberin- hamartin complex, thus resulting in loss of cell growth regulation.
- the role of the tuberin- hamartin complex as a key regulator of multiple signaling pathways is consistent with the wide spectrum of clinical findings among patients with tuberous sclerosis complex that involve multiple organ systems, including neurological and neurobehavioral abnormalities such as seizures, intellectual disability, and developmental delay (Au et al., J. Child Neurol. 2004, 19, 699-709).
- RIC Pre -mRNA Retained Intron Containing Pre-mRNA
- the methods of the present invention exploit the presence of retained- intron-containing pre-mRNA (RIC pre-mRNA) transcribed from the TSC2 gene and encoding tuberin protein, in the cell nucleus.
- Splicing of the identified TSC2 RIC pre-mRNA species to produce mature, fully-spliced, TSC2 mRNA is induced using ASOs that stimulate splicing out of the retained introns.
- the resulting mature TSC2 mRNA can be exported to the cytoplasm and translated, thereby increasing the amount of tuberin protein in the patient's cells and alleviating symptoms of tuberous sclerosis complex.
- This method described further below, is known as Targeted Augmentation of Nuclear Gene Output (TANGO).
- RNA sequencing visualized in the UCSC genome browser, showed TSC2 transcripts expressed in HCN (human cortical neurons) cells and localized in either the cytoplasmic or nuclear fraction. In both fractions, reads were not observed for the majority of the introns. However, higher read density was detected for introns 4, 25, 26, 31, and 32 in the nuclear fraction compared to the cytoplasmic fraction indicating that splicing efficiency of introns 4, 25, 26, 31, and 32 is low, resulting in intron retention.
- the retained-intron containing pre-mRNA transcripts accumulate primarily in the nucleus and not translated into the tuberin protein.
- the read density for intron 26 in AST is shown in detail in the lower panel indicating 51% intron retention as calculated by bioinformatic analysis.
- the percent intron retention (PIR) value for intron 26 was obtained by averaging four values (87, 83, 20, and 12), each determined in renal epithelial cells using one of four different algorithms.
- the read density for intron 31 in AST is shown in detail in the lower panel indicating 43% intron retention.
- the percent intron retention (PIR) value for intron 31 was obtained by averaging four values (78, 71, 16, and 8), each determined in renal epithelial cells using one of four different algorithms.
- Introns 4 and 32 were not mapped.
- Analysis of the ENCODE data (described by, e.g., Tilgner, et al., 2012, "Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for IncRNAs," Genome Research 22(9): 1616-25) to identify intron retention events did not identify intron 25, 26, or 31 as retained, and did not map introns 4 and 32.
- Table 1 provides a non-limiting list of target sequences of a TSC2 RIC pre-mRNA transcript by sequence ID, and ASOs by sequence ID, useful for increasing production of tuberin protein by targeting a region of a TSC2 RIC pre-mRNA.
- ASOs useful for these purposes are identified, using, e.g., methods described herein.
- TSC2 NM 001318831 SEQ ID NOs: 3346-
- TSC2 NM 001318827 SEQ ID NOs: 4646-
- the ASOs disclosed herein target a RIC pre-mRNA transcribed from a TSC2 genomic sequence. In some embodiments, the ASO targets a RIC pre-mRNA transcript from a TSC2 genomic sequence comprising a retained intron. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 1. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 1 comprising a retained intron. In some embodiments, the ASOs disclosed herein target a TSC2 RIC pre-mRNA sequence.
- the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 3, 24, 29 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM_001318829. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 4, 25, 26, 31, 32 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM 000548. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 4, 25, 30 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM_001077183.
- the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 4, 25, 26, 31, or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 22, 27, 28 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM_001318831. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 4, 25, 30 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM 001318832. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 3, 24, 29 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM_001318827.
- the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 2. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 2 comprising a retained intron 24, a retained intron 3, a retained intron 29, or a combination thereof. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 3. In some embodiments, the ASO targets a TSC2 RIC pre- mRNA sequence according to SEQ ID NO: 3 comprising a retained intron 32, a retained intron 25, a retained intron 26, a retained intron 4, a retained intron 31 or a combination thereof.
- the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 4. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 4 comprising a retained intron 25, a retained intron 4, a retained intron 30 or a combination thereof. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 5. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 5 comprising a retained intron 25, a retained intron 26, a retained intron 4, a retained intron 31 or a combination thereof.
- the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 6. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 6 comprising a retained intron 27, a retained intron 28, a retained intron 22 or a combination thereof. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 7. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 7 comprising a retained intron 25, a retained intron 4, a retained intron 30 or a combination thereof.
- the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 8 . In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 8 comprising a retained intron 24, a retained intron 3, a retained intron 29 or a combination thereof. In some embodiments, the ASOs disclosed herein target SEQ ID NOs: 5097-5105. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 9-5096.
- the ASO targets exon 24 or exon 25 of a TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the intron numbering correspond to the mRNA sequence at M 001318829.
- the ASO targets an exon 24 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
- the ASO targets an exon 24 sequence about 2 to about 75 or about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
- the ASO targets an exon 25 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24. In some embodiments, the ASO targets an exon 25 sequence about 2 to about 150 or about 2 to about 147 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre- mRNA comprising the retained intron 24.
- the ASO targets intron 24 in a TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the intron numbering correspond to the mRNA sequence at NM 001318829.
- the ASO targets an intron 24 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
- the ASO targets an intron 24 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
- the ASO targets an intron 24 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence about 16 to about 496 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
- the ASO targets exon 3 or exon 4 of a TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the intron numbering correspond to the mRNA sequence at NM 001318829.
- the ASO targets an exon 3 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
- the ASO targets an exon 3 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
- the ASO targets an exon 4 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments, the ASO targets an exon 4 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
- the ASO targets intron 3 in a TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the intron numbering correspond to the mRNA sequence at
- the ASO targets an intron 3 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments, the ASO targets an intron 3 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments, the ASO targets an intron 3 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments,
- the ASO targets an intron 3 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
- the ASO targets exon 29 or exon 30 of a TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the intron numbering correspond to the mRNA sequence at NM 001318829.
- the ASO targets an exon 29 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets an exon 29 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets an exon 30 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29. In some embodiments, the ASO targets an exon 30 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets intron 29 in a TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the intron numbering correspond to the mRNA sequence at NM 001318829.
- the ASO targets an intron 29 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets an intron 29 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets an intron 29 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29. In some embodiments, the ASO targets an intron 29 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets exon 32 or exon 33 of a TSC2 RIC pre-mRNA comprising a retained intron 32, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
- the ASO targets an exon 32 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
- the ASO targets an exon 32 sequence about 4 to about 49 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
- the ASO targets an exon 33 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32. In some embodiments, the ASO targets an exon 33 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
- the ASO targets intron 32 in a TSC2 RIC pre-mRNA comprising a retained intron 32, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
- the ASO targets an intron 32 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
- the ASO targets an intron 32 sequence about 6 to about 495 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
- the ASO targets an intron 32 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32. In some embodiments, the ASO targets an intron 32 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
- the ASO targets exon 25 or exon 26 of a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
- the ASO targets an exon 25 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an exon 26 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 112 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets intron 25 in a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
- the ASO targets an intron 25 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an intron 25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 16 to about 498 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets exon 26 or exon 27 of a TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
- the ASO targets an exon 26 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets an exon 26 sequence about 4 to about 111 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets an exon 27 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26. In some embodiments, the ASO targets an exon 27 sequence about 2 to about 147 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets intron 26 in a TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the intron numbering correspond to the mRNA sequence at NM 000548.
- the ASO targets an intron 26 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets an intron 26 sequence about 6 to about 500 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets an intron 26 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26. In some embodiments, the ASO targets an intron 26 sequence about 16 to about 496 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets exon 4 or exon 5 of a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
- the ASO targets an exon 4 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an exon 5 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets intron 4 in a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at M 000548.
- the ASO targets an intron 4 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an intron 4 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an intron 4 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an intron 4 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets exon 31 or exon 32 of a TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
- the ASO targets an exon 31 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets an exon 31 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets an exon 32 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31. In some embodiments, the ASO targets an exon 32 sequence about 2 to about 52 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets intron 31 in a TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the intron numbering correspond to the mRNA sequence at NM 000548.
- the ASO targets an intron 31 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets an intron 31 sequence about 6 to about 331 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets an intron 31 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31. In some embodiments, the ASO targets an intron 31 sequence about 16 to about 333 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets exon 25 or exon 26 of a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM_001077183.
- the ASO targets an exon 25 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an ex on 26 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an ex on 26 sequence about 2 to about 142 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets intron 25 in a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM OO 1077183.
- the ASO targets an intron 25 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an intron 25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 16 to about 499 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets exon 4 or exon 5 of a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at NM_001077183.
- the ASO targets an exon 4 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an exon 5 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets intron 4 in a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at
- the ASO targets an intron 4 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some
- the ASO targets an intron 4 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets exon 30 or exon 31 of a TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the intron numbering correspond to the mRNA sequence at NM_001077183.
- the ASO targets an exon 30 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets an exon 30 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30. In some embodiments, the ASO targets an exon 31 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30. In some embodiments, the ASO targets an exon 31 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets intron 30 in a TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the intron numbering correspond to the mRNA sequence at NM OO 1077183.
- the ASO targets an intron 30 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets an intron 30 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets an intron 30 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30. In some embodiments, the ASO targets an intron 30 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets exon 25 or exon 26 of a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM_001114382.
- the ASO targets an exon 25 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an exon 26 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 112 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets intron 25 in a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
- the ASO targets an intron 25 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an intron 25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 16 to about 498 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets exon 26 or exon 27 of a TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
- the ASO targets an exon 26 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets an exon 26 sequence about 4 to about 111 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets an exon 27 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26. In some embodiments, the ASO targets an exon 27 sequence about 2 to about 147 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets intron 26 in a TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
- the ASO targets an intron 26 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets an intron 26 sequence about 6 to about 500 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets an intron 26 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26. In some embodiments, the ASO targets an intron 26 sequence about 16 to about 496 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
- the ASO targets exon 4 or exon 5 of a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
- the ASO targets an exon 4 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an exon 5 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets intron 4 in a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at
- the ASO targets an intron 4 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some
- the ASO targets an intron 4 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets exon 31 or exon 32 of a TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
- the ASO targets an exon 31 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets an exon 31 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets an exon 32 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31. In some embodiments, the ASO targets an exon 32 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets intron 31 in a TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
- the ASO targets an intron 31 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets an intron 31 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets an intron 31 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31. In some embodiments, the ASO targets an intron 31 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
- the ASO targets exon 27 or exon 28 of a TSC2 RIC pre-mRNA comprising a retained intron 27, wherein the intron numbering correspond to the mRNA sequence at NM 001318831.
- the ASO targets an exon 27 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron
- the ASO targets an exon 27 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 27. In some embodiments, the ASO targets an exon 28 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 27. In some embodiments, the ASO targets an exon 28 sequence about 2 to about 52 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 27.
- the ASO targets intron 27 in a TSC2 RIC pre-mRNA comprising a retained intron 27, wherein the intron numbering correspond to the mRNA sequence at
- the ASO targets an intron 27 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 27. In some embodiments, the ASO targets an intron 27 sequence about 6 to about 331 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 27. In some embodiments, the ASO targets an intron 27 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 27. In some embodiments, the ASO targets an intron 27 sequence about 16 to about 333 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 27.
- the ASO targets exon 28 or exon 29 of a TSC2 RIC pre-mRNA comprising a retained intron 28, wherein the intron numbering correspond to the mRNA sequence at NM 001318831.
- the ASO targets an exon 28 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron
- the ASO targets an exon 28 sequence about 4 to about 49 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28. In some embodiments, the ASO targets an exon 29 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28. In some embodiments, the ASO targets an exon 29 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28.
- the ASO targets intron 28 in a TSC2 RIC pre-mRNA comprising a retained intron 28, wherein the intron numbering correspond to the mRNA sequence at M 001318831.
- the ASO targets an intron 28 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28.
- the ASO targets an intron 28 sequence about 6 to about 495 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28.
- the ASO targets an intron 28 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28. In some embodiments, the ASO targets an intron 28 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28.
- the ASO targets exon 22 or exon 23 of a TSC2 RIC pre-mRNA comprising a retained intron 22, wherein the intron numbering correspond to the mRNA sequence at NM 001318831.
- the ASO targets an exon 22 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
- the ASO targets an exon 22 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
- the ASO targets an exon 23 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22. In some embodiments, the ASO targets an exon 23 sequence about 2 to about 142 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
- the ASO targets intron 22 in a TSC2 RIC pre-mRNA comprising a retained intron 22, wherein the intron numbering correspond to the mRNA sequence at NM 001318831.
- the ASO targets an intron 22 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
- the ASO targets an intron 22 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
- the ASO targets an intron 22 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22. In some embodiments, the ASO targets an intron 22 sequence about 16 to about 499 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
- the ASO targets exon 25 or exon 26 of a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
- the ASO targets an exon 25 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an ex on 26 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 142 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets intron 25 in a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
- the ASO targets an intron 25 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets an intron 25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 16 to about 499 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
- the ASO targets exon 4 or exon 5 of a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
- the ASO targets an exon 4 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets an exon 5 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets intron 4 in a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at
- the ASO targets an intron 4 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
- the ASO targets exon 30 or exon 31 of a TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
- the ASO targets an exon 30 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets an exon 30 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets an exon 31 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30. In some embodiments, the ASO targets an exon 31 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets intron 30 in a TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
- the ASO targets an intron 30 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets an intron 30 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets an intron 30 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30. In some embodiments, the ASO targets an intron 30 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
- the ASO targets exon 24 or exon 25 of a TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the intron numbering correspond to the mRNA sequence at NM_001318827.
- the ASO targets an exon 24 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
- the ASO targets an exon 24 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
- the ASO targets an exon 25 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24. In some embodiments, the ASO targets an exon 25 sequence about 2 to about 147 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24. [0088] In some embodiments, the ASO targets intron 24 in a TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the intron numbering correspond to the mRNA sequence at NM 001318827.
- the ASO targets an intron 24 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence about 16 to about 496 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
- the ASO targets exon 3 or exon 4 of a TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the intron numbering correspond to the mRNA sequence at NM 001318827.
- the ASO targets an exon 3 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
- the ASO targets an exon 3 sequence about 4 to about 69 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
- the ASO targets an exon 4 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments, the ASO targets an exon 4 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
- the ASO targets intron 3 in a TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the intron numbering correspond to the mRNA sequence at
- the ASO targets an intron 3 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments, the ASO targets an intron 3 sequence about 6 to about 499 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments, the ASO targets an intron 3 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments,
- the ASO targets an intron 3 sequence about 16 to about 497 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
- the ASO targets exon 29 or exon 30 of a TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the intron numbering correspond to the mRNA sequence at NM 001318827.
- the ASO targets an exon 29 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets an ex on 29 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets an exon 30 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29. In some embodiments, the ASO targets an exon 30 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets intron 29 in a TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the intron numbering correspond to the mRNA sequence at NM 001318827.
- the ASO targets an intron 29 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets an intron 29 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the ASO targets an intron 29 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29. In some embodiments, the ASO targets an intron 29 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
- the degree of intron retention can be represented using the metric percent intron retention (PIR), the percentage of transcripts in which a given intron is retained.
- PIR metric percent intron retention
- PIR can be calculated as the percentage of the average number of reads mapping to the exon-intron junctions, over the sum of the average of the exon-intron junction reads plus the exon-exon junction reads.
- the methods described herein are used to increase the production of a functional tuberin protein.
- the term “functional” refers to the amount of activity or function of a tuberin protein that is necessary to eliminate any one or more symptoms of tuberous sclerosis complex.
- the methods are used to increase the production of a partially functional tuberin protein.
- the term “partially functional” refers to any amount of activity or function of the tuberin protein that is less than the amount of activity or function that is necessary to eliminate or prevent any one or more symptoms of a disease or condition.
- a partially functional protein or RNA will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%), 85%, at least 90%, or at least 95% less activity relative to the fully functional protein or RNA.
- the method is a method of increasing the expression of the tuberin protein by cells of a subject having a RIC pre-mRNA encoding the tuberin protein, wherein the subject has tuberous sclerosis complex caused by a deficient amount of activity of tuberin protein, and wherein the deficient amount of the tuberin protein is caused by haploinsufficiency of the tuberin protein.
- the subject has a first allele encoding a functional tuberin protein, and a second allele from which the tuberin protein is not produced.
- the subject has a first allele encoding a functional tuberin protein, and a second allele encoding a nonfunctional tuberin protein.
- the subject has a first allele encoding a functional tuberin protein, and a second allele encoding a partially functional tuberin protein.
- the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional tuberin protein), thereby inducing constitutive splicing of the retained intron from the RIC pre- mRNA, and causing an increase in the level of mature mRNA encoding functional tuberin protein, and an increase in the expression of the tuberin protein in the cells of the subject.
- the subject has a first allele encoding a functional tuberin protein, and a second allele encoding a partially functional tuberin protein, and the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional tuberin protein) or a targeted portion of the RIC pre-mRNA transcribed from the second allele (encoding partially functional tuberin protein), thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mature mRNA encoding the tuberin protein, and an increase in the expression of functional or partially functional tuberin protein in the cells of the subject.
- the method is a method of using an ASO to increase the expression of a protein or functional RNA.
- an ASO is used to increase the expression of tuberin protein in cells of a subject having a RIC pre-mRNA encoding tuberin protein, wherein the subject has a deficiency, e.g., tuberous sclerosis complex, in the amount or function of a tuberin protein.
- the RIC pre-mRNA transcript that encodes the protein that is causative of the disease or condition is targeted by the ASOs described herein.
- a RIC pre-mRNA transcript that encodes a protein that is not causative of the disease is targeted by the ASOs.
- a disease or condition that is the result of a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting a RIC pre-mRNA that encodes a second protein, thereby increasing production of the second protein.
- the function of the second protein is able to compensate for the mutation or deficiency of the first protein (which is causative of the disease or condition).
- the subject has:
- the tuberin protein is produced at a reduced level compared to production from a wild-type allele
- tuberin protein is produced in a form having reduced function
- the tuberin protein is produced at a reduced level compared to production from a wild-type allele
- tuberin protein is produced in a form having reduced function
- the RIC pre-mRNA is transcribed from the first allele and/or the second allele.
- the ASO binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele or the second allele, thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mRNA encoding tuberin protein and an increase in the expression of the target protein or functional RNA in the cells of the subject.
- the target protein or functional RNA having an increase in expression level resulting from the constitutive splicing of the retained intron from the RIC pre- mRNA is either in a form having reduced function compared to the equivalent wild-type protein (partially-functional), or having full function compared to the equivalent wild-type protein (fully-functional).
- the level of mRNA encoding tuberin protein is increased 1.1 to 10-fold, when compared to the amount of mRNA encoding TSC2 that is produced in a control cell, e.g., one that is not treated with the anti sense oligomer or one that is treated with an anti sense oligomer that does not bind to the targeted portion of the TSC2 RIC pre-mRNA.
- condition caused by a deficient amount or activity of tuberin protein is not a condition caused by alternative or aberrant splicing of the retained intron to which the
- the condition caused by a deficient amount or activity of the tuberin protein is not a condition caused by alternative or aberrant splicing of any retained intron in a RIC pre-mRNA encoding the tuberin protein.
- alternative or aberrant splicing may occur in a pre-mRNA transcribed from the gene, however the compositions and methods of the invention do not prevent or correct this alternative or aberrant splicing.
- a subject treated using the methods of the invention expresses a partially functional tuberin protein from one allele, wherein the partially functional tuberin protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion.
- a subject treated using the methods of the invention expresses a nonfunctional tuberin protein from one allele, wherein the nonfunctional tuberin protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, a partial gene deletion, in one allele.
- a subject treated using the methods of the invention has a TSC2 whole gene deletion, in one allele.
- the subject has a TSC2 missense mutation selected from R611Q/W and R905W.
- the subject has TSC2 deletion mutation of at least 6 amino acids in exon 38.
- the subject has TSC2 substitution mutation of arginine 611 for tryptophan or glutamine.
- Targeted Augmentation of Nuclear Gene Output is used in the methods of the invention to increase expression of a tuberin protein.
- a retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding tuberin protein is present in the nucleus of a cell.
- Cells having a TSC2 RIC pre-mRNA that comprises a retained intron, an exon flanking the 5' splice site, and an exon flanking the 3' splice site, encoding the tuberin protein are contacted with antisense oligomers (ASOs) that are
- Hybridization of the ASOs to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3' splice site) of the retained intron and subsequently increases target protein production.
- pre-mRNA and “pre-mRNA transcript” may be used interchangeably and refer to any pre-mRNA species that contains at least one intron.
- pre-mRNA or pre-mRNA transcripts comprise a 5'-7-methylguanosine cap and/or a poly-A tail.
- pre-mRNA or pre-mRNA transcripts comprise both a 5'-7-methylguanosine cap and a poly-A tail. In some embodiments, the pre-mRNA transcript does not comprise a 5 '-7- methylguanosine cap and/or a poly-A tail.
- a pre-mRNA transcript is a non-productive messenger RNA (mRNA) molecule if it is not translated into a protein (or transported into the cytoplasm from the nucleus).
- mRNA non-productive messenger RNA
- RIC pre-mRNA is a pre- mRNA transcript that contains at least one retained intron.
- the RIC pre-mRNA contains a retained intron, an ex on flanking the 5' splice site of the retained intron, an exon flanking the 3 ' splice site of the retained intron, and encodes the target protein.
- An "RIC pre-mRNA encoding a target protein” is understood to encode the target protein when fully spliced.
- a "retained intron” is any intron that is present in a pre-mRNA transcript when one or more other introns, such as an adjacent intron, encoded by the same gene have been spliced out of the same pre- mRNA transcript.
- the retained intron is the most abundant intron in RIC pre-mRNA encoding the target protein.
- the retained intron is the most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns.
- an antisense oligomer targeted to the most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds.
- a mature mRNA encoding the target protein is thereby produced.
- the terms "mature mRNA,” and "fully-spliced mRNA,” are used
- the targeted region is in a retained intron that is the most abundant intron in a RIC pre-mRNA encoding the tuberin protein.
- the most abundant retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 4, 25, 26, 31, 32, 25 and 26, or 31 and 32.
- the most abundant retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 26. In embodiments, the most abundant retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 31. In some embodiments, the most abundant retained intron in a RIC pre- mRNA encoding the tuberin protein is intron 26, and the second most abundant retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 31.
- a retained intron is an intron that is identified as a retained intron based on a determination of at least about 5%, at least about 10%, at least about 15%, at least about 20%), at least about 25%, at least about 30%>, at least about 35%, at least about 40%, at least about 45%), or at least about 50%, retention.
- a retained intron is an intron that is identified as a retained intron based on a determination of about 5% to about 100%, about 5% to about 95%), about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 65%, about 10% to about 60%, about 10% to about 5
- the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited feature (e.g. in the case of an antisense oligomer, a defined nucleobase sequence) but not the exclusion of any other features.
- the term “comprising” is inclusive and does not exclude additional, unrecited features (e.g. in the case of an antisense oligomer, the presence of additional, unrecited nucleobases).
- compositions and methods comprising
- comprising may be replaced with “consisting essentially of " or “consisting of.”
- the phrase “consisting essentially of is used herein to require the specified feature(s) (e.g. nucleobase sequence) as well as those which do not materially affect the character or function of the claimed invention.
- the term “consisting” is used to indicate the presence of the recited feature (e.g. nucleobase sequence) alone (so that in the case of an antisense oligomer consisting of a specified nucleobase sequence, the presence of additional, unrecited nucleobases is excluded).
- the targeted region is in a retained intron that is the second most abundant intron in a RIC pre-mRNA encoding the tuberin protein.
- the second most abundant retained intron may be targeted rather than the most abundant retained intron due to the uniqueness of the nucleotide sequence of the second most abundant retained intron, ease of ASO design to target a particular nucleotide sequence, and/or amount of increase in protein production resulting from targeting the intron with an ASO.
- the retained intron is the second most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns.
- an antisense oligomer targeted to the second most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds.
- fully-spliced (mature) RNA encoding the target protein is thereby produced.
- the second most retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 4, 25, 26, 31, 32, 25 and 26, or 31 and 32.
- the second most abundant retained intron in a RIC pre- mRNA encoding the tuberin protein is intron 31.
- an ASO is complementary to a targeted region that is within a non- retained intron in a RIC pre-mRNA.
- the targeted portion of the RIC pre- mRNA is within: the region +6 to +100 relative to the 5' splice site of the non-retained intron; or the region -16 to -100 relative to the 3' splice site of the non-retained intron.
- the targeted portion of the RIC pre-mRNA is within the region +100 relative to the 5' splice site of the non-retained intron to -100 relative to the 3' splice site of the non-retained intron.
- RNA encoding the target protein is thereby produced.
- the retained intron of the RIC pre-mRNA is an inefficiently spliced intron.
- "inefficiently spliced” may refer to a relatively low frequency of splicing at a splice site adjacent to the retained intron (5' splice site or 3' splice site) as compared to the frequency of splicing at another splice site in the RIC pre-mRNA.
- inefficiently spliced may also refer to the relative rate or kinetics of splicing at a splice site, in which an "inefficiently spliced" intron may be spliced or removed at a slower rate as compared to another intron in a RIC pre-mRNA.
- the 9-nucleotide sequence at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron is identical to the corresponding wild-type sequence.
- the 16 nucleotide sequence at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site is identical to the corresponding wild-type sequence.
- wild-type sequence refers to the nucleotide sequence for the TSC2 gene in the published reference genome deposited in the NCBI repository of biological and scientific information.
- wild-type sequence refers to the canonical sequence for the TSC2 gene found at NCBI Gene ID 7249.
- nucleotide position denoted with an "e” indicates the nucleotide is present in the sequence of an exon (e.g., the exon flanking the 5' splice site or the exon flanking the 3' splice site).
- the methods involve contacting cells with an ASO that is complementary to a portion of a pre-mRNA encoding tuberin protein, resulting in increased expression of TSC2.
- contacting or administering to cells refers to any method of providing an ASO in immediate proximity with the cells such that the ASO and the cells interact.
- a cell that is contacted with an ASO will take up or transport the ASO into the cell.
- the method involves contacting a condition or disease-associated or condition or disease-relevant cell with any of the ASOs described herein.
- the ASO may be further modified or attached (e.g., covalently attached) to another molecule to target the ASO to a cell type, enhance contact between the ASO and the condition or disease-associated or condition or disease-relevant cell, or enhance uptake of the ASO.
- the term “increasing protein production” or “increasing expression of a target protein” means enhancing the amount of protein that is translated from an mRNA in a cell.
- a “target protein” may be any protein for which increased expression/production is desired.
- contacting a cell that expresses a TSC2 RIC pre-mRNA with an ASO that is complementary to a targeted portion of the TSC2 RIC pre-mRNA transcript results in a measurable increase in the amount of the tuberin protein (e.g., a target protein) encoded by the pre-mRNA.
- Methods of measuring or detecting production of a protein will be evident to one of skill in the art and include any known method, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.
- contacting cells with an ASO that is complementary to a targeted portion of a TSC2 RIC pre-mRNA transcript results in an increase in the amount of tuberin protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment.
- the total amount of tuberin protein produced by the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 1.1 to
- contacting cells with an ASO that is complementary to a targeted portion of a TSC2 RIC pre-mRNA transcript results in an increase in the amount of mRNA encoding TSC2, including the mature mRNA encoding the target protein.
- the amount of mRNA encoding tuberin protein, or the mature mRNA encoding the tuberin protein is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment.
- the total amount of the mRNA encoding tuberin protein, or the mature mRNA encoding tuberin protein produced in the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8- fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 6-fold, about 3 to about
- a control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the TSC2 RIC pre-mRNA.
- the methods and antisense oligonucleotide compositions provided herein are useful for increasing the expression of tuberin protein in cells, for example, in a subject having Tuberous
- Sclerosis Complex caused by a deficiency in the amount or activity of tuberin protein, by increasing the level of mRNA encoding tuberin protein, or the mature mRNA encoding tuberin protein.
- the methods and compositions as described herein induce the constitutive splicing of a retained intron from a TSC2 RIC pre-mRNA transcript encoding tuberin protein, thereby increasing the level of mRNA encoding tuberin protein, or the mature mRNA encoding tuberin protein and increasing the expression of tuberin protein.
- Constitutive splicing of a retained intron from a RIC pre-mRNA correctly removes the retained intron from the RIC pre-mRNA, wherein the retained intron has wild-type splice sequences.
- Constitutive splicing does not encompass splicing of a retained intron from a RIC pre-mRNA transcribed from a gene or allele having a mutation that causes alternative splicing or aberrant splicing of a pre-mRNA transcribed from the gene or allele.
- constitutive splicing of a retained intron does not correct aberrant splicing in or influence alternative splicing of a pre-mRNA to result in an increased expression of a target protein or functional RNA.
- constitutive splicing correctly removes a retained intron from a TSC2 RIC pre-mRNA, wherein the TSC2 RIC pre-mRNA is transcribed from a wild-type gene or allele, or a polymorphic gene or allele, that encodes a fully-functional target protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
- constitutive splicing of a retained intron from a TSC2 RIC pre- mRNA encoding tuberin protein correctly removes a retained intron from a TSC2 RIC pre- mRNA encoding tuberin protein, wherein the TSC2 RIC pre-mRNA is transcribed from a gene or allele from which the target gene or functional RNA is produced at a reduced level compared to production from a wild-type allele, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
- the correct removal of the constitutively spliced retained intron results in production of target protein or functional RNA that is functional when compared to an equivalent wild-type protein or functional RNA.
- constitutive splicing correctly removes a retained intron from a TSC2 RIC pre-mRNA, wherein the TSC2 RIC pre-mRNA is transcribed from a gene or allele that encodes a target protein or functional RNA produced in a form having reduced function compared to an equivalent wild-type protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
- the correct removal of the constitutively spliced retained intron results in production of partially functional target protein, or functional RNA that is partially functional when compared to an equivalent wild-type protein or functional RNA.
- an antisense oligomer as described herein or used in any method described herein does not increase the amount of mRNA encoding tuberin protein or the amount of tuberin protein by modulating alternative splicing or aberrant splicing of a pre-mRNA transcribed from the TSC2 gene.
- Modulation of alternative splicing or aberrant splicing can be measured using any known method for analyzing the sequence and length of RNA species, e.g., by RT-PCR and using methods described elsewhere herein and in the literature.
- modulation of alternative or aberrant splicing is determined based on an increase or decrease in the amount of the spliced species of interest of at least 10% or 1.1-fold. In embodiments, modulation is determined based on an increase or decrease at a level that is at least 10% to 100% or 1.1 to 10-fold, as described herein regarding determining an increase in mRNA encoding tuberin protein in the methods of the invention.
- the method is a method wherein the TSC2 RIC pre-mRNA was produced by partial splicing of a wild-type TSC2 pre-mRNA. In embodiments, the method is a method wherein the TSC2 RIC pre-mRNA was produced by partial splicing of a full-length wild-type TSC2 pre-mRNA. In embodiments, the TSC2 RIC pre-mRNA was produced by partial splicing of a full-length TSC2 pre-mRNA.
- a full-length TSC2 pre- mRNA may have a polymorphism in a splice site of the retained intron that does not impair correct splicing of the retained intron as compared to splicing of the retained intron having the wild-type splice site sequence.
- the mRNA encoding tuberin protein is a full-length mature mRNA, or a wild-type mature mRNA.
- a full-length mature mRNA may have a polymorphism that does not affect the activity of the target protein or the functional RNA encoded by the mature mRNA, as compared to the activity of tuberin protein encoded by the wild-type mature mRNA.
- compositions comprising antisense oligomers that enhances splicing by binding to a targeted portion of a TSC2 RIC pre-mRNA.
- ASO antisense oligomer
- the terms "ASO” and "antisense oligomer” are used interchangeably and refer to an oligomer such as a polynucleotide, comprising nucleobases that hybridizes to a target nucleic acid (e.g., a TSC2 RIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U).
- the ASO may have exact sequence complementary to the target sequence or near complementarity (e.g., sufficient complementarity to bind the target sequence and enhancing splicing at a splice site).
- ASOs are designed so that they bind (hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid).
- Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre- mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre-mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause "off-target” effects is limited.
- Any antisense oligomers known in the art for example in PCT Application No. PCT/US2014/054151, published as WO 2015/035091, titled "Reducing Nonsense-Mediated mRNA Decay,” can be used to practice the methods described herein.
- ASOs "specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of a RIC pre-mRNA.
- hybridization occurs with a Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C.
- Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C.
- Such hybridization preferably corresponds to stringent hybridization conditions.
- the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
- Oligomers such as oligonucleotides, are "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded
- a double-stranded polynucleotide can be "complementary" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
- Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules.
- the sequence of an antisense oligomer (ASO) need not be 100%) complementary to that of its target nucleic acid to hybridize.
- ASOs can comprise at least 70%>, at least 75%>, at least 80%>, at least 85%>, at least 90%>, at least 95%, at least 96%>, at least 97%>, at least 98%>, or at least 99%> sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted.
- an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent
- the remaining non-complementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
- Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
- An ASO need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize.
- ASOs described herein comprise nucleobases that are complementary to
- ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
- PNA peptide nucleic acid
- the ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding.
- naturally occurring nucleotides includes deoxyribonucleotides and ribonucleotides.
- modified nucleotides includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be evident to one of skill in the art and can be found, for example, in U. S. Patent No.
- the nucleobase of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA.
- modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5- m ethyl cytosine, and 5 -hydroxymethoyl cytosine.
- the ASOs described herein also comprise a backbone structure that connects the components of an oligomer.
- backbone structure and “oligomer linkages” may be used interchangeably and refer to the connection between monomers of the ASO.
- the backbone comprises a 3 '-5 ' phosphodiester linkage connecting sugar moieties of the oligomer.
- the backbone structure or oligomer linkages of the ASOs described herein may include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et al., Nucleic Acids Res.
- the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.
- PNA peptide nucleic acid
- the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.
- the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is random. In embodiments, the stereochemistry at each of the
- an ASO used in the methods of the invention comprises an ASO having phosphorus internucleotide linkages that are not random.
- a composition used in the methods of the invention comprises a pure diastereomeric ASO.
- a composition used in the methods of the invention comprises an ASO that has diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%), at least about 96%>, at least about 97%>, at least about 98%>, at least about 99%>, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100% , or about 99% to about 100%.
- the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus internucleotide linkages.
- Rp and Sp are required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability.
- an ASO used in the methods of the invention comprises about 5-100% Rp, at least about 5% Rp, at least about 10%) Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30%) Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70%) Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90%) Rp, or at least about 95% Rp, with the remainder Sp, or about 100% Rp.
- an ASO used in the methods of the invention comprises about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55%) Rp, with the remainder Sp.
- an ASO used in the methods of the invention comprises about 5-100%> Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70%) Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90%) Sp, or at least about 95% Sp, with the remainder Rp, or about 100% Sp.
- an ASO used in the methods of the invention comprises about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder Rp.
- Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring.
- modified sugar moieties include 2' substitutions such as 2'-0-methyl (2'-0-Me), 2'-0-methoxyethyl (2'MOE), 2'-0-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-0-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars.
- the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE.
- the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA).
- the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO).
- the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification.
- the sugar moiety comprises 2' 4' -constrained 2'0-methyloxyethyl (cMOE) modifications.
- the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications.
- the sugar moiety comprises tricycloDNA (tcDNA) modifications.
- the sugar moiety comprises ethylene nucleic acid (ENA) modifications.
- the sugar moiety comprises MCE modifications. Modifications are known in the art and described in the literature, e.g., by Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications,” Nucleic Acid Therapeutics 24(1): 37-47, incorporated by reference for this purpose herein.
- each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'O-methyl modification.
- Such modifications that are present on each of the monomer components of an ASO are referred to as “uniform modifications.”
- a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos).
- Combinations of different modifications to an ASO are referred to as “mixed modifications” or “mixed chemistries.”
- the ASO comprises one or more backbone modification. In some embodiments, the ASO comprises one or more sugar moiety modification. In some
- the ASO comprises one or more backbone modification and one or more sugar moiety modification.
- the ASO comprises 2'MOE modifications and a phosphorothioate backbone.
- the ASO comprises a phosphorodiamidate morpholino (PMO).
- the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs or any component of an ASO (e.g., a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO.
- an ASO or one or more component of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (i.e., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and modulate the half-life of the ASO.
- RNase H cellular nucleases
- the ASOs are comprised of 2'-0-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
- MOE 2-methoxyethyl
- ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein. See e.g., Geary et al., J Pharmacol Exp Ther. 2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001; 296(3):898-904.
- ASOs may be obtained from a commercial source.
- the left-hand end of single-stranded nucleic acid e.g., pre- mRNA transcript, oligonucleotide, ASO, etc.
- sequences is the 5' end and the left-hand direction of single or double-stranded nucleic acid sequences is referred to as the 5' direction.
- the right-hand end or direction of a nucleic acid sequence is the 3' end or direction.
- nucleotides that are upstream of a reference point in a nucleic acid may be designated by a negative number, while nucleotides that are downstream of a reference point may be designated by a positive number.
- a reference point e.g., an exon- ex on junction in mRNA
- a nucleotide that is directly adjacent and upstream of the reference point is designated “minus one,” e.g., while a nucleotide that is directly adjacent and downstream of the reference point is designated “plus one,” e.g., "+1.”
- the ASOs are complementary to (and bind to) a targeted portion of a TSC2 RIC pre-mRNA that is downstream (in the 3' direction) of the 5' splice site of the retained intron in a TSC2 RIC pre-mRNA (e.g., the direction designated by positive numbers relative to the 5' splice site) (FIG. 1).
- the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region +6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100 relative to the 5' splice site of the retained intron.
- the ASO is not complementary to nucleotides +1 to +5 relative to the 5' splice site (the first five nucleotides located downstream of the 5' splice site).
- the ASOs may be complementary to a targeted portion of a TSC2 RIC pre-mRNA that is within the region between nucleotides +6 and +50 relative to the 5' splice site of the retained intron.
- the ASOs are complementary to a targeted portion that is within the region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +20 relative to 5' splice site of the retained intron.
- the ASOs are complementary to a targeted region of a TSC2 RIC pre-mRNA that is upstream (5' relative) of the 3 ' splice site of the retained intron in a TSC2 RIC pre-mRNA (e.g., in the direction designated by negative numbers) (FIG. 1).
- a TSC2 RIC pre-mRNA that is upstream (5' relative) of the 3 ' splice site of the retained intron in a TSC2 RIC pre-mRNA (e.g., in the direction designated by negative numbers) (FIG. 1).
- the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region -16 to -500, -16 to -400, -16 to -300, -6 to -200, -16 to -100, relative to the 3' splice site of the retained intron.
- the ASO is not complementary to nucleotides -1 to -15 relative to the 3' splice site (the first 15 nucleotides located upstream of the 3' splice site).
- the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region -16 to -50 relative to the 3' splice site of the retained intron.
- the ASOs are complementary to a targeted portion that is within the region -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30 relative to 3' splice site of the retained intron.
- the targeted portion of the TSC2 RIC pre-mRNA is within the region +100 relative to the 5' splice site of the retained intron to -100 relative to the 3' splice site of the retained intron.
- the ASOs are complementary to a targeted portion of a TSC2 RIC pre-mRNA that is within the ex on flanking the 5' splice site (upstream) of the retained intron (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 5' splice site of the retained intron. In some embodiments, the ASOs are not complementary to nucleotides -le to - 3e relative to the 5' splice site of the retained intron.
- the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region -4e to-lOOe, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4 to -40e, -4e to -30e, or - 4e to -20e relative to the 5' splice site of the retained intron.
- the ASOs are complementary to a targeted portion of a TSC2 RIC pre-mRNA that is within the exon flanking the 3' splice site (downstream) of the retained intron (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion to the TSC2 RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 3' splice site of the retained intron. In some embodiments, the ASOs are not complementary to nucleotide +le relative to the 3' splice site of the retained intron.
- the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region+2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to +30e, or +2 to +20e relative to the 3' splice site of the retained intron.
- the ASOs may be of any length suitable for specific binding and effective enhancement of splicing. In some
- the ASOs consist of 8 to 50 nucleobases.
- the ASO may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length.
- the ASOs consist of more than 50 nucleobases.
- the ASO is from 8 to 50 nucleobases, 8 to 40 nucleobases,
- nucleobases 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30 nucleo
- ASOs are 30 nucleotides in length. In some embodiments, the ASOs are 29 nucleotides in length. In some embodiments, the ASOs are 28 nucleotides in length. In some embodiments, the ASOs are 27 nucleotides in length. In some embodiments, the ASOs are 26 nucleotides in length. In some embodiments, the ASOs are 25 nucleotides in length. In some embodiments, the ASOs are 24 nucleotides in length. In some embodiments, the ASOs are 23 nucleotides in length. In some embodiments, the ASOs are 22 nucleotides in length. In some embodiments, the ASOs are 21 nucleotides in length.
- two or more ASOs with different chemistries but complementary to the same targeted portion of the RIC pre-mRNA are used. In some embodiments, two or more ASOs that are complementary to different targeted portions of the RIC pre-mRNA are used.
- the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide.
- moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
- the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N- Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a
- Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker.
- Linkers can include a bivalent or trivalent branched linker.
- the conjugate is attached to the 3' end of the antisense oligonucleotide.
- the nucleic acid to be targeted by an ASO is a TSC2 RIC pre- mRNA expressed in a cell, such as a eukaryotic cell.
- a cell may refer to a population of cells.
- the cell is in a subject.
- the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a condition or disease-relevant cell or a cell line. In some embodiments, the cell is in vitro ⁇ e.g., in cell culture).
- compositions or formulations comprising the antisense oligonucleotide of the described compositions and for use in any of the described methods can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature.
- a pharmaceutical composition or formulation for treating a subject comprises an effective amount of any antisense oligomer as described above, or a pharmaceutically acceptable salt, solvate, hydrate or ester thereof, and a pharmaceutically acceptable diluent.
- the antisense oligomer of a pharmaceutical formulation may further comprise a pharmaceutically acceptable excipient, diluent or carrier.
- salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the free base function with a suitable organic acid.
- suitable organic acid examples include salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange.
- salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi sulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, peroxine sodium
- pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
- compositions are formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
- the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media.
- Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium
- a pharmaceutical formulation or composition of the present invention includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome-containing formulation (e.g., cationic or noncationic liposomes).
- the pharmaceutical composition or formulation of the present invention may comprise one or more penetration enhancer, carrier, excipients or other active or inactive ingredients as appropriate and well known to those of skill in the art or described in the published literature.
- liposomes also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation lifetimes.
- a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
- a surfactant is included in the pharmaceutical formulation or compositions.
- the present invention employs a penetration enhancer to effect the efficient delivery of the antisense oligonucleotide, e.g., to aid diffusion across cell membranes and /or enhance the permeability of a lipophilic drug.
- the penetration enhancers are a surfactant, fatty acid, bile salt, chelating agent, or non-chelating non-surfactant.
- the pharmaceutical formulation comprises multiple antisense oligonucleotides.
- the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.
- the antisense oligonucleotide is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. For example, delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, "Adenoviral -vector-mediated gene transfer into medullary motor neurons,” incorporated herein by reference.
- vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra
- Delivery of vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," incorporated herein by reference.
- the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
- the antisense oligonucleotide is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor.
- the antisense oligonucleotide is linked with a viral vector, e.g., to render the antisense compound more effective or increase transport across the blood-brain barrier.
- osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g.,, meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-
- the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide.
- moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
- the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N- Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a
- Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker.
- Linkers can include a bivalent or trivalent branched linker.
- the conjugate is attached to the 3' end of the antisense oligonucleotide.
- compositions provided herein may be administered to an individual.
- “Individual” may be used interchangeably with “subject” or "patient.”
- An individual may be a mammal, for example a human or animal such as a non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep.
- the individual is a human.
- the individual is a fetus, an embryo, or a child.
- the individual may be another eukaryotic organism, such as a plant.
- the compositions provided herein are administered to a cell ex vivo.
- the compositions provided herein are administered to an individual as a method of treating a disease or disorder.
- the individual has a genetic disease, such as any of the diseases described herein.
- the individual is at risk of having the disease, such as any of the diseases described herein.
- the individual is at increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If an individual is "at an increased risk" of having a disease or disorder caused insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment.
- an individual may be at an increased risk of having such a disease or disorder because of family history of the disease.
- individuals at an increased risk of having such a disease or disorder benefit from prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder).
- Suitable routes for administration of ASOs of the present invention may vary depending on cell type to which delivery of the ASOs is desired. Multiple tissues and organs are affected by tuberous sclerosis complex, with the brain, kidney, skin, lung and heart being the most significantly affected tissues.
- the ASOs of the present invention may be administered to patients topically to the skin or by pulmonary delivery to the lung.
- the ASOs of the present invention may be administered to patients parenterally, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
- delivery is to the brain, kidney, skin, lung or heart.
- a fetus is treated in utero, e.g., by administering the ASO composition to the fetus directly or indirectly (e.g., via the mother).
- ASOs that specifically hybridize to different nucleotides within the target region of the pre-mRNA may be screened to identify (determine) ASOs that improve the rate and/or extent of splicing of the target intron.
- the ASO may block or interfere with the binding site(s) of a splicing repressor(s)/silencer.
- Any method known in the art may be used to identify (determine) an ASO that when hybridized to the target region of the intron results in the desired effect (e.g., enhanced splicing, protein or functional RNA production). These methods also can be used for identifying ASOs that enhance splicing of the retained intron by binding to a targeted region in an exon flanking the retained intron, or in a non-retained intron. An example of a method that may be used is provided below. [0166] A round of screening, referred to as an ASO "walk” may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA.
- the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 5' splice site of the retained intron (e.g., a portion of sequence of the ex on located upstream of the target/retained intron) to approximately 100 nucleotides downstream of the 5' splice site of the target/retained intron and/or from approximately 100 nucleotides upstream of the 3 ' splice site of the retained intron to approximately 100 nucleotides downstream of the 3 ' splice site of the target/retained intron (e.g., a portion of sequence of the ex on located
- a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 5' splice site of the target/retained intron.
- a second ASO is designed to specifically hybridize to nucleotides +1 1 to +25 relative to the 5' splice site of the target/retained intron.
- ASOs are designed as such spanning the target region of the pre-mRNA.
- the ASOs can be tiled more closely, e.g., every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled from 100 nucleotides downstream of the 5 ' splice site, to 100 nucleotides upstream of the 3 ' splice site.
- One or more ASOs, or a control ASO are delivered, for example by transfection, into a disease-relevant cell line that expresses the target pre-mRNA (e.g., the RIC pre-mRNA described elsewhere herein).
- the splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described herein (see “Identification of intron-retention events").
- a reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced.
- the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein.
- the amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.
- a second round of screening referred to as an ASO "micro-walk” may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA.
- the ASOs used in the ASO micro-walk are tiled every 1 nucleotide to further refine the nucleotide acid sequence of the pre-mRNA that when hybridized with an ASO results in enhanced splicing.
- Regions defined by ASOs that promote splicing of the target intron are explored in greater detail by means of an ASO "micro-walk", involving ASOs spaced in 1-nt steps, as well as longer ASOs, typically 18-25 nt.
- the ASO micro-walk is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that is not expected to hybridize to the target region), for example by transfection, into a disease-relevant cell line that expresses the target pre-mRNA.
- the splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)- PCR using primers that span the splice junction, as described herein (see “Identification of intron-retention events").
- a reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced.
- the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein.
- the amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.
- ASOs that when hybridized to a region of a pre-mRNA result in enhanced splicing and increased protein production may be tested in vivo using animal models, for example transgenic mouse models in which the full-length human gene has been knocked-in or in humanized mouse models of disease. Suitable routes for administration of ASOs may vary depending on the disease and/or the cell types to which delivery of the ASOs is desired. ASOs may be
- the cells, tissues, and/or organs of the model animals may be assessed to determine the effect of the ASO treatment by for example evaluating splicing (efficiency, rate, extent) and protein production by methods known in the art and described herein.
- the animal models may also be any phenotypic or behavioral indication of the disease or disease severity.
- Example 1 Identification of intron retention events in TSC2 transcripts by RNAseq using next generation sequencing
- FIG. 3 shows the mapped reads visualized using the UCSC genome browser (operated by the UCSC Genome Informatics Group (Center for Biomolecular Science & Engineering, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064) and described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome Browser database: 2015 update," Nucleic Acids Research 43, Database Issue, doi: 10.1093/nar/gkul 177) and the coverage and number of reads can be inferred by the peak signals.
- the height of the peaks indicates the level of expression given by the density of the reads in a particular region.
- TSC2 drawn to scale
- UCSC genome browser below the read signals
- An ASO walk was designed to target intron 4 using the method described herein (FIG. 4; Table 2).
- a region immediately downstream of the 5' splice site of intron 4 spanning nucleotides +6 to +58 and a region immediately upstream of the 3' splice site of intron 4 spanning nucleotides -16 to -68 of the intron were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals.
- An ASO walk was designed to target introns 25 and 26 using the method described herein (FIG. 6; Table 2).
- a region immediately downstream of the intron 25 5' splice site spanning nucleotides +6 to +58 and a region immediately upstream of intron 26 3' splice site spanning nucleotides -16 to -68 of the intron were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals.
- the splice site intronic regions flanking alternative exon 26 are not targeted to avoid affecting the inclusion level of exon 26.
- An ASO walk was designed to target introns 31 and 32 using the method described herein (FIG. 8; Table 2).
- a region immediately downstream of the 5' splice site of intron 31 spanning nucleotides +6 to +58 and a region immediately upstream 3' splice site of intron 32 spanning nucleotides -18 to -68 were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals (with the exception of 2 ASOs, TSC2-IVS32-33 and TSC2- IVS32-51).
- the splice site intronic regions flanking alternative exon 32 are not targeted to avoid affecting the inclusion level of exon 32.
- Example 5 Improved splicing efficiency via ASO-targeting of TSC2 intron 4, 25, 26, 31 or 32 increases transcript levels
- RT-PCR products were evaluated using radioactive RT-PCR and RT-qPCR.
- ARPE-19 cells a human retinal epithelium cell line (American Type Culture Collection (ATCC), USA), or Huh-7, a human hepatoma cell line (NIBIOHN, Japan), or SK-N-AS, a human neuroblastoma cell line (ATCC) were mock- transfected, or transfected with the targeting ASOs described in FIG. 10, FIG. 12, FIG. 15 and Tables 1-2.
- RNAiMax transfection reagent (Thermo Fisher) according to vendor's specifications. Briefly, ASOs were plated in 96-well tissue culture plates and combined with RNAiMax diluted in Opti-MEM. Cells were detached using trypsin and resuspended in full medium, and approximately 25,000 cells were added the ASO- transfection mixture. Transfection experiments were carried out in triplicate plate
- Taqman assays were carried out according to vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher).
- Target gene assay values were normalized to RPL32 (deltaCt) and plate-matched mock transfected samples (delta-delta Ct), generating fold- change over mock quantitation (2 A -(delta-deltaCt). Average fold-change over mock of the three plate replicates was plotted (FIG. 10, FIG. 12 and FIG. 15). In FIG. 10, FIG. 12 and FIG. 15, several ASOs were identified to increase the target gene expression, indicating an increase in splicing at the respective target intron.
- TSC2-IVS31+26 cagugggagcagagcccg 31 4900
- TSC2-IVS31+31 caggccagugggagcaga 31 4901
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Oncology (AREA)
- Pathology (AREA)
- Plant Pathology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562267212P | 2015-12-14 | 2015-12-14 | |
PCT/US2016/066705 WO2017106375A1 (fr) | 2015-12-14 | 2016-12-14 | Oligomères antisens pour le traitement de la sclérose tubéreuse de bourneville |
Publications (2)
Publication Number | Publication Date |
---|---|
EP3390635A1 true EP3390635A1 (fr) | 2018-10-24 |
EP3390635A4 EP3390635A4 (fr) | 2019-05-01 |
Family
ID=59057534
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP16876615.2A Withdrawn EP3390635A4 (fr) | 2015-12-14 | 2016-12-14 | Oligomères antisens pour le traitement de la sclérose tubéreuse de bourneville |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP3390635A4 (fr) |
JP (2) | JP7051683B2 (fr) |
CA (1) | CA3005131A1 (fr) |
WO (1) | WO2017106375A1 (fr) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB201410693D0 (en) | 2014-06-16 | 2014-07-30 | Univ Southampton | Splicing modulation |
CN107109411B (zh) | 2014-10-03 | 2022-07-01 | 冷泉港实验室 | 核基因输出的定向增加 |
IL308174A (en) | 2015-10-09 | 2024-01-01 | Univ Southampton | Gene expression modulation and dysregulated protein expression scanning |
EP3390636B1 (fr) | 2015-12-14 | 2021-05-19 | Cold Spring Harbor Laboratory | Oligomères antisens destinés au traitement du syndrome de dravet |
US11096956B2 (en) | 2015-12-14 | 2021-08-24 | Stoke Therapeutics, Inc. | Antisense oligomers and uses thereof |
CN118325899A (zh) | 2017-01-23 | 2024-07-12 | 瑞泽恩制药公司 | Hsd17b13变体及其应用 |
MX2019012169A (es) | 2017-04-11 | 2019-12-11 | Regeneron Pharma | Ensayos para evaluar la actividad de moduladores de miembros de la familia de hidroxiesteroide (17-beta) deshidrogenasa (hsd17b). |
GB2599884B (en) | 2017-08-25 | 2022-08-31 | Stoke Therapeutics Inc | Antisense oligomers for treatment of conditions and diseases |
MX2020003561A (es) | 2017-10-11 | 2020-08-03 | Regeneron Pharma | Inhibicion de la hsd17b13 en el tratamiento de la enfermedad hepatica en pacientes que expresan la variacion pnpla3 i148m. |
JP2021523227A (ja) | 2018-05-04 | 2021-09-02 | ストーク セラピューティクス,インク. | コレステリルエステル蓄積症の処置のための方法及び組成物 |
AU2020334067A1 (en) * | 2019-08-19 | 2022-03-17 | Stoke Therapeutics, Inc. | Compositions and methods for modulating splicing and protein expression |
KR20230022409A (ko) | 2020-05-11 | 2023-02-15 | 스톡 테라퓨틱스, 인크. | 병태 및 질환의 치료를 위한 opa1 안티센스 올리고머 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011020118A1 (fr) * | 2009-08-14 | 2011-02-17 | Theramind Research, Llc | Méthodes et compositions permettant de traiter la sclérose tubéreuse de bourneville |
WO2011031786A2 (fr) * | 2009-09-08 | 2011-03-17 | Laboratory Corporation Of America Holdings | Compositions et procédés pour diagnostiquer des troubles du spectre autistique |
EP3041958B1 (fr) * | 2013-09-04 | 2019-12-04 | Cold Spring Harbor Laboratory | Réduction de dégradation d'arnm à médiation non-sens |
GB201410693D0 (en) * | 2014-06-16 | 2014-07-30 | Univ Southampton | Splicing modulation |
CN107109411B (zh) * | 2014-10-03 | 2022-07-01 | 冷泉港实验室 | 核基因输出的定向增加 |
JP7049249B2 (ja) * | 2015-12-14 | 2022-04-06 | コールド スプリング ハーバー ラボラトリー | 中枢神経系疾患の処置のための組成物および方法 |
-
2016
- 2016-12-14 EP EP16876615.2A patent/EP3390635A4/fr not_active Withdrawn
- 2016-12-14 WO PCT/US2016/066705 patent/WO2017106375A1/fr active Application Filing
- 2016-12-14 CA CA3005131A patent/CA3005131A1/fr active Pending
- 2016-12-14 JP JP2018529258A patent/JP7051683B2/ja active Active
-
2022
- 2022-03-29 JP JP2022053948A patent/JP2022088533A/ja active Pending
Also Published As
Publication number | Publication date |
---|---|
JP7051683B2 (ja) | 2022-04-11 |
EP3390635A4 (fr) | 2019-05-01 |
CA3005131A1 (fr) | 2017-06-22 |
WO2017106375A1 (fr) | 2017-06-22 |
JP2019500350A (ja) | 2019-01-10 |
JP2022088533A (ja) | 2022-06-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3390636B1 (fr) | Oligomères antisens destinés au traitement du syndrome de dravet | |
EP3390642B1 (fr) | Compositions et procédés pour le traitement de rétinite pigmentaire 13 | |
EP3390635A1 (fr) | Oligomères antisens pour le traitement de la sclérose tubéreuse de bourneville | |
AU2015327836B2 (en) | Targeted augmentation of nuclear gene output | |
JP7049247B2 (ja) | 腎臓病の処置のための組成物と方法 | |
EP3389671A1 (fr) | Oligomères antisens pour le traitement du syndrome d'alagille | |
EP3389672A1 (fr) | Compositions et procédés de traitement de maladies hépatiques | |
EP3389782A1 (fr) | Oligomères antisens pour le traitement de la polykystose rénale | |
CA3131591A1 (fr) | Oligomeres antisens pour le traitement d'etats pathologiques et autres maladies | |
US20240117353A1 (en) | Compositions for treatment of conditions and diseases associated with polycystin expression | |
WO2023235509A2 (fr) | Oligomères antisens pour le traitement de pathologies et de maladies fondées sur la dégradation des arn non-sens | |
WO2024097138A1 (fr) | Oligomères antisens pour le traitement de pathologies et de maladies fondées sur la dégradation des arn à médiation non-sens | |
WO2022271699A2 (fr) | Oligomères antisens pour le traitement d'états et de maladies fondées sur la dégradation des arnm non-sens |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20180706 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: BA ME |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
A4 | Supplementary search report drawn up and despatched |
Effective date: 20190401 |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: C12Q 1/6883 20180101ALI20190326BHEP Ipc: A61K 31/7088 20060101ALI20190326BHEP Ipc: C12N 15/113 20100101AFI20190326BHEP |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1262746 Country of ref document: HK |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: KRAINER, ADRIAN Inventor name: NASH, HUW M. Inventor name: AZNAREZ, ISABEL |
|
17Q | First examination report despatched |
Effective date: 20200506 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
P01 | Opt-out of the competence of the unified patent court (upc) registered |
Effective date: 20230922 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20240222 |