WO2013143700A2 - Artificial nucleic acid molecules comprising a 5'top utr - Google Patents
Artificial nucleic acid molecules comprising a 5'top utr Download PDFInfo
- Publication number
- WO2013143700A2 WO2013143700A2 PCT/EP2013/000938 EP2013000938W WO2013143700A2 WO 2013143700 A2 WO2013143700 A2 WO 2013143700A2 EP 2013000938 W EP2013000938 W EP 2013000938W WO 2013143700 A2 WO2013143700 A2 WO 2013143700A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- utr
- nucleic acid
- sequence
- acid molecule
- Prior art date
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 525
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 347
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 347
- 108020003589 5' Untranslated Regions Proteins 0.000 claims abstract description 461
- 108020005345 3' Untranslated Regions Proteins 0.000 claims abstract description 383
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 330
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 238
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 224
- 108700026244 Open Reading Frames Proteins 0.000 claims abstract description 217
- 101150114197 TOP gene Proteins 0.000 claims abstract description 142
- 108010088751 Albumins Proteins 0.000 claims abstract description 65
- 238000001415 gene therapy Methods 0.000 claims abstract description 24
- 239000002773 nucleotide Substances 0.000 claims description 260
- 125000003729 nucleotide group Chemical group 0.000 claims description 259
- 239000013598 vector Substances 0.000 claims description 155
- 210000004027 cell Anatomy 0.000 claims description 151
- 239000012634 fragment Substances 0.000 claims description 145
- 102000004169 proteins and genes Human genes 0.000 claims description 123
- 241000282414 Homo sapiens Species 0.000 claims description 89
- 230000014616 translation Effects 0.000 claims description 81
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 74
- 102000002278 Ribosomal Proteins Human genes 0.000 claims description 72
- 108010000605 Ribosomal Proteins Proteins 0.000 claims description 72
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 71
- 238000001890 transfection Methods 0.000 claims description 58
- 108020004414 DNA Proteins 0.000 claims description 53
- 239000008194 pharmaceutical composition Substances 0.000 claims description 50
- 108060001084 Luciferase Proteins 0.000 claims description 44
- 238000000034 method Methods 0.000 claims description 43
- 230000001965 increasing effect Effects 0.000 claims description 42
- 108091034057 RNA (poly(A)) Proteins 0.000 claims description 41
- -1 RPL32 Proteins 0.000 claims description 41
- 108700026220 vif Genes Proteins 0.000 claims description 41
- 101000975753 Homo sapiens Acid ceramidase Proteins 0.000 claims description 39
- 101000718108 Homo sapiens Androgen-induced gene 1 protein Proteins 0.000 claims description 37
- 102100024005 Acid ceramidase Human genes 0.000 claims description 33
- 102000008186 Collagen Human genes 0.000 claims description 32
- 108010035532 Collagen Proteins 0.000 claims description 32
- 229920001436 collagen Polymers 0.000 claims description 32
- 230000008488 polyadenylation Effects 0.000 claims description 31
- 102000009027 Albumins Human genes 0.000 claims description 30
- 102100037965 60S ribosomal protein L21 Human genes 0.000 claims description 29
- 102100027573 ATP synthase subunit alpha, mitochondrial Human genes 0.000 claims description 29
- 108091081024 Start codon Proteins 0.000 claims description 25
- 238000000338 in vitro Methods 0.000 claims description 24
- 210000004962 mammalian cell Anatomy 0.000 claims description 24
- 101100451537 Caenorhabditis elegans hsd-1 gene Proteins 0.000 claims description 23
- 101150087698 alpha gene Proteins 0.000 claims description 21
- 241000251556 Chordata Species 0.000 claims description 20
- 101150042997 21 gene Proteins 0.000 claims description 19
- 102100026468 Androgen-induced gene 1 protein Human genes 0.000 claims description 19
- 101000936262 Homo sapiens ATP synthase subunit alpha, mitochondrial Proteins 0.000 claims description 19
- 239000002671 adjuvant Substances 0.000 claims description 19
- 101150007523 32 gene Proteins 0.000 claims description 18
- 101150066375 35 gene Proteins 0.000 claims description 18
- 101000861049 Homo sapiens Cytochrome c oxidase subunit 6C Proteins 0.000 claims description 18
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical class N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 18
- 229960005486 vaccine Drugs 0.000 claims description 18
- KVUXYQHEESDGIJ-UHFFFAOYSA-N 10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthrene-3,16-diol Chemical compound C1CC2CC(O)CCC2(C)C2C1C1CC(O)CC1(C)CC2 KVUXYQHEESDGIJ-UHFFFAOYSA-N 0.000 claims description 17
- 101150033839 4 gene Proteins 0.000 claims description 17
- 101710088194 Dehydrogenase Proteins 0.000 claims description 17
- 238000010367 cloning Methods 0.000 claims description 17
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 16
- 101150007503 rps1 gene Proteins 0.000 claims description 16
- 238000011144 upstream manufacturing Methods 0.000 claims description 16
- 101150084750 1 gene Proteins 0.000 claims description 15
- 102100026926 60S ribosomal protein L4 Human genes 0.000 claims description 15
- 101150008822 rpsA gene Proteins 0.000 claims description 14
- 238000002255 vaccination Methods 0.000 claims description 14
- 101710170662 ATP synthase subunit alpha, mitochondrial Proteins 0.000 claims description 13
- 101100527655 Arabidopsis thaliana RPL4D gene Proteins 0.000 claims description 11
- 101100469270 Candida albicans (strain SC5314 / ATCC MYA-2876) RPL10A gene Proteins 0.000 claims description 11
- 108091026898 Leader sequence (mRNA) Proteins 0.000 claims description 11
- 101100304908 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) RPL5 gene Proteins 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 11
- 101150060526 rpl1 gene Proteins 0.000 claims description 11
- 101150009248 rpl4 gene Proteins 0.000 claims description 11
- 101150079275 rplA gene Proteins 0.000 claims description 11
- 238000006467 substitution reaction Methods 0.000 claims description 11
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 10
- 238000003780 insertion Methods 0.000 claims description 9
- 230000037431 insertion Effects 0.000 claims description 9
- 102100037563 40S ribosomal protein S2 Human genes 0.000 claims description 8
- 101000840051 Homo sapiens Ubiquitin-60S ribosomal protein L40 Proteins 0.000 claims description 8
- 102100028462 Ubiquitin-60S ribosomal protein L40 Human genes 0.000 claims description 8
- 239000013600 plasmid vector Substances 0.000 claims description 8
- 108091035707 Consensus sequence Proteins 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 7
- 238000012217 deletion Methods 0.000 claims description 7
- 230000037430 deletion Effects 0.000 claims description 7
- 108060003196 globin Proteins 0.000 claims description 7
- 102100026112 60S acidic ribosomal protein P2 Human genes 0.000 claims description 6
- 101000691878 Homo sapiens 60S acidic ribosomal protein P2 Proteins 0.000 claims description 6
- 102000018146 globin Human genes 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 6
- 102100021308 60S ribosomal protein L23 Human genes 0.000 claims description 5
- 210000003705 ribosome Anatomy 0.000 claims description 5
- 102100023912 40S ribosomal protein S12 Human genes 0.000 claims description 4
- 102100023415 40S ribosomal protein S20 Human genes 0.000 claims description 4
- 102100037710 40S ribosomal protein S21 Human genes 0.000 claims description 4
- 102100037513 40S ribosomal protein S23 Human genes 0.000 claims description 4
- 102100033449 40S ribosomal protein S24 Human genes 0.000 claims description 4
- 102100022721 40S ribosomal protein S25 Human genes 0.000 claims description 4
- 102100027337 40S ribosomal protein S26 Human genes 0.000 claims description 4
- 102100022681 40S ribosomal protein S27 Human genes 0.000 claims description 4
- 102100023679 40S ribosomal protein S28 Human genes 0.000 claims description 4
- 102100031928 40S ribosomal protein S29 Human genes 0.000 claims description 4
- 102100033409 40S ribosomal protein S3 Human genes 0.000 claims description 4
- 102100022600 40S ribosomal protein S3a Human genes 0.000 claims description 4
- 102100024088 40S ribosomal protein S7 Human genes 0.000 claims description 4
- 102100037663 40S ribosomal protein S8 Human genes 0.000 claims description 4
- 102100027271 40S ribosomal protein SA Human genes 0.000 claims description 4
- 102100021546 60S ribosomal protein L10 Human genes 0.000 claims description 4
- 102100025643 60S ribosomal protein L12 Human genes 0.000 claims description 4
- 102100021206 60S ribosomal protein L19 Human genes 0.000 claims description 4
- 102100037685 60S ribosomal protein L22 Human genes 0.000 claims description 4
- 102100023247 60S ribosomal protein L23a Human genes 0.000 claims description 4
- 102100035322 60S ribosomal protein L24 Human genes 0.000 claims description 4
- 102100028348 60S ribosomal protein L26 Human genes 0.000 claims description 4
- 102100025601 60S ribosomal protein L27 Human genes 0.000 claims description 4
- 102100021927 60S ribosomal protein L27a Human genes 0.000 claims description 4
- 102100021660 60S ribosomal protein L28 Human genes 0.000 claims description 4
- 102100021671 60S ribosomal protein L29 Human genes 0.000 claims description 4
- 102100038237 60S ribosomal protein L30 Human genes 0.000 claims description 4
- 102100023777 60S ribosomal protein L31 Human genes 0.000 claims description 4
- 102100040637 60S ribosomal protein L34 Human genes 0.000 claims description 4
- 102100022276 60S ribosomal protein L35a Human genes 0.000 claims description 4
- 102100022048 60S ribosomal protein L36 Human genes 0.000 claims description 4
- 102100031002 60S ribosomal protein L36a Human genes 0.000 claims description 4
- 102100040131 60S ribosomal protein L37 Human genes 0.000 claims description 4
- 102100036126 60S ribosomal protein L37a Human genes 0.000 claims description 4
- 102100030982 60S ribosomal protein L38 Human genes 0.000 claims description 4
- 102100035988 60S ribosomal protein L39 Human genes 0.000 claims description 4
- 102100040623 60S ribosomal protein L41 Human genes 0.000 claims description 4
- 102100035841 60S ribosomal protein L7 Human genes 0.000 claims description 4
- 102100036630 60S ribosomal protein L7a Human genes 0.000 claims description 4
- 102100035931 60S ribosomal protein L8 Human genes 0.000 claims description 4
- 102100034003 FAU ubiquitin-like and ribosomal protein S30 Human genes 0.000 claims description 4
- 101001097953 Homo sapiens 40S ribosomal protein S23 Proteins 0.000 claims description 4
- 101000678929 Homo sapiens 40S ribosomal protein S25 Proteins 0.000 claims description 4
- 101000862491 Homo sapiens 40S ribosomal protein S26 Proteins 0.000 claims description 4
- 101000678466 Homo sapiens 40S ribosomal protein S27 Proteins 0.000 claims description 4
- 101000623076 Homo sapiens 40S ribosomal protein S28 Proteins 0.000 claims description 4
- 101000679249 Homo sapiens 40S ribosomal protein S3a Proteins 0.000 claims description 4
- 101000732165 Homo sapiens 40S ribosomal protein S4, X isoform Proteins 0.000 claims description 4
- 101000694288 Homo sapiens 40S ribosomal protein SA Proteins 0.000 claims description 4
- 101001115494 Homo sapiens 60S ribosomal protein L23a Proteins 0.000 claims description 4
- 101001080179 Homo sapiens 60S ribosomal protein L26 Proteins 0.000 claims description 4
- 101000753696 Homo sapiens 60S ribosomal protein L27a Proteins 0.000 claims description 4
- 101001110988 Homo sapiens 60S ribosomal protein L35a Proteins 0.000 claims description 4
- 101001127203 Homo sapiens 60S ribosomal protein L36a Proteins 0.000 claims description 4
- 101001127258 Homo sapiens 60S ribosomal protein L36a-like Proteins 0.000 claims description 4
- 101000671735 Homo sapiens 60S ribosomal protein L37 Proteins 0.000 claims description 4
- 101001092424 Homo sapiens 60S ribosomal protein L37a Proteins 0.000 claims description 4
- 101001127039 Homo sapiens 60S ribosomal protein L38 Proteins 0.000 claims description 4
- 101000691203 Homo sapiens 60S ribosomal protein L4 Proteins 0.000 claims description 4
- 101000674326 Homo sapiens 60S ribosomal protein L41 Proteins 0.000 claims description 4
- 101000853617 Homo sapiens 60S ribosomal protein L7 Proteins 0.000 claims description 4
- 101000853243 Homo sapiens 60S ribosomal protein L7a Proteins 0.000 claims description 4
- 101000732045 Homo sapiens FAU ubiquitin-like and ribosomal protein S30 Proteins 0.000 claims description 4
- 102100023341 Ubiquitin-40S ribosomal protein S27a Human genes 0.000 claims description 4
- 102000004314 ribosomal protein S14 Human genes 0.000 claims description 4
- 108090000850 ribosomal protein S14 Proteins 0.000 claims description 4
- 102100039980 40S ribosomal protein S18 Human genes 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 239000013603 viral vector Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 239000003102 growth factor Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 101000661708 Homo sapiens 60S ribosomal protein L21 Proteins 0.000 claims 9
- 102100026744 40S ribosomal protein S10 Human genes 0.000 claims 1
- 102100023779 40S ribosomal protein S5 Human genes 0.000 claims 1
- 102100033714 40S ribosomal protein S6 Human genes 0.000 claims 1
- 102100033731 40S ribosomal protein S9 Human genes 0.000 claims 1
- 102100033416 60S acidic ribosomal protein P1 Human genes 0.000 claims 1
- 102100022406 60S ribosomal protein L10a Human genes 0.000 claims 1
- 102100031854 60S ribosomal protein L14 Human genes 0.000 claims 1
- 102100040540 60S ribosomal protein L3 Human genes 0.000 claims 1
- 102100026750 60S ribosomal protein L5 Human genes 0.000 claims 1
- 102100040924 60S ribosomal protein L6 Human genes 0.000 claims 1
- 102100041029 60S ribosomal protein L9 Human genes 0.000 claims 1
- 108091033380 Coding strand Proteins 0.000 claims 1
- 101000639726 Homo sapiens 28S ribosomal protein S12, mitochondrial Proteins 0.000 claims 1
- 101001119189 Homo sapiens 40S ribosomal protein S10 Proteins 0.000 claims 1
- 101000682687 Homo sapiens 40S ribosomal protein S12 Proteins 0.000 claims 1
- 101000811259 Homo sapiens 40S ribosomal protein S18 Proteins 0.000 claims 1
- 101001098029 Homo sapiens 40S ribosomal protein S2 Proteins 0.000 claims 1
- 101001114932 Homo sapiens 40S ribosomal protein S20 Proteins 0.000 claims 1
- 101001097814 Homo sapiens 40S ribosomal protein S21 Proteins 0.000 claims 1
- 101000656669 Homo sapiens 40S ribosomal protein S24 Proteins 0.000 claims 1
- 101000704060 Homo sapiens 40S ribosomal protein S29 Proteins 0.000 claims 1
- 101000656561 Homo sapiens 40S ribosomal protein S3 Proteins 0.000 claims 1
- 101000622644 Homo sapiens 40S ribosomal protein S5 Proteins 0.000 claims 1
- 101000656896 Homo sapiens 40S ribosomal protein S6 Proteins 0.000 claims 1
- 101000690200 Homo sapiens 40S ribosomal protein S7 Proteins 0.000 claims 1
- 101001097439 Homo sapiens 40S ribosomal protein S8 Proteins 0.000 claims 1
- 101000657066 Homo sapiens 40S ribosomal protein S9 Proteins 0.000 claims 1
- 101000712357 Homo sapiens 60S acidic ribosomal protein P1 Proteins 0.000 claims 1
- 101001108634 Homo sapiens 60S ribosomal protein L10 Proteins 0.000 claims 1
- 101000755323 Homo sapiens 60S ribosomal protein L10a Proteins 0.000 claims 1
- 101000575173 Homo sapiens 60S ribosomal protein L12 Proteins 0.000 claims 1
- 101000704267 Homo sapiens 60S ribosomal protein L14 Proteins 0.000 claims 1
- 101001117935 Homo sapiens 60S ribosomal protein L15 Proteins 0.000 claims 1
- 101001105789 Homo sapiens 60S ribosomal protein L19 Proteins 0.000 claims 1
- 101001097555 Homo sapiens 60S ribosomal protein L22 Proteins 0.000 claims 1
- 101000675833 Homo sapiens 60S ribosomal protein L23 Proteins 0.000 claims 1
- 101000660926 Homo sapiens 60S ribosomal protein L24 Proteins 0.000 claims 1
- 101000719728 Homo sapiens 60S ribosomal protein L27 Proteins 0.000 claims 1
- 101000676271 Homo sapiens 60S ribosomal protein L28 Proteins 0.000 claims 1
- 101000676246 Homo sapiens 60S ribosomal protein L29 Proteins 0.000 claims 1
- 101000673985 Homo sapiens 60S ribosomal protein L3 Proteins 0.000 claims 1
- 101001101319 Homo sapiens 60S ribosomal protein L30 Proteins 0.000 claims 1
- 101001113162 Homo sapiens 60S ribosomal protein L31 Proteins 0.000 claims 1
- 101000672659 Homo sapiens 60S ribosomal protein L34 Proteins 0.000 claims 1
- 101001110263 Homo sapiens 60S ribosomal protein L36 Proteins 0.000 claims 1
- 101000716179 Homo sapiens 60S ribosomal protein L39 Proteins 0.000 claims 1
- 101000691083 Homo sapiens 60S ribosomal protein L5 Proteins 0.000 claims 1
- 101000673524 Homo sapiens 60S ribosomal protein L6 Proteins 0.000 claims 1
- 101000853659 Homo sapiens 60S ribosomal protein L8 Proteins 0.000 claims 1
- 101000672886 Homo sapiens 60S ribosomal protein L9 Proteins 0.000 claims 1
- 101000711369 Homo sapiens Probable ribosome biogenesis protein RLP24 Proteins 0.000 claims 1
- 101001115218 Homo sapiens Ubiquitin-40S ribosomal protein S27a Proteins 0.000 claims 1
- 238000011239 genetic vaccination Methods 0.000 abstract description 22
- 235000018102 proteins Nutrition 0.000 description 107
- 108090000765 processed proteins & peptides Proteins 0.000 description 67
- 230000014509 gene expression Effects 0.000 description 64
- 108020004705 Codon Proteins 0.000 description 51
- 102000036639 antigens Human genes 0.000 description 47
- 108091007433 antigens Proteins 0.000 description 47
- 239000000427 antigen Substances 0.000 description 46
- 238000013518 transcription Methods 0.000 description 44
- 230000035897 transcription Effects 0.000 description 44
- 150000001413 amino acids Chemical group 0.000 description 43
- 230000006870 function Effects 0.000 description 42
- 239000005089 Luciferase Substances 0.000 description 38
- 102000004196 processed proteins & peptides Human genes 0.000 description 36
- 229940024606 amino acid Drugs 0.000 description 30
- 235000001014 amino acid Nutrition 0.000 description 30
- 125000002091 cationic group Chemical group 0.000 description 25
- 238000012986 modification Methods 0.000 description 23
- 244000052769 pathogen Species 0.000 description 23
- 238000013519 translation Methods 0.000 description 23
- 230000000977 initiatory effect Effects 0.000 description 21
- 230000004048 modification Effects 0.000 description 21
- 108090000327 ribosomal protein L21 Proteins 0.000 description 20
- 108010033040 Histones Proteins 0.000 description 19
- 150000001875 compounds Chemical class 0.000 description 19
- 230000028993 immune response Effects 0.000 description 19
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 18
- 230000000694 effects Effects 0.000 description 18
- 230000000087 stabilizing effect Effects 0.000 description 18
- 102100040768 60S ribosomal protein L32 Human genes 0.000 description 17
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 17
- 108091027974 Mature messenger RNA Proteins 0.000 description 17
- 108010025325 ribosomal protein L32 Proteins 0.000 description 17
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 16
- 206010028980 Neoplasm Diseases 0.000 description 16
- 230000003308 immunostimulating effect Effects 0.000 description 16
- 150000003230 pyrimidines Chemical class 0.000 description 16
- 102000053602 DNA Human genes 0.000 description 15
- 108090000128 Lipoxygenases Proteins 0.000 description 15
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 15
- 239000002719 pyrimidine nucleotide Substances 0.000 description 15
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 14
- 230000002163 immunogen Effects 0.000 description 14
- 230000001717 pathogenic effect Effects 0.000 description 14
- 108091026890 Coding region Proteins 0.000 description 13
- 201000010099 disease Diseases 0.000 description 13
- 239000007788 liquid Substances 0.000 description 13
- 238000011282 treatment Methods 0.000 description 13
- 108091005902 Hemoglobin subunit alpha Proteins 0.000 description 12
- 108020004566 Transfer RNA Proteins 0.000 description 12
- 229920000642 polymer Polymers 0.000 description 12
- 210000001744 T-lymphocyte Anatomy 0.000 description 11
- 230000033289 adaptive immune response Effects 0.000 description 11
- 239000000654 additive Substances 0.000 description 11
- 210000000987 immune system Anatomy 0.000 description 11
- 238000001727 in vivo Methods 0.000 description 11
- 230000015788 innate immune response Effects 0.000 description 11
- 229920001184 polypeptide Polymers 0.000 description 11
- 230000002028 premature Effects 0.000 description 11
- 108091008146 restriction endonucleases Proteins 0.000 description 11
- 108091023045 Untranslated Region Proteins 0.000 description 10
- 210000005006 adaptive immune system Anatomy 0.000 description 10
- 230000000996 additive effect Effects 0.000 description 10
- 239000000178 monomer Substances 0.000 description 10
- 241000894007 species Species 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 230000001225 therapeutic effect Effects 0.000 description 10
- 239000001226 triphosphate Substances 0.000 description 10
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 9
- 230000004913 activation Effects 0.000 description 9
- 210000005007 innate immune system Anatomy 0.000 description 9
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 8
- 230000027455 binding Effects 0.000 description 8
- 230000033228 biological regulation Effects 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 8
- 230000002500 effect on skin Effects 0.000 description 8
- 210000002950 fibroblast Anatomy 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 235000000346 sugar Nutrition 0.000 description 8
- 239000003981 vehicle Substances 0.000 description 8
- 102100027685 Hemoglobin subunit alpha Human genes 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 239000003446 ligand Substances 0.000 description 7
- 238000001638 lipofection Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 230000002195 synergetic effect Effects 0.000 description 7
- 230000002103 transcriptional effect Effects 0.000 description 7
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 6
- 101000763579 Homo sapiens Toll-like receptor 1 Proteins 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 229920002873 Polyethylenimine Polymers 0.000 description 6
- 108091036066 Three prime untranslated region Proteins 0.000 description 6
- 102100027010 Toll-like receptor 1 Human genes 0.000 description 6
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 230000001419 dependent effect Effects 0.000 description 6
- 230000001747 exhibiting effect Effects 0.000 description 6
- 210000005260 human cell Anatomy 0.000 description 6
- 230000035800 maturation Effects 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 230000001575 pathological effect Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 102000002689 Toll-like receptor Human genes 0.000 description 5
- 108020000411 Toll-like receptor Proteins 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 210000003719 b-lymphocyte Anatomy 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 5
- 210000004443 dendritic cell Anatomy 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 108700010070 Codon Usage Proteins 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 4
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 4
- 101000763537 Homo sapiens Toll-like receptor 10 Proteins 0.000 description 4
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 description 4
- 101000831496 Homo sapiens Toll-like receptor 3 Proteins 0.000 description 4
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 4
- 101000669460 Homo sapiens Toll-like receptor 5 Proteins 0.000 description 4
- 101000669406 Homo sapiens Toll-like receptor 6 Proteins 0.000 description 4
- 101000669402 Homo sapiens Toll-like receptor 7 Proteins 0.000 description 4
- 101000800483 Homo sapiens Toll-like receptor 8 Proteins 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 108091036407 Polyadenylation Proteins 0.000 description 4
- 108050001924 Ribosomal protein L23 Proteins 0.000 description 4
- 102000008235 Toll-Like Receptor 9 Human genes 0.000 description 4
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 4
- 102100027009 Toll-like receptor 10 Human genes 0.000 description 4
- 102100024333 Toll-like receptor 2 Human genes 0.000 description 4
- 102100024324 Toll-like receptor 3 Human genes 0.000 description 4
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 4
- 102100039357 Toll-like receptor 5 Human genes 0.000 description 4
- 102100039387 Toll-like receptor 6 Human genes 0.000 description 4
- 102100039390 Toll-like receptor 7 Human genes 0.000 description 4
- 102100033110 Toll-like receptor 8 Human genes 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 241000269370 Xenopus <genus> Species 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 108010029483 alpha 1 Chain Collagen Type I Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 229920006317 cationic polymer Polymers 0.000 description 4
- 229940000425 combination drug Drugs 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000010668 complexation reaction Methods 0.000 description 4
- 210000002865 immune cell Anatomy 0.000 description 4
- 230000001771 impaired effect Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 230000002035 prolonged effect Effects 0.000 description 4
- 239000002213 purine nucleotide Substances 0.000 description 4
- 150000003212 purines Chemical class 0.000 description 4
- 125000000714 pyrimidinyl group Chemical group 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 230000006641 stabilisation Effects 0.000 description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- 102100026357 40S ribosomal protein S13 Human genes 0.000 description 3
- 102100024113 40S ribosomal protein S15a Human genes 0.000 description 3
- 101710198769 40S ribosomal protein S15a Proteins 0.000 description 3
- 102100033051 40S ribosomal protein S19 Human genes 0.000 description 3
- 102100022289 60S ribosomal protein L13a Human genes 0.000 description 3
- 102100024406 60S ribosomal protein L15 Human genes 0.000 description 3
- 102100021690 60S ribosomal protein L18a Human genes 0.000 description 3
- 101710187808 60S ribosomal protein L19 Proteins 0.000 description 3
- 101710187788 60S ribosomal protein L22 Proteins 0.000 description 3
- 101710187872 60S ribosomal protein L36 Proteins 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical group N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 3
- 208000035657 Abasia Diseases 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- 208000023275 Autoimmune disease Diseases 0.000 description 3
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 3
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 3
- 108010092160 Dactinomycin Proteins 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- 102100031334 Elongation factor 2 Human genes 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- 108060002716 Exonuclease Proteins 0.000 description 3
- 101000681240 Homo sapiens 60S ribosomal protein L13a Proteins 0.000 description 3
- 102000002265 Human Growth Hormone Human genes 0.000 description 3
- 108010000521 Human Growth Hormone Proteins 0.000 description 3
- 239000000854 Human Growth Hormone Substances 0.000 description 3
- 102000000589 Interleukin-1 Human genes 0.000 description 3
- 108010002352 Interleukin-1 Proteins 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 108010038807 Oligopeptides Proteins 0.000 description 3
- 102000015636 Oligopeptides Human genes 0.000 description 3
- 102000004285 Ribosomal Protein L3 Human genes 0.000 description 3
- 108090000894 Ribosomal Protein L3 Proteins 0.000 description 3
- 108090000986 Ribosomal protein L10 Proteins 0.000 description 3
- 102000013817 Ribosomal protein L13 Human genes 0.000 description 3
- 108050003655 Ribosomal protein L13 Proteins 0.000 description 3
- 102000004387 Ribosomal protein L14 Human genes 0.000 description 3
- 108090000985 Ribosomal protein L14 Proteins 0.000 description 3
- 108090000983 Ribosomal protein L15 Proteins 0.000 description 3
- 102000003926 Ribosomal protein L18 Human genes 0.000 description 3
- 108090000343 Ribosomal protein L18 Proteins 0.000 description 3
- 108050009586 Ribosomal protein L28 Proteins 0.000 description 3
- 108090000180 Ribosomal protein L31 Proteins 0.000 description 3
- 102000004209 Ribosomal protein L5 Human genes 0.000 description 3
- 108090000776 Ribosomal protein L5 Proteins 0.000 description 3
- 102000004394 Ribosomal protein S10 Human genes 0.000 description 3
- 108090000928 Ribosomal protein S10 Proteins 0.000 description 3
- 108050001197 Ribosomal protein S13 Proteins 0.000 description 3
- 108090000904 Ribosomal protein S2 Proteins 0.000 description 3
- 102000003861 Ribosomal protein S6 Human genes 0.000 description 3
- 108090000221 Ribosomal protein S6 Proteins 0.000 description 3
- 102000004282 Ribosomal protein S9 Human genes 0.000 description 3
- 108090000878 Ribosomal protein S9 Proteins 0.000 description 3
- 230000024932 T cell mediated immunity Effects 0.000 description 3
- 101001110004 Tetrahymena thermophila 60S acidic ribosomal protein P1 Proteins 0.000 description 3
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 3
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 3
- 230000003044 adaptive effect Effects 0.000 description 3
- 230000003172 anti-dna Effects 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 230000002238 attenuated effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000007969 cellular immunity Effects 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 229960000640 dactinomycin Drugs 0.000 description 3
- 239000000412 dendrimer Substances 0.000 description 3
- 229920000736 dendritic polymer Polymers 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 102000013165 exonuclease Human genes 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000002349 favourable effect Effects 0.000 description 3
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 3
- 229940029575 guanosine Drugs 0.000 description 3
- 230000028996 humoral immune response Effects 0.000 description 3
- 230000004727 humoral immunity Effects 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000015654 memory Effects 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000012261 overproduction Methods 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 229920000962 poly(amidoamine) Polymers 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 108010013519 rat ribosomal protein L8 Proteins 0.000 description 3
- 108010019034 ribosomal protein L18a Proteins 0.000 description 3
- 108010025463 ribosomal protein L24 Proteins 0.000 description 3
- 108010025498 ribosomal protein L29 Proteins 0.000 description 3
- 108010025327 ribosomal protein L30 Proteins 0.000 description 3
- 108010025396 ribosomal protein L34 Proteins 0.000 description 3
- 108010025387 ribosomal protein L39 Proteins 0.000 description 3
- 102000004291 ribosomal protein L6 Human genes 0.000 description 3
- 108090000892 ribosomal protein L6 Proteins 0.000 description 3
- 108010037046 ribosomal protein L7-L12 Proteins 0.000 description 3
- 102000004346 ribosomal protein L9 Human genes 0.000 description 3
- 108090000907 ribosomal protein L9 Proteins 0.000 description 3
- 108010092841 ribosomal protein S12 Proteins 0.000 description 3
- 108010088974 ribosomal protein S15a Proteins 0.000 description 3
- 108010093046 ribosomal protein S19 Proteins 0.000 description 3
- 108010092942 ribosomal protein S20 Proteins 0.000 description 3
- 108010092936 ribosomal protein S21 Proteins 0.000 description 3
- 108010011179 ribosomal protein S27a Proteins 0.000 description 3
- 108010093173 ribosomal protein S29 Proteins 0.000 description 3
- 108010033804 ribosomal protein S3 Proteins 0.000 description 3
- 102000004337 ribosomal protein S5 Human genes 0.000 description 3
- 108090000902 ribosomal protein S5 Proteins 0.000 description 3
- 108010033405 ribosomal protein S7 Proteins 0.000 description 3
- 108010033800 ribosomal protein S8 Proteins 0.000 description 3
- 108010067528 ribosomal proteins L27 Proteins 0.000 description 3
- 238000011105 stabilization Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 2
- 102100031571 40S ribosomal protein S16 Human genes 0.000 description 2
- 102100023587 ATP synthase F(0) complex subunit C2, mitochondrial Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- 108010083359 Antigen Receptors Proteins 0.000 description 2
- 101100305156 Brugia malayi rpp-2 gene Proteins 0.000 description 2
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 2
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- 208000017667 Chronic Disease Diseases 0.000 description 2
- 108091062157 Cis-regulatory element Proteins 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 2
- 102100027896 Cytochrome b-c1 complex subunit 7 Human genes 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- 102100022462 Eukaryotic initiation factor 4A-II Human genes 0.000 description 2
- 101710139370 Eukaryotic translation elongation factor 2 Proteins 0.000 description 2
- 102100040015 Eukaryotic translation initiation factor 2 subunit 3 Human genes 0.000 description 2
- 102100035045 Eukaryotic translation initiation factor 3 subunit C Human genes 0.000 description 2
- 102100033132 Eukaryotic translation initiation factor 3 subunit E Human genes 0.000 description 2
- 102100034255 Eukaryotic translation initiation factor 3 subunit F Human genes 0.000 description 2
- 102100037115 Eukaryotic translation initiation factor 3 subunit H Human genes 0.000 description 2
- 102100037110 Eukaryotic translation initiation factor 3 subunit K Human genes 0.000 description 2
- 102100038085 Eukaryotic translation initiation factor 3 subunit L Human genes 0.000 description 2
- 101000905797 Homo sapiens ATP synthase F(0) complex subunit C2, mitochondrial Proteins 0.000 description 2
- 101000875027 Homo sapiens Collagen alpha-1(X) chain Proteins 0.000 description 2
- 101001060428 Homo sapiens Cytochrome b-c1 complex subunit 7 Proteins 0.000 description 2
- 101001044475 Homo sapiens Eukaryotic initiation factor 4A-II Proteins 0.000 description 2
- 101000959829 Homo sapiens Eukaryotic translation initiation factor 2 subunit 3 Proteins 0.000 description 2
- 101000877285 Homo sapiens Eukaryotic translation initiation factor 3 subunit C Proteins 0.000 description 2
- 101000851079 Homo sapiens Eukaryotic translation initiation factor 3 subunit E Proteins 0.000 description 2
- 101000925825 Homo sapiens Eukaryotic translation initiation factor 3 subunit F Proteins 0.000 description 2
- 101000881778 Homo sapiens Eukaryotic translation initiation factor 3 subunit H Proteins 0.000 description 2
- 101000881804 Homo sapiens Eukaryotic translation initiation factor 3 subunit K Proteins 0.000 description 2
- 101000810389 Homo sapiens Eukaryotic translation initiation factor 3 subunit L Proteins 0.000 description 2
- 101001109719 Homo sapiens Nucleophosmin Proteins 0.000 description 2
- 101000979623 Homo sapiens Nucleoside diphosphate kinase B Proteins 0.000 description 2
- 101001045218 Homo sapiens Peroxisomal multifunctional enzyme type 2 Proteins 0.000 description 2
- 101000653679 Homo sapiens Translationally-controlled tumor protein Proteins 0.000 description 2
- 101000595682 Homo sapiens Tubulin beta-1 chain Proteins 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 2
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- 102000003820 Lipoxygenases Human genes 0.000 description 2
- 244000137850 Marrubium vulgare Species 0.000 description 2
- 101100368144 Mus musculus Synb gene Proteins 0.000 description 2
- 101100481579 Mus musculus Tlr11 gene Proteins 0.000 description 2
- 101100481580 Mus musculus Tlr12 gene Proteins 0.000 description 2
- 101100481581 Mus musculus Tlr13 gene Proteins 0.000 description 2
- 102100031789 Myeloid-derived growth factor Human genes 0.000 description 2
- 102100022678 Nucleophosmin Human genes 0.000 description 2
- 102100023258 Nucleoside diphosphate kinase B Human genes 0.000 description 2
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 102100022587 Peroxisomal multifunctional enzyme type 2 Human genes 0.000 description 2
- 206010057249 Phagocytosis Diseases 0.000 description 2
- 108010012887 Poly(A)-Binding Protein I Proteins 0.000 description 2
- 102000019200 Poly(A)-Binding Protein I Human genes 0.000 description 2
- 229920001212 Poly(beta amino esters) Polymers 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Chemical compound OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- 102100025234 Receptor of activated protein C kinase 1 Human genes 0.000 description 2
- 108010044157 Receptors for Activated C Kinase Proteins 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 102000004093 Ribosomal protein S15 Human genes 0.000 description 2
- 108090000530 Ribosomal protein S15 Proteins 0.000 description 2
- 108091081021 Sense strand Proteins 0.000 description 2
- 101710192266 Tegument protein VP22 Proteins 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- 210000000447 Th1 cell Anatomy 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical class O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102100029887 Translationally-controlled tumor protein Human genes 0.000 description 2
- 102100036084 Tubulin beta-1 chain Human genes 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102100040247 Tumor necrosis factor Human genes 0.000 description 2
- DBFUQOZREOHGAV-UAKXSSHOSA-N [[(2r,3s,4r,5r)-5-(4-amino-5-bromo-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=C(Br)C(N)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 DBFUQOZREOHGAV-UAKXSSHOSA-N 0.000 description 2
- YIJVOACVHQZMKI-JXOAFFINSA-N [[(2r,3s,4r,5r)-5-(4-amino-5-methyl-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O=C1N=C(N)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 YIJVOACVHQZMKI-JXOAFFINSA-N 0.000 description 2
- VEWJOCYCKIZKKV-GBNDHIKLSA-N [[(2r,3s,4r,5s)-5-(2,4-dioxo-1h-pyrimidin-5-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1C1=CNC(=O)NC1=O VEWJOCYCKIZKKV-GBNDHIKLSA-N 0.000 description 2
- 125000002015 acyclic group Chemical group 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000001588 bifunctional effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 230000004700 cellular uptake Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 230000024203 complement activation Effects 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 210000003162 effector t lymphocyte Anatomy 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 102000034356 gene-regulatory proteins Human genes 0.000 description 2
- 108091006104 gene-regulatory proteins Proteins 0.000 description 2
- 208000016361 genetic disease Diseases 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000000122 growth hormone Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 102000044123 human COL10A1 Human genes 0.000 description 2
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 239000012678 infectious agent Substances 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 238000007917 intracranial administration Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000007919 intrasynovial administration Methods 0.000 description 2
- 238000007913 intrathecal administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 210000002751 lymph Anatomy 0.000 description 2
- 210000003563 lymphoid tissue Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 108091035155 miR-10a stem-loop Proteins 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- DAZSWUUAFHBCGE-KRWDZBQOSA-N n-[(2s)-3-methyl-1-oxo-1-pyrrolidin-1-ylbutan-2-yl]-3-phenylpropanamide Chemical compound N([C@@H](C(C)C)C(=O)N1CCCC1)C(=O)CCC1=CC=CC=C1 DAZSWUUAFHBCGE-KRWDZBQOSA-N 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 229960003104 ornithine Drugs 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000008782 phagocytosis Effects 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 2
- 210000002729 polyribosome Anatomy 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 230000037425 regulation of transcription Effects 0.000 description 2
- 230000009712 regulation of translation Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 108010092955 ribosomal protein S16 Proteins 0.000 description 2
- 108090000842 ribosomal protein S18 Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000009469 supplementation Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 238000003151 transfection method Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 230000001960 triggered effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- CPCWCRHPTDMVFU-UHFFFAOYSA-N (2-hydroxy-4,5-dioctadecoxypentyl)-dimethylazanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCCOCC(CC(O)C[NH+](C)C)OCCCCCCCCCCCCCCCCCC CPCWCRHPTDMVFU-UHFFFAOYSA-N 0.000 description 1
- OFMQLVRLOGHAJI-FGHAYEPSSA-N (4r,7s,10s,13r,16s,19r)-n-[(2s,3r)-1-amino-3-hydroxy-1-oxobutan-2-yl]-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-10-[3-(diaminomethylideneamino)propyl]-7-[(1r)-1-hydroxyethyl]-16-[(4-hydroxyphenyl)methyl]-13-(1h-indol-3-ylmethyl)-3,3-dimethyl-6,9,12,15,18 Chemical compound C([C@H]1C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(=O)N[C@@H](C(SSC[C@@H](C(=O)N1)NC(=O)[C@H](N)CC=1C=CC=CC=1)(C)C)C(=O)N[C@@H]([C@H](O)C)C(N)=O)[C@@H](C)O)C1=CC=C(O)C=C1 OFMQLVRLOGHAJI-FGHAYEPSSA-N 0.000 description 1
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 description 1
- MPCAJMNYNOGXPB-UHFFFAOYSA-N 1,5-anhydrohexitol Chemical class OCC1OCC(O)C(O)C1O MPCAJMNYNOGXPB-UHFFFAOYSA-N 0.000 description 1
- KHWCHTKSEGGWEX-RRKCRQDMSA-N 2'-deoxyadenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(O)=O)O1 KHWCHTKSEGGWEX-RRKCRQDMSA-N 0.000 description 1
- NCMVOABPESMRCP-SHYZEUOFSA-N 2'-deoxycytosine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 NCMVOABPESMRCP-SHYZEUOFSA-N 0.000 description 1
- LTFMZDNNPPEQNG-KVQBGUIXSA-N 2'-deoxyguanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1C[C@H](O)[C@@H](COP(O)(O)=O)O1 LTFMZDNNPPEQNG-KVQBGUIXSA-N 0.000 description 1
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 1
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 1
- WALUVDCNGPQPOD-UHFFFAOYSA-M 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCC WALUVDCNGPQPOD-UHFFFAOYSA-M 0.000 description 1
- OULMZHKWILMJEK-UHFFFAOYSA-N 2-[4-(2,3-dihexadecoxypropoxy)-4-oxobutanoyl]oxyethyl-trimethylazanium Chemical compound CCCCCCCCCCCCCCCCOCC(COC(=O)CCC(=O)OCC[N+](C)(C)C)OCCCCCCCCCCCCCCCC OULMZHKWILMJEK-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
- ASJSAQIRZKANQN-UHFFFAOYSA-N 2-deoxypentose Chemical compound OCC(O)C(O)CC=O ASJSAQIRZKANQN-UHFFFAOYSA-N 0.000 description 1
- HYRQVGHJHUTCJL-UHFFFAOYSA-M 3,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCOC(CC[N+](C)(C)CCO)OCCCCCCCCCCCCCC HYRQVGHJHUTCJL-UHFFFAOYSA-M 0.000 description 1
- LZINOQJQXIEBNN-UHFFFAOYSA-N 4-hydroxybutyl dihydrogen phosphate Chemical compound OCCCCOP(O)(O)=O LZINOQJQXIEBNN-UHFFFAOYSA-N 0.000 description 1
- 102100039882 40S ribosomal protein S17 Human genes 0.000 description 1
- IWFHOSULCAJGRM-UAKXSSHOSA-N 5-bromouridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@@H](O)[C@@H]1N1C(=O)NC(=O)C(Br)=C1 IWFHOSULCAJGRM-UAKXSSHOSA-N 0.000 description 1
- XYVLZAYJHCECPN-UHFFFAOYSA-L 6-aminohexyl phosphate Chemical compound NCCCCCCOP([O-])([O-])=O XYVLZAYJHCECPN-UHFFFAOYSA-L 0.000 description 1
- 102100023990 60S ribosomal protein L17 Human genes 0.000 description 1
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- 108700031308 Antennapedia Homeodomain Proteins 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000537222 Betabaculovirus Species 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 102100028737 CAP-Gly domain-containing linker protein 1 Human genes 0.000 description 1
- 108010029697 CD40 Ligand Proteins 0.000 description 1
- 102100032937 CD40 ligand Human genes 0.000 description 1
- 101100530643 Caenorhabditis elegans rps-19 gene Proteins 0.000 description 1
- 101100093804 Caenorhabditis elegans rps-6 gene Proteins 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 208000031404 Chromosome Aberrations 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
- 102000005221 Cleavage Stimulation Factor Human genes 0.000 description 1
- 108010081236 Cleavage Stimulation Factor Proteins 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 102100035431 Complement factor I Human genes 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699662 Cricetomys gambianus Species 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 102100032620 Cytotoxic granule associated RNA binding protein TIA1 Human genes 0.000 description 1
- 101710086368 Cytotoxic granule associated RNA binding protein TIA1 Proteins 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- XULFJDKZVHTRLG-JDVCJPALSA-N DOSPA trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F.CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCNC(=O)C(CCCNCCCN)NCCCN)OCCCCCCCC\C=C/CCCCCCCC XULFJDKZVHTRLG-JDVCJPALSA-N 0.000 description 1
- 108700006830 Drosophila Antp Proteins 0.000 description 1
- 239000004150 EU approved colour Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150021185 FGF gene 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
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 101000767052 Homo sapiens CAP-Gly domain-containing linker protein 1 Proteins 0.000 description 1
- 101000866749 Homo sapiens Elongation factor 2 Proteins 0.000 description 1
- 101100443149 Homo sapiens HSD17B4 gene Proteins 0.000 description 1
- 101001002470 Homo sapiens Interferon lambda-1 Proteins 0.000 description 1
- 101000853002 Homo sapiens Interleukin-25 Proteins 0.000 description 1
- 101000853000 Homo sapiens Interleukin-26 Proteins 0.000 description 1
- 101000998139 Homo sapiens Interleukin-32 Proteins 0.000 description 1
- 101001128431 Homo sapiens Myeloid-derived growth factor Proteins 0.000 description 1
- 101000609211 Homo sapiens Polyadenylate-binding protein 2 Proteins 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102100026720 Interferon beta Human genes 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 102000003814 Interleukin-10 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108090000176 Interleukin-13 Proteins 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 102000003812 Interleukin-15 Human genes 0.000 description 1
- 101800003050 Interleukin-16 Proteins 0.000 description 1
- 102000049772 Interleukin-16 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 102100030704 Interleukin-21 Human genes 0.000 description 1
- 102100030703 Interleukin-22 Human genes 0.000 description 1
- 108010065637 Interleukin-23 Proteins 0.000 description 1
- 102000013264 Interleukin-23 Human genes 0.000 description 1
- 102100036679 Interleukin-26 Human genes 0.000 description 1
- 108010066979 Interleukin-27 Proteins 0.000 description 1
- 102100039064 Interleukin-3 Human genes 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 102100021596 Interleukin-31 Human genes 0.000 description 1
- 101710181613 Interleukin-31 Proteins 0.000 description 1
- 108010067003 Interleukin-33 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 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
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-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
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-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
- 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
- 108010063045 Lactoferrin Proteins 0.000 description 1
- 102100032241 Lactotransferrin Human genes 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 108090000362 Lymphotoxin-beta Proteins 0.000 description 1
- 102100026894 Lymphotoxin-beta Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091008060 MIR10A Proteins 0.000 description 1
- 108010047702 MPG peptide Proteins 0.000 description 1
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 102000013967 Monokines Human genes 0.000 description 1
- 108010050619 Monokines Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101000681246 Mus musculus 60S ribosomal protein L13a Proteins 0.000 description 1
- 101001026869 Mus musculus F-box/LRR-repeat protein 3 Proteins 0.000 description 1
- NSTPXGARCQOSAU-VIFPVBQESA-N N-formyl-L-phenylalanine Chemical compound O=CN[C@H](C(=O)O)CC1=CC=CC=C1 NSTPXGARCQOSAU-VIFPVBQESA-N 0.000 description 1
- 102000012064 NLR Proteins Human genes 0.000 description 1
- 108091005686 NOD-like receptors Proteins 0.000 description 1
- 241000737052 Naso hexacanthus Species 0.000 description 1
- 101100036257 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) rpl-30 gene Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700022034 Opsonin Proteins Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010088535 Pep-1 peptide Proteins 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 241000577979 Peromyscus spicilegus Species 0.000 description 1
- 101000622060 Photinus pyralis Luciferin 4-monooxygenase Proteins 0.000 description 1
- 102100039427 Polyadenylate-binding protein 2 Human genes 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 108010049404 Prokaryotic Initiation Factor-3 Proteins 0.000 description 1
- 102100035703 Prostatic acid phosphatase Human genes 0.000 description 1
- NUQJULCGNZMBEF-UHFFFAOYSA-N Prostratin Natural products COC(=O)C12CC(C)C3(O)C(C=C(CO)CC4(O)C3C=C(C)C4=O)C1C2(C)C NUQJULCGNZMBEF-UHFFFAOYSA-N 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 206010037075 Protozoal infections Diseases 0.000 description 1
- 108091005685 RIG-I-like receptors Proteins 0.000 description 1
- 108020005144 RNA 5' Terminal Oligopyrimidine Sequence Proteins 0.000 description 1
- 230000021839 RNA stabilization Effects 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 239000008156 Ringer's lactate solution Substances 0.000 description 1
- 102100027720 SH2 domain-containing protein 1A Human genes 0.000 description 1
- 108010045517 Serum Amyloid P-Component Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- RZCIEJXAILMSQK-JXOAFFINSA-N TTP Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 RZCIEJXAILMSQK-JXOAFFINSA-N 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical class O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 1
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- CAEFEWVYEZABLA-UUOKFMHZSA-N XTP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 CAEFEWVYEZABLA-UUOKFMHZSA-N 0.000 description 1
- JCAQMQLAHNGVPY-UUOKFMHZSA-N [(2r,3s,4r,5r)-3,4-dihydroxy-5-(2,2,4-trioxo-1h-imidazo[4,5-c][1,2,6]thiadiazin-7-yl)oxolan-2-yl]methyl dihydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(NS(=O)(=O)NC2=O)=C2N=C1 JCAQMQLAHNGVPY-UUOKFMHZSA-N 0.000 description 1
- NYDLOCKCVISJKK-WRBBJXAJSA-N [3-(dimethylamino)-2-[(z)-octadec-9-enoyl]oxypropyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(CN(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC NYDLOCKCVISJKK-WRBBJXAJSA-N 0.000 description 1
- GKVHYBAWZAYQDO-XVFCMESISA-N [[(2r,3s,4r,5r)-3,4-dihydroxy-5-(2-oxo-4-sulfanylidenepyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@@H](O)[C@@H]1N1C(=O)NC(=S)C=C1 GKVHYBAWZAYQDO-XVFCMESISA-N 0.000 description 1
- KHYOUGAATNYCAZ-XVFCMESISA-N [[(2r,3s,4r,5r)-3,4-dihydroxy-5-(4-oxo-2-sulfanylidenepyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@@H](O)[C@@H]1N1C(=S)NC(=O)C=C1 KHYOUGAATNYCAZ-XVFCMESISA-N 0.000 description 1
- ABOQIBZHFFLOGM-UAKXSSHOSA-N [[(2r,3s,4r,5r)-3,4-dihydroxy-5-(5-iodo-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@@H](O)[C@@H]1N1C(=O)NC(=O)C(I)=C1 ABOQIBZHFFLOGM-UAKXSSHOSA-N 0.000 description 1
- QTWNSBVFPSAMPO-IOSLPCCCSA-N [[(2r,3s,4r,5r)-3,4-dihydroxy-5-(6-imino-1-methylpurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=NC=2C(=N)N(C)C=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O QTWNSBVFPSAMPO-IOSLPCCCSA-N 0.000 description 1
- LCQWKKZWHQFOAH-IOSLPCCCSA-N [[(2r,3s,4r,5r)-3,4-dihydroxy-5-[6-(methylamino)purin-9-yl]oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O LCQWKKZWHQFOAH-IOSLPCCCSA-N 0.000 description 1
- CABDYDUZLRXGTB-UUOKFMHZSA-N [[(2r,3s,4r,5r)-5-(2,6-diaminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C12=NC(N)=NC(N)=C2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O CABDYDUZLRXGTB-UUOKFMHZSA-N 0.000 description 1
- YWHNPOKVSACYOQ-KQYNXXCUSA-N [[(2r,3s,4r,5r)-5-(2-amino-1-methyl-6-oxopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=NC=2C(=O)N(C)C(N)=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O YWHNPOKVSACYOQ-KQYNXXCUSA-N 0.000 description 1
- NCKFQXVRKKNRBB-SHUUEZRQSA-N [[(2r,3s,4r,5r)-5-(3,5-dioxo-1,2,4-triazin-2-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=N1 NCKFQXVRKKNRBB-SHUUEZRQSA-N 0.000 description 1
- WJUFDWJKJXOYSB-XVFCMESISA-N [[(2r,3s,4r,5r)-5-(4-amino-2-sulfanylidenepyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound S=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 WJUFDWJKJXOYSB-XVFCMESISA-N 0.000 description 1
- ZPZGYYNOHSQDQC-UAKXSSHOSA-N [[(2r,3s,4r,5r)-5-(4-amino-5-iodo-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=C(I)C(N)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 ZPZGYYNOHSQDQC-UAKXSSHOSA-N 0.000 description 1
- GVVRDIINMFAFEO-KCGFPETGSA-N [[(2r,3s,4r,5r)-5-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=CC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O GVVRDIINMFAFEO-KCGFPETGSA-N 0.000 description 1
- UOVXAGVICVPZQP-SHUUEZRQSA-N [[(2r,3s,4r,5r)-5-(5-amino-3-oxo-1,2,4-triazin-2-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O=C1N=C(N)C=NN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 UOVXAGVICVPZQP-SHUUEZRQSA-N 0.000 description 1
- PQISXOFEOCLOCT-UUOKFMHZSA-N [[(2r,3s,4r,5r)-5-(6-amino-8-azidopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound [N-]=[N+]=NC1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O PQISXOFEOCLOCT-UUOKFMHZSA-N 0.000 description 1
- WDPOFPOWJQWIPX-UUOKFMHZSA-N [[(2r,3s,4r,5r)-5-(7-aminotriazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound N1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O WDPOFPOWJQWIPX-UUOKFMHZSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229960003190 adenosine monophosphate Drugs 0.000 description 1
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 description 1
- 230000009824 affinity maturation Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000013602 bacteriophage vector Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 108010025307 buforin II Proteins 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000002837 carbocyclic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 108010059385 chemotactic factor inactivator Proteins 0.000 description 1
- 231100000005 chromosome aberration Toxicity 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- IERHLVCPSMICTF-XVFCMESISA-N cytidine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 IERHLVCPSMICTF-XVFCMESISA-N 0.000 description 1
- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000003467 diminishing effect Effects 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
- 230000006806 disease prevention Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000463 effect on translation Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000012178 germinal center formation Effects 0.000 description 1
- 125000003976 glyceryl group Chemical group [H]C([*])([H])C(O[H])([H])C(O[H])([H])[H] 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 1
- 235000013928 guanylic acid Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- PHNWGDTYCJFUGZ-UHFFFAOYSA-L hexyl phosphate Chemical compound CCCCCCOP([O-])([O-])=O PHNWGDTYCJFUGZ-UHFFFAOYSA-L 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 102000047408 human ASAH1 Human genes 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000000677 immunologic agent Substances 0.000 description 1
- 229940124541 immunological agent Drugs 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 108010074108 interleukin-21 Proteins 0.000 description 1
- 108010074109 interleukin-22 Proteins 0.000 description 1
- 108090000237 interleukin-24 Proteins 0.000 description 1
- 102000003898 interleukin-24 Human genes 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000005210 lymphoid organ Anatomy 0.000 description 1
- 102000004356 mRNA Cleavage and Polyadenylation Factors Human genes 0.000 description 1
- 108010042176 mRNA Cleavage and Polyadenylation Factors Proteins 0.000 description 1
- 230000034701 macropinocytosis Effects 0.000 description 1
- 210000001806 memory b lymphocyte Anatomy 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical group CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 108091064399 miR-10b stem-loop Proteins 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 229940006093 opthalmologic coloring agent diagnostic Drugs 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 108010043655 penetratin Proteins 0.000 description 1
- MCYTYTUNNNZWOK-LCLOTLQISA-N penetratin Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 MCYTYTUNNNZWOK-LCLOTLQISA-N 0.000 description 1
- 230000004526 pharmaceutical effect Effects 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- 229920001559 poly(2-methyloxazoline)-block-poly(dimethylsiloxane) Polymers 0.000 description 1
- 102000028499 poly(A) binding Human genes 0.000 description 1
- 108091023021 poly(A) binding Proteins 0.000 description 1
- 229920000083 poly(allylamine) Polymers 0.000 description 1
- 229920001308 poly(aminoacid) Polymers 0.000 description 1
- 229920000333 poly(propyleneimine) Polymers 0.000 description 1
- 229920002246 poly[2-(dimethylamino)ethyl methacrylate] polymer Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 108010011110 polyarginine Proteins 0.000 description 1
- 229920002851 polycationic polymer Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 108040000983 polyphosphate:AMP phosphotransferase activity proteins Proteins 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- REQCZEXYDRLIBE-UHFFFAOYSA-N procainamide Chemical compound CCN(CC)CCNC(=O)C1=CC=C(N)C=C1 REQCZEXYDRLIBE-UHFFFAOYSA-N 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 108010025578 ribosomal protein L17 Proteins 0.000 description 1
- 108010093121 ribosomal protein S17 Proteins 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000003019 stabilising effect Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000005451 thionucleotide Substances 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 239000012049 topical pharmaceutical composition Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 108010062760 transportan Proteins 0.000 description 1
- PBKWZFANFUTEPS-CWUSWOHSSA-N transportan Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(N)=O)[C@@H](C)CC)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CN)[C@@H](C)O)C1=CC=C(O)C=C1 PBKWZFANFUTEPS-CWUSWOHSSA-N 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
-
- 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
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/10—Vectors comprising a special translation-regulating system regulates levels of translation
- C12N2840/105—Vectors comprising a special translation-regulating system regulates levels of translation enhancing translation
Definitions
- the invention relates to artificial nucleic acid molecules comprising a 5'UTR element derived from the 5'UTR of a TOP gene, an open reading frame, and optionally a 3'UTR element, a poly(A) sequence and/or a polyadenylation signal.
- the invention relates further to a vector comprising a 5'UTR element derived from the 5'UTR of a TOP gene, to a pharmaceutical composition comprising the artificial nucleic acid molecule or the vector, and to a kit comprising the artificial nucleic acid molecule, the vector and/or the pharmaceutical composition, preferably for use in the field of gene therapy and/or genetic vaccination.
- Gene therapy and genetic vaccination belong to the most promising and quickly developing methods of modern medicine.
- Pathologically altered gene expression may result in lack or overproduction of essential gene products, for example, signalling factors such as hormones, housekeeping factors, metabolic enzymes, structural proteins or the like. Altered gene expression may not only be due to mis- regulation of transcription and/or translation, but also due to mutations within the ORF coding for a particular protein. Pathological mutations may be caused by e.g. chromosomal aberration, or by more specific mutations, such as point or frame-shift-mutations, all of them resulting in limited functionality and, potentially, total loss of function of the gene product.
- misregulation of transcription or translation may also occur, if mutations affect genes encoding proteins which are involved in the transcriptional or translational machinery of the cell. Such mutations may lead to pathological up- or down-regulation of genes which are - as such - functional. Genes encoding gene products which exert such regulating functions, may be, e.g., transcription factors, signal receptors, messenger proteins or the like. However, loss of function of such genes encoding regulatory proteins may, under certain circumstances, be reversed by artificial introduction of other factors acting further downstream of the impaired gene product. Such gene defects may also be compensated by gene therapy via substitution of the affected gene itself.
- vaccines may be subdivided into “first”, “second” and “third” generation vaccines.
- First generation vaccines are, typically, whole-organism vaccines. They are based on either live and attenuated or killed pathogens, e.g. viruses, bacteria or the like. The major drawback of live and attenuated vaccines is the risk for a reversion to life- threatening variants. Thus, although attenuated, such pathogens may still intrinsically bear unpredictable risks. Killed pathogens may not be as effective as desired for generating a specific immune response. In order to minimize these risks, “second generation” vaccines were developed. These are, typically, subunit vaccines, consisting of defined antigens or recombinant protein components which are derived from pathogens.
- Genetic vaccines i.e. vaccines for genetic vaccination, are usually understood as "third generation” vaccines. They are typically composed of genetically engineered nucleic acid molecules which allow expression of peptide or protein (antigen) fragments characteristic for a pathogen or a tumor antigen in vivo. Genetic vaccines are expressed upon administration to a patient and uptake by competent cells. Expression of the administered nucleic acids results in production of the encoded proteins. In the event these proteins are recognized as foreign by the patient's immune system, an immune response is triggered.
- both methods, gene therapy and genetic vaccination are essentially based on the administration of nucleic acid molecules to a patient and subsequent transcription and/or translation of the encoded genetic information.
- genetic vaccination or gene therapy may also comprise methods which include isolation of specific body cells from a patient to be treated, subsequent in vitro transfection of such cells, and re-administration of the treated cells to the patient.
- DNA as well as RNA may be used as nucleic acid molecules for administration in the context of gene therapy or genetic vaccination.
- DNA is known to be relatively stable and easy to handle.
- the use of DNA bears the risk of undesired insertion of the administered DNA-fragments into the patient's genome potentially resulting in loss of function of the impaired genes.
- the undesired generation of anti-DNA antibodies has emerged.
- Another drawback is the limited expression level of the encoded peptide or protein that is achievable upon DNA administration and its transcription/translation.
- the expression level of the administered DNA will be dependent on the presence of specific transcription factors which regulate DNA transcription. In the absence of such factors, DNA transcription will not yield satisfying amounts of RNA. As a result, the level of translated peptide or protein obtained is limited.
- RNA is considered to be a rather unstable molecular species which may readily be degraded by ubiquitous RNAses.
- RNA-degradation contributes to the regulation of the RNA half-life time. That effect was considered and proven to fine tune the regulation of eukaryotic gene expression (Friedel et a/., conserveed principles of mammalian transcriptional regulation revealed by RNA half-life, Nucleic Acid Research, 2009, 1 -12). Accordingly, each naturally occurring mRNA has its individual half-life depending on the gene from which the mRNA is derived. It contributes to the regulation of the expression level of this gene. Unstable RNAs are important to realize transient gene expression at distinct points in time. However, long-lived RNAs may be associated with accumulation of distinct proteins or continuous expression of genes.
- mRNAs may also be dependent on environmental factors, such as hormonal treatment, as has been shown, e.g., for insulin-like growth factor I, actin, and albumin mRNA Gohnson et a/., Newly synthesized RNA: Simultaneous measurement in intact cells of transcription rates and RNA stability of insulin-like growth factor I, actin, and albumin in growth hormone-stimulated hepatocytes, Proc. Natl. Acad. Sci., Vol. 88, pp. 5287-5291 , 1991 ).
- RNA For gene therapy and genetic vaccination, usually stable RNA is desired. This is, on the one hand, due to the fact that the product encoded by the RNA-sequence shall accumulate in vivo. On the other hand, the RNA has to maintain its structural and functional integrity when prepared for a suitable dosage form, in the course of its storage, and when administered. Thus, considerable attention was dedicated to provide stable RNA molecules for gene therapy or genetic vaccination in order to prevent them from being subject to early degradation or decay. It has been reported that the G/C-content of nucleic acid molecules may influence their stability.
- nucleic acids comprising an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides.
- WO02/098443 provides a pharmaceutical composition containing an mRNA that is stabilised by sequence modifications in the translated region. Such a sequence modification takes advantage of the degeneracy of the genetic code. Accordingly, codons which contain a less favourable combination of nucleotides (less favourable in terms of RNA stability) may be substituted by alternative codons without altering the encoded amino acid sequence.
- This method of RNA stabilization is limited by the provisions of the specific nucleotide sequence of each single RNA molecule which is not allowed to leave the space of the desired amino acid sequence. Also, that approach is restricted to coding regions of the RNA.
- mRNA stabilisation As an alternative option for mRNA stabilisation, it has been found that naturally occurring eukaryotic mRNA molecules contain characteristic stabilising elements. For example, they may comprise so-called untranslated regions (UTR) at their 5'-end (5'UTR) and/or at their 3'- end (3'UTR) as well as other structural features, such as a 5'-cap structure or a 3'- poly(A) tail. Both, 5'UTR and 3'UTR are typically transcribed from the genomic DNA and are, thus, an element of the premature mRNA.
- UTR untranslated regions
- Characteristic structural features of mature mRNA such as the 5'-cap and the 3'-poly(A) tail (also called poly(A) tail or poly(A) sequence) are usually added to the transcribed (premature) mRNA during mRNA processing.
- a 3'-poly(A) tail is typically a monotonous sequence stretch of adenine nucleotides added to the 3 '-end of the transcribed mRNA. It may comprise up to about 400 adenine nucleotides. It was found that the length of such a 3'-poly(A) tail is a potentially critical element for the stability of the individual mRNA.
- RNA, 8, pp. 1526-1537, 2002 may be an important factor for the well-known stability of ⁇ -globin mRNA (Rodgers et a/., Regulated ⁇ -globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping, RNA, 8, pp. 1526-1537, 2002).
- ⁇ -globin mRNA The 3'UTR of ⁇ -globin mRNA is obviously involved in the formation of a specific ribonucleoprotein-complex, the a-complex, whose presence correlates with mRNA stability in vitro (Wang et a/., An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro, Molecular and Cellular biology, Vol 19, No. 7, July 1999, p. 4552-4560).
- proteins belonging to the translational apparatus may be regulated not only at the transcriptional but also at the translational level.
- translation of proteins encoded by so called 'TOP-genes' may be down-regulated by translational repression.
- 'TOP-gene' relates to a gene corresponding to an mRNA that is characterized by the presence of a TOP sequence at the 5'end and in most cases by a growth-associated translation regulation (ladevaia et a/., All translation elongation factors and the e, f, and h subunits of translation initiation factor 3 are encoded by 5'-terminal oligopyrimidine (TOP) mRNAs; RNA, 2008, 14:1 730-1 736).
- TOP 5'-terminal oligopyrimidine
- a TOP sequence - also called the '5'-terminal oligopyrimidine tract' - typically consists of a C residue at the cap site, followed by an uninterrupted sequence of up to 13 or even more pyrimidines (Avni et a/., Vertebrate mRNAs with a 5'-terminal pyrimidine tract are Candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element, Molecular and Cellular Biology, 1994, p. 3822-3833).
- TOP sequences are reported to be present in many mRNAs encoding components of the translational machinery and to be responsible for selective repression of the translation of these TOP containing mRNAs due to growth arrest (Meyuhas, eta/., Translational Control of Ribosomal Protein mRNAs in Eukaryotes, Translational Control. Cold Spring Harbor Monograph Archive. Cold Spring Harbor Laboratory Press, 1996, p. 363-388). These TOP sequences are thought to serve as a cis-regulatory element which inhibits the binding of translational regulatory proteins or the translational machinery itself. As a result, the translation of these genes is inhibited at the growth arrest of cells.
- mRNAs of TOP genes which are normally associated with polysomes, change their status into the translational ly inactive 'sub-polysome' while most non-TOP mRNAs stay in the'polysome' state (Yamashita et a/., Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics. Nucleic Acids Res. 2008 Jun;36(1 1 ):3707-15. doi: 10.1093/nar/gkn248. Epub 2008 May 14).
- oligopyrimidine tract at the 5'end of the 5'UTR was required for translational repression of TOP genes.
- the oligopyrimidine tract at the 5' end of mammalian ribosomal protein mRNAs is required for their translational control (Levy et a/., Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):331 9-23).
- miRNA miR-10a positively controls the translation of ribosomal proteins by binding downstream of the TOP motif present in the 5'UTRs of TOP genes. Such an enhancement of translation was dependent on the presence of the TOP motif in the 5'UTR.
- nucleic acid molecules which may be suitable for application in gene therapy and/or genetic vaccination.
- Another object of the present invention is to provide nucleic acid molecules coding for such a superior mRNA species which may be amenable for use in gene therapy and/or genetic vaccination.
- nucleic acid molecules coding for such a superior mRNA species which may be amenable for use in gene therapy and/or genetic vaccination.
- it is the object of the present invention to provide improved nucleic acid species which overcome the above discussed disadvantages of the prior art by a cost-effective and straight-forward approach.
- the object underlying the present invention is solved by the claimed subject-matter.
- Adaptive immune response The adaptive immune response is typically understood to be an antigen-specific response of the immune system. Antigen specificity allows for the generation of responses that are tailored to specific pathogens or pathogen-infected cells. The ability to mount these tailored responses is usually maintained in the body by "memory cells". Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it.
- the first step of an adaptive immune response is the activation of naive antigen-specific T cells or different immune cells able to induce an antigen-specific immune response by antigen-presenting cells.
- This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing.
- the three cell types that may serve as antigen-presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses.
- Dendritic cells may take up antigens by phagocytosis and macropinocytosis and may become stimulated by contact with e.g. a foreign antigen to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells.
- Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express MHC molecules.
- the unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells.
- MHC-molecules are, typically, responsible for presentation of an antigen to T-cells. Therein, presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells.
- T cells recognize an antigen by their T cell receptors which do not recognize and bind the antigen directly, but instead recognize short peptide fragments e.g. of pathogen-derived protein antigens, e.g. so-called epitopes, which are bound to MHC molecules on the surfaces of other cells.
- the adaptive immune system is essentially dedicated to eliminate or prevent pathogenic growth. It typically regulates the adaptive immune response by providing the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered.
- the system is highly adaptable because of somatic hypermutation (a process of accelerated somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte.
- Adjuvant/adjuvant component in the broadest sense is typically a pharmacological and/or immunological agent that may modify, e.g. enhance, the effect of other agents, such as a drug or vaccine. It is to be interpreted in a broad sense and refers to a broad spectrum of substances. Typically, these substances are able to increase the immunogenicity of antigens.
- adjuvants may be recognized by the innate immune systems and, e.g., may elicit an innate immune response. "Adjuvants" typically do not elicit an adaptive immune response. Insofar, "adjuvants" do not qualify as antigens. Their mode of action is distinct from the effects triggered by antigens resulting in an adaptive immune response.
- Antigen refers typically to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response.
- an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells.
- An artificial nucleic acid molecule may typically be understood to be a nucleic acid molecule, e.g. a DNA or an RNA, that does not occur naturally.
- an artificial nucleic acid molecule may be understood as a non- natural nucleic acid molecule.
- Such nucleic acid molecule may be non-natural due to its individual sequence (which does not occur naturally) and/or due to other modifications, e.g. structural modifications of nucleotides which do not occur naturally.
- An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule or a hybrid-molecule comprising DNA and RNA portions.
- artificial nucleic acid molecules may be designed and/or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence).
- an artificial sequence is usually a sequence that may not occur naturally, i.e. it differs from the wild type sequence by at least one nucleotide.
- the term 'wild type' may be understood as a sequence occurring in nature.
- the term 'artificial nucleic acid molecule' is not restricted to mean 'one single molecule' but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.
- Bicistronic RNA, multicistronic RNA A bicistronic or multicistronic RNA is typically an RNA, preferably an mRNA, that typically may have two (bicistronic) or more (multicistronic) open reading frames (ORF).
- An open reading frame in this context is a sequence of codons that is translatable into a peptide or protein.
- Carrier / polymeric carrier A carrier in the context of the invention may typically be a compound that facilitates transport and/or complexation of another compound (cargo).
- a polymeric carrier is typically a carrier that is formed of a polymer.
- a carrier may be associated to its cargo by covalent or non-covalent interaction.
- a carrier may transport nucleic acids, e.g. RNA or DNA, to the target cells.
- the carrier may - for some embodiments - be a cationic component.
- Cationic component typically refers to a charged molecule, which is positively charged (cation) at a pH value typically from 1 to 9, preferably at a pH value of or below 9 (e.g. from 5 to 9), of or below 8 (e.g. from 5 to 8), of or below 7 (e.g. from 5 to 7), most preferably at a physiological pH, e.g. from 7.3 to 7.4.
- a cationic component may be any positively charged compound or polymer, preferably a cationic peptide or protein which is positively charged under physiological conditions, particularly under physiological conditions in vivo.
- a 'cationic peptide or protein' may contain at least one positively charged amino acid, or more than one positively charged amino acid, e.g. selected from Arg, His, Lys or Orn. Accordingly, 'polycationic' components are also within the scope exhibiting more than one positive charge under the conditions given.
- a 5'-cap is an entity, typically a modified nucleotide entity, which generally
- a 5 '-cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide.
- the 5'-cap is linked to the 5'-terminus via a 5'-5'-triphosphate linkage.
- a 5'-cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap, typically the 5'-end of an RNA.
- 5'cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, 1 -(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3
- Cellular immunity relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T- lymphocytes, and the release of various cytokines in response to an antigen.
- cellular immunity is not based on antibodies, but on the activation of cells of the immune system.
- a cellular immune response may be characterized e.g. by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in cells, e.g. specific immune cells like dendritic cells or other cells, displaying epitopes of foreign antigens on their surface.
- Such cells may be virus-infected or infected with intracellular bacteria, or cancer cells displaying tumor antigens. Further characteristics may be activation of macrophages and natural killer cells, enabling them to destroy pathogens and stimulation of cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.
- DNA is the usual abbreviation for deoxy-ribonucleic-acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually deoxy- adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine- monophosphate and deoxy-cytidine-monophosphate monomers which are - by themselves - composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerize by a characteristic backbone structure.
- the backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i.e. deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer.
- the specific order of the monomers i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the DNA-sequence.
- DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g. by A/T-base-pairing and G/C-base-pairing.
- Epitopes can be distinguished in T cell epitopes and B cell epitopes.
- T cell epitopes or parts of the proteins in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g.
- B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form.
- Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides.
- antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
- Fragment of a sequence A fragment of a sequence may typically be a shorter portion of a full-length sequence of e.g. a nucleic acid molecule or an amino acid sequence.
- a fragment typically, consists of a sequence that is identical to the corresponding stretch within the full-length sequence.
- a preferred fragment of a sequence in the context of the present invention consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the total (i.e. full- length) molecule from which the fragment is derived.
- a G/C-modified nucleic acid may typically be a nucleic acid, preferably an artificial nucleic acid molecule as defined herein, based on a modified wild- type sequence comprising a preferably increased number of guanosine and/or cytosine nucleotides as compared to the wild-type sequence. Such an increased number may be generated by substitution of codons containing adenosine or thymidine nucleotides by codons containing guanosine or cytosine nucleotides. If the enriched G/C content occurs in a coding region of DNA or RNA, it makes use of the degeneracy of the genetic code. Accordingly, the codon substitutions preferably do not alter the encoded amino acid residues, but exclusively increase the G/C content of the nucleic acid molecule.
- Gene therapy may typically be understood to mean a treatment of a patient's body or isolated elements of a patient's body, for example isolated tissues/cells, by nucleic acids encoding a peptide or protein. It typically may comprise at least one of the steps of a) administration of a nucleic acid, preferably an artificial nucleic acid molecule as defined herein, directly to the patient - by whatever administration route - or in vitro to isolated cells/tissues of the patient, which results in transfection of the patient's cells either in vivo/ex vivo or in vitro, b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administration of isolated, transfected cells to the patient, if the nucleic acid has not been administered directly to the patient.
- a nucleic acid preferably an artificial nucleic acid molecule as defined herein
- Genetic vaccination may typically be understood to be vaccination by administration of a nucleic acid molecule encoding an antigen or an immunogen or fragments thereof.
- the nucleic acid molecule may be administered to a subject's body or to isolated cells of a subject. Upon transfection of certain cells of the body or upon transfection of the isolated cells, the antigen or immunogen may be expressed by those cells and subsequently presented to the immune system, eliciting an adaptive, i.e. antigen- specific immune response.
- genetic vaccination typically comprises at least one of the steps of a) administration of a nucleic acid, preferably an artificial nucleic acid molecule as defined herein, to a subject, preferably a patient, or to isolated cells of a subject, preferably a patient, which usually results in transfection of the subject's cells either in vivo or in vitro, b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administration of isolated, transfected cells to the subject, preferably the patient, if the nucleic acid has not been administered directly to the patient.
- Heterologous sequence Two sequences are typically understood to be 'heterologous' if they are not derivable from the same gene. I.e., although heterologous sequences may be derivable from the same organism, they naturally (in nature) do not occur in the same nucleic acid molecule, such as in the same mRNA.
- Humoral immunity refers typically to antibody production and optionally to accessory processes accompanying antibody production.
- a humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation.
- Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.
- an immunogen may be typically understood to be a compound that is able to stimulate an immune response.
- an immunogen is a peptide, polypeptide, or protein.
- an immunogen in the sense of the present invention is the product of translation of a provided nucleic acid molecule, preferably an artificial nucleic acid molecule as defined herein.
- an immunogen elicits at least an adaptive immune response.
- an immunostimulatory composition may be typically understood to be a composition containing at least one component which is able to induce an immune response or from which a component which is able to induce an immune response is derivable. Such immune response may be preferably an innate immune response or a combination of an adaptive and an innate immune response.
- an immunostimulatory composition in the context of the invention contains at least one artificial nucleic acid molecule, more preferably an RNA, for example an mRNA molecule.
- the immunostimulatory component, such as the mRNA may be complexed with a suitable carrier.
- the immunostimulatory composition may comprise an mRNA/carrier-complex.
- the immunostimulatory composition may comprise an adjuvant and/or a suitable vehicle for the immunostimulatory component, such as the mRNA.
- Immune response An immune response may typically be a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response), or a combination thereof.
- the immune system may protect organisms from infection. If a pathogen succeeds in passing a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system.
- the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered.
- the immune system comprises the innate and the adaptive immune system. Each of these two parts typically contains so called humoral and cellular components.
- Immunostimulatory RNA in the context of the invention may typically be an RNA that is able to induce an innate immune response. It usually does not have an open reading frame and thus does not provide a peptide-antigen or immunogen but elicits an immune response e.g. by binding to a specific kind of Toll-like- receptor (TLR) or other suitable receptors.
- TLR Toll-like- receptor
- mRNAs having an open reading frame and coding for a peptide/protein may induce an innate immune response and, thus, may be immunostimulatory RNAs.
- the innate immune system also known as non-specific (or unspecific) immune system, typically comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system may recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host.
- the innate immune system may be, e.g., activated by ligands of Toll-like receptors (TLRs) or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1 , IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-1 7, IL-18, IL-19, IL- 20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31 , IL-32, IL-33, IFN- alpha, IFN-beta, IFN-gamma, GM-C
- the pharmaceutical composition according to the present invention may comprise one or more such substances.
- a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system; and/or acting as a physical and chemical barrier to infectious agents.
- a cloning site is typically understood to be a segment of a nucleic acid molecule, which is suitable for insertion of a nucleic acid sequence, e.g., a nucleic acid sequence comprising an open reading frame. Insertion may be performed by any molecular biological method known to the one skilled in the art, e.g. by restriction and ligation.
- a cloning site typically comprises one or more restriction enzyme recognition sites (restriction sites). These one or more restrictions sites may be recognized by restriction enzymes which cleave the DNA at these sites.
- a cloning site which comprises more than one restriction site may also be termed a multiple cloning site (MCS) or a polylinker.
- MCS multiple cloning site
- Nucleic acid molecule is a molecule comprising, preferably consisting of nucleic acid components.
- the term nucleic acid molecule preferably refers to DNA or RNA molecules. It is preferably used synonymous with the term "polynucleotide".
- a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone.
- the term "nucleic acid molecule” also encompasses modified nucleic acid molecules, such as base-modified, sugar-modified or backbone-modified etc. DNA or RNA molecules.
- Open reading frame in the context of the invention may typically be a sequence of several nucleotide triplets which may be translated into a peptide or protein.
- An open reading frame preferably contains a start codon, i.e. a combination of three subsequent nucleotides coding usually for the amino acid methionine (ATG or AUG), at its 5'-end and a subsequent region which usually exhibits a length which is a multiple of 3 nucleotides.
- An ORF is preferably terminated by a stop-codon (e.g., TAA, TAG, TGA). Typically, this is the only stop-codon of the open reading frame.
- an open reading frame in the context of the present invention is preferably a nucleotide sequence, consisting of a number of nucleotides that may be divided by three, which starts with a start codon (e.g. ATG or AUG) and which preferably terminates with a stop codon (e.g., TAA, TGA, or TAG or UAA, UAG, UGA, respectively).
- the open reading frame may be isolated or it may be incorporated in a longer nucleic acid sequence, for example in a vector or an mRNA.
- An open reading frame may also be termed 'protein coding region'.
- a peptide or polypeptide is typically a polymer of amino acid monomers, linked by peptide bonds. It typically contains less than 50 monomer units. Nevertheless, the term peptide is not a disclaimer for molecules having more than 50 monomer units. Long peptides are also called polypeptides, typically having between 50 and 600 monomeric units.
- a pharmaceutically effective amount in the context of the invention is typically understood to be an amount that is sufficient to induce a pharmaceutical effect, such as an immune response, altering a pathological level of an expressed peptide or protein, or substituting a lacking gene product, e.g., in case of a pathological situation.
- Protein A protein typically comprises one or more peptides or polypeptides.
- a protein is typically folded into 3-dimensional form, which may be required for to protein to exert its biological function.
- a poly(A) sequence also called poly(A) tail or 3'-poly(A) tail, is typically understood to be a sequence of adenine nucleotides, e.g., of up to about 400 adenine nucleotides, e.g. from about 20 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenine nucleotides.
- a poly(A) sequence is typically located at the 3'end of an mRNA.
- a poly(A) sequence may be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector.
- Polyadenylation is typically understood to be the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule, e.g. to a premature mRNA. Polyadenylation may be induced by a so called polyadenylation signal. This signal is preferably located within a stretch of nucleotides at the 3'-end of a nucleic acid molecule, such as an RNA molecule, to be polyadenylated.
- a polyadenylation signal typically comprises a hexamer consisting of adenine and uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA.
- RNA maturation from pre-mRNA to mature mRNA comprises the step of polyadenylation.
- a restriction site also termed 'restriction enzyme recognition site', is a nucleotide sequence recognized by a restriction enzyme.
- a restriction site is typically a short, preferably palindromic nucleotide sequence, e.g. a sequence comprising 4 to 8 nucleotides.
- a restriction site is preferably specifically recognized by a restriction enzyme.
- the restriction enzyme typically cleaves a nucleotide sequence comprising a restriction site at this site. In a double-stranded nucleotide sequence, such as a double-stranded DNA sequence, the restriction enzyme typically cuts both strands of the nucleotide sequence.
- RNA is the usual abbreviation for ribonucleic-acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually adenosine- monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine- monophosphate monomers which are connected to each other along a so-called backbone.
- the backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer.
- the specific succession of the monomers is called the RNA-sequence.
- RNA may be obtainable by transcription of a DNA-sequence, e.g., inside a cell.
- transcription is typically performed inside the nucleus or the mitochondria.
- transcription of DNA usually results in the so-called premature RNA which has to be processed into so-called messenger-RNA, usually abbreviated as mRNA.
- Processing of the premature RNA e.g. in eukaryotic organisms, comprises a variety of different posttranscriptional-modifications such as splicing, 5'- capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA.
- the mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein.
- a mature mRNA comprises a 5'-cap, a 5'UTR, an open reading frame, a 3'UTR and a poly(A) sequence.
- sequence of a nucleic acid molecule The sequence of a nucleic acid molecule is typically understood to be the particular and individual order, i.e. the succession of its nucleotides.
- the sequence of a protein or peptide is typically understood to be the order, i.e. the succession of its amino acids.
- Sequence identity Two or more sequences are identical if they exhibit the same length and order of nucleotides or amino acids.
- the percentage of identity typically describes the extent to which two sequences are identical, i.e. it typically describes the percentage of nucleotides that correspond in their sequence position with identical nucleotides of a reference-sequence.
- the sequences to be compared are considered to exhibit the same length, i.e. the length of the longest sequence of the sequences to be compared. This means that a first sequence consisting of 8 nucleotides is 80% identical to a second sequence consisting of 10 nucleotides comprising the first sequence.
- identity of sequences preferably relates to the percentage of nucleotides of a sequence which have the same position in two or more sequences having the same length. Gaps are usually regarded as non-identical positions, irrespective of their actual position in an alignment.
- a stabilized nucleic acid molecule is a nucleic acid molecule, preferably a DNA or RNA molecule that is modified such, that it is more stable to disintegration or degradation, e.g., by environmental factors or enzymatic digest, such as by an exo- or endonuclease degradation, than the nucleic acid molecule without the modification.
- a stabilized nucleic acid molecule in the context of the present invention is stabilized in a cell, such as a prokaryotic or eukaryotic cell, preferably in a mammalian cell, such as a human cell.
- the stabilization effect may also be exerted outside of cells, e.g. in a buffer solution etc., for example, in a manufacturing process for a pharmaceutical composition comprising the stabilized nucleic acid molecule.
- Transfection refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably into eukaryotic cells.
- nucleic acid molecules such as DNA or RNA (e.g. mRNA) molecules
- the term 'transfection' encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, preferably into eukaryotic cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g.
- the introduction is non-viral.
- a vaccine is typically understood to be a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen.
- the antigen or immunogen may be derived from any material that is suitable for vaccination.
- the antigen or immunogen may be derived from a pathogen, such as from bacteria or virus particles etc., or from a tumor or cancerous tissue.
- the antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response.
- Vector The term 'vector' refers to a nucleic acid molecule, preferably to an artificial nucleic acid molecule.
- a vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame.
- Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc.
- a storage vector is a vector which allows the convenient storage of a nucleic acid molecule, for example, of an mRNA molecule.
- the vector may comprise a sequence corresponding, e.g., to a desired mRNA sequence or a part thereof, such as a sequence corresponding to the open reading frame and the 3'UTR of an mRNA.
- An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.
- an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a promoter sequence, e.g.
- a cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector.
- a cloning vector may be, e.g., a plasmid vector or a bacteriophage vector.
- a transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors.
- a vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector.
- a vector is a DNA molecule.
- a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication.
- a vector in the context of the present application is a plasmid vector.
- Vehicle A vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound.
- it may be a physiologically acceptable liquid which is suitable for storing, transporting, and/or administering a pharmaceutically active compound.
- 3'-untranslated region (3'UTR) A 3'UTR is typically the part of an mRNA which is located between the protein coding region (i.e.
- a 3'UTR of the mRNA is not translated into an amino acid sequence.
- the 3'UTR sequence is generally encoded by the gene which is transcribed into the respective mRNA during the gene expression process.
- the genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns.
- the pre-mature mRNA is then further processed into mature mRNA in a maturation process.
- This maturation process comprises the steps of 5'capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3'-end, such as polyadenylation of the 3'-end of the premature mRNA and optional endo- or exonuclease cleavages etc.
- a 3'UTR corresponds to the sequence of a mature mRNA which is located 3' to the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and which extends to the 5'-side of the poly(A) sequence, preferably to the nucleotide immediately 5' to the poly(A) sequence.
- the 3'UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3'UTR sequence, or a DNA sequence which corresponds to such RNA sequence.
- a 3'UTR of a gene such as "a 3'UTR of an albumin gene” is the sequence which corresponds to the 3'UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
- the term "3'UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 3'UTR.
- a 5'-untranslated region is typically understood to be a particular section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Typically, the 5'UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame.
- the 5'UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites or a 5'-Terminal Oligopyrimidine Tract.
- the 5'UTR may be posttranscriptionally modified, for example by addition of a 5'-cap.
- a 5'UTR corresponds to the sequence of a mature mRNA which is located between the 5'cap and the start codon.
- the 5'UTR corresponds to the sequence which extends from a nucleotide located 3' to the 5'-cap, preferably from the nucleotide located immediately 3' to the 5'cap, to a nucleotide located 5' to the start codon of the protein coding region, preferably to the nucleotide located immediately 5' to the start codon of the protein coding region.
- the nucleotide located immediately 3' to the 5'cap of a mature mRNA typically corresponds to the transcriptional start site.
- the term “corresponds to” means that the 5'UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 5'UTR sequence, or a DNA sequence which corresponds to such RNA sequence.
- a 5'UTR of a gene such as "a 5'UTR of a TOP gene” is the sequence which corresponds to the 5'UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
- the term “5'UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 5'UTR.
- TOP 5'Terminal Oligopyrimidine Tract
- the 5'terminal oligopyrimidine tract (TOP) is typically a stretch of pyrimidine nucleotides located at the 5' terminal region of a nucleic acid molecule, such as the 5' terminal region of certain mRNA molecules or the 5' terminal region of a functional entity, e.g. the transcribed region, of certain genes.
- the sequence starts with a cytidine, which usually corresponds to the transcriptional start site, and is followed by a stretch of usually about 3 to 30 pyrimidine nucleotides, more often 3 to 1 5 pyrimidine nucleotides.
- the TOP may comprise 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 1 6, 1 7, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides.
- Messenger RNA that contains a 5'-terminal oligopyrimidine tract is often referred to as TOP mRNA. Accordingly, genes that provide such messenger RNAs are referred to as TOP genes.
- TOP sequences have, for example, been found in genes and mRNAs encoding peptide elongation factors and ribosomal proteins.
- TOP motif In the context of the present invention, a TOP motif is a nucleic acid sequence which corresponds to a 5'TOP as defined above. Thus, a TOP motif in the context of the present invention is preferably a stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.
- the TOP-motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, most preferably at least 8 pyrimidine nucleotides, wherein the stretch of pyrimidine nucleotides prefera- bly starts at its 5'end with a cytosine nucleotide.
- the TOP- motif preferably starts at its 5'end with the transcriptional start site and ends one nucleotide 5' to the first purin residue in said gene or mRNA.
- a TOP motif in the sense of the present invention is preferably located at the 5'end of a sequence which represents a 5'UTR or at the 5'end of a sequence which codes for a 5'UTR.
- TOP motif a stretch of 3 or more pyrimidine nucleotides is called "TOP motif" in the sense of the present invention if this stretch is located at the 5'end of a respective sequence, such as the artificial nucleic acid molecule according to the present invention, the 5'UTR element of the artificial nucleic acid molecule according to the present invention, or the nucleic acid sequence which is derived from the 5'UTR of a TOP gene as described herein.
- a stretch of 3 or more pyrimidine nucleotides which is not located at the 5'-end of a 5'UTR or a 5'UTR element but anywhere within a 5'UTR or a 5'UTR element is preferably not referred to as "TOP motif".
- TOP genes are typically characterised by the presence of a 5' terminal oli- gopyrimidine tract. Furthermore, most TOP genes are characterized by a growth -associated translational regulation. However, also TOP genes with a tissue specific translational regulation are known.
- the 5'UTR of a TOP gene corresponds to the sequence of a 5'UTR of a mature mRNA derived from a TOP gene, which preferably extends from the nucleotide located 3' to the 5'cap to the nucleotide located 5' to the start codon.
- a 5'UTR of a TOP gene typically does not comprise any start codons, preferably no upstream AUGs (uAUGs) or upstream open reading frames (uORFs).
- upstream AUGs and upstream open reading frames are typically understood to be AUGs and open reading frames that occur 5' of the start codon (AUG) of the open reading frame that should be translated.
- the 5'UTRs of TOP genes are generally rather short.
- the lengths of 5'UTRs of TOP genes may vary between 20 nucleotides up to 500 nucleotides, and are typically less than about 200 nucleotides, preferably less than about 150 nucleotides, more preferably less than about 100 nucleotides.
- Exemplary 5'UTRs of TOP genes in the sense of the present invention are the nucleic acid sequences extending from the nucleotide at position 5 to the nucleotide located immediately 5' to the start codon (e.g. the ATG) in the sequences according to SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422.
- the present invention relates to an artificial nucleic acid molecule comprising a. at least one 5'-untranslated region element (5'UTR element) which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene or which is derived from a variant of the 5'UTR of a TOP gene; and b. at least one open reading frame (ORF).
- an artificial nucleic acid molecule may be DNA or RNA.
- the artificial nucleic acid molecule is DNA it may be used for providing RNA, preferably an mRNA with a corresponding sequence as is described further below.
- the inventive artificial nucleic acid molecule is particularly useful in gene therapy and genetic vaccination because it may provide increased and/or prolonged protein production of the protein encoded by the open reading frame. It is preferred, if the components (a) and (b) are heterologous, such that the inventive nucleic acid molecule does not occur naturally, but is an artificial chimeric recombinant nucleic acid molecule.
- '5'UTR element' preferably refers to a nucleic acid sequence which represents a 5'UTR of an artificial nucleic acid sequence, such as an artificial mRNA, or which codes for a 5'UTR of an artificial nucleic acid molecule.
- a 5'UTR element may be the 5'UTR of an mRNA, preferably of an artificial mRNA, or it may be the transcription template for a 5'UTR of an mRNA.
- a 5'UTR element preferably is a nucleic acid sequence which corresponds to the 5'UTR of an mRNA, preferably to the 5'UTR of an artificial mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct.
- a 5'UTR element in the sense of the present invention functions as a 5'UTR or codes for a nucleotide sequence that fulfils the function of a 5'UTR.
- the term '5'UTR element' may also refer to a fragment or part of a 5'UTR of an artificial nucleic acid sequence, such as an artificial mRNA, or which codes for a part or fragment of a 5'UTR of an artificial nucleic acid molecule.
- the 5'UTR element in the sense of the present invention may be comprised in the 5'UTR of an artificial nucleic acid sequence, such as an artificial mRNA, or which codes for a 5'UTR of an artificial nucleic acid molecule.
- the 5'UTR element comprises or consists of a nucleic acid sequence that is derived from the 5'UTR of a TOP gene or from a variant of the 5'UTR of a TOP gene.
- nucleic acid sequence which is derived from the 5'UTR of a TOP gene' preferably refers to a nucleic acid sequence which is based on the 5'UTR sequence of a TOP gene or on a fragment thereof. This term includes sequences corresponding to the entire 5'UTR sequence, i.e. the full length 5'UTR sequence of a TOP gene, and sequences corresponding to a fragment of the 5'UTR sequence of a TOP gene.
- a fragment of a 5'UTR of a TOP gene consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length 5'UTR of a TOP gene, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length 5'UTR of a TOP gene.
- a fragment in the sense of the present invention, is preferably a functional fragment as described herein.
- a particularly preferred fragment of a 5'UTR of a TOP gene is a 5'UTR of a TOP gene lacking the 5'TOP motif, which typically corresponds to a pyrimidine stretch of 3 to 30 pyrimidine nucleotides at the 5' terminus of the 5'UTR of a TOP gene.
- the 5'UTR (comprised by the inventive nucleic acid molecule) starts with the first nucleotide following the most 3'-terminal nucleotide of the 5'TOP motif.
- the 5' UTR (of the TOP gene) employed in the inventive nucleic acid may consist of the nucleotide sequence located upstream of the 5'terminus of the 5'TOP motif and/or of the nucleotide sequence located downstream of the 3'terminus of the 5'TOP motif.
- the 5' motif of a 5'UTR of a TOP gene may be rendered dysfunctional by e.g.
- Another way of rendering the 5' TOP motif dysfunctional is the deletion of one or more pyrimidine nucleotides of the 5'TOP motif sequence (either at the termini and/or within the 5'TOP motif).
- the 5'UT of a TOP gene will not be derived from the 5'UTR of ribosomal proteins (rp) mRNA (in particular not from mammalian 5'UTR of rp mRNA, more specifically not from rpP2 (e.g. rat rpP2), rpL32, rpL30, rpL13a (e.g. mouse transplantation antigen P198), rpS20, rpS6, rpLI 2 or rpS1 6 mRNA or not from an rpS19 mRNA (e.g. from Xenopus).
- rp ribosomal proteins
- the 5'UTR of a TOP gene is not derived from the 5'UTR of a EF1 alpha or (hamster) EF2 mRNA.
- the 5'UTRs of these afore-mentioned rp mRNAs are specifically not used, if they are linked to reporter genes in the ORF of the inventive nucleic acid. If e.g. the 5'UTR of rpS1 6 mRNA is used for the inventive nucleic acid, that 5'UTR will either not contain the 5'TOP motif sequence (composed ot the oligonucleotide (CCTTTTCC or CCUUUUCC) or will contain a dysfunctional variant therof by e.g.
- the dysfunctional mutants may e.g. contain one or more purine nucleotides within the 5'TOP motif sequence thereby lacking the translational control function exerted by the 5'TOP motif, e.g. by abolishing its interaction with other regulatory compounds, e.g. miRNA or interaction with granule-associated proteins TIA-1 and TIAR.
- '5'UTR of a TOP gene' preferably refers to the 5'UTR of a naturally occurring TOP gene.
- the terms 'variant of the 5'UTR of a TOP gene' and 'variant thereof in the context of a 5'UTR of a TOP gene refers to a variant of the 5'UTR of a naturally occurring TOP gene, preferably to a variant of the 5'UTR of a vertebrate TOP gene, preferably to a variant of the 5'UTR of a mammalian TOP gene, more preferably to a variant of the 5'UTR of a human TOP gene.
- Such variant may be a modified 5'UTR of a TOP gene.
- a variant 5'UTR may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the naturally occurring 5'UTR from which the variant is derived.
- a variant of a 5'UTR of a TOP gene is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the naturally occurring 5'UTR the variant is derived from.
- the variant is a functional variant as described herein.
- a nucleic acid sequence that is derived from a variant of the 5'UTR of a TOP gene preferably refers to a nucleic acid sequence which is based on a variant of a 5'UTR sequence of a TOP gene or on a fragment thereof. This term includes sequences corresponding to the entire variant 5'UTR sequence, i.e. the full length variant 5'UTR sequence of a TOP gene, and sequences corresponding to a fragment of the variant 5'UTR sequence of a TOP gene.
- a fragment of a variant of the 5'UTR of a TOP gene consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length variant 5'UTR of a TOP gene, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length variant 5'UTR of a TOP gene.
- a fragment of a variant in the sense of the present invention, is preferably a functional fragment as described herein.
- the 5'UTR element of the artificial nucleic acid molecule may comprise or consist of a fragment of the 5'UTR of a TOP gene or of a fragment of a variant of the 5'UTR of a TOP gene or may comprise or consist of the entire 5'UTR of a TOP gene or may comprise or consist of a variant of the 5'UTR of a TOP gene.
- the 5'UTR element is preferably suitable for increasing protein production from the artificial nucleic acid molecule.
- the at least one 5'UTR element is functionally linked to the ORF.
- the 5'UTR element is associated with the ORF such that it may exert a function, such as a protein production increasing function for the protein encoded by the ORF or a stabilizing function on the artificial nucleic acid molecule.
- the 5'UTR element and the ORF are associated in 5'- 3' direction.
- the artificial nucleic acid molecule comprises the structure 5'-5'UTR element-(optional)linker-ORF-3', wherein the linker may be present or absent.
- the linker may be one or more nucleotides, such as a stretch of 1 -50 or 1 -20 nucleotides, e.g., comprising or consisting of one or more restriction enzyme recognition sites (restriction sites).
- the 5'UTR element and the at least one open reading frame are heterologous.
- the term 'heterologous' in this context means that the open reading frame and the 5'UTR element are not occurring naturally (in nature) in this combination.
- the 5'UTR element is derived from a different gene than the open reading frame.
- the ORF may be derived from a different gene than the 5'UTR element, e.g. encoding a different protein or the same protein but of a different species etc.
- the ORF does not encode the protein which is encoded by the gene from which the 5'UTR element is derived.
- the 5'UTR element preferably the artificial nucleic acid molecule, does not comprise a complete TOP-motif or 5'TOP sequence.
- the 5'UTR element preferably the artificial nucleic acid molecule, does not comprise the complete TOP-motif of the TOP gene from which the nucleic acid sequence of the 5'UTR element is derived.
- the 5'UTR element or the artificial nucleic acid molecule according to the present invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine residues of the TOP-motif or 5'TOP, preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine residues of the TOP-motif located at the 3'side of the TOP-motif or 5'TOP.
- the 5'UTR element may comprise or consist of a nucleic acid sequence which starts at its 5'end with a pyrimidine residue that corresponds to residue 2, 3, 4, 5, 6, 7, 8, 9, 10 etc. of the TOP-motif or 5'TOP of the TOP gene from which the nucleic acid sequence of the 5'UTR element is derived.
- the 5'UTR element preferably the artificial nucleic acid molecule according to the present invention, does not comprise a TOP-motif or a 5'TOP.
- the nucleic acid sequence of the 5'UTR element which is derived from a 5'UTR of a TOP gene starts at its 5'-end with a nucleotide located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 downstream of the 5'terminal oligopyrimidine tract (TOP) of the 5'UTR of a TOP gene.
- Position 1 downstream of the 5'terminal oligopyrimidine tract (TOP) is the first purine based nucleotide 3' of the TOP-motif or the 5'TOP.
- position 1 downstream of the 5'terminal oligopyrimidine tract is the first nucleotide following the 3'- end of the 5'terminal oligopyrimidine tract in 5'-3'-direction.
- position 2 downstream of the 5'TOP is the second nucleotide following the end of the 5'terminal oligopyrimidine tract, position 3 the third nucleotide and so on.
- the 5'UTR element preferably starts 5, 10, 15, 20, 25, 30, 40 or 50 nucleotides downstream of the transcriptional start site of the 5'UTR of a TOP gene.
- the nucleic acid sequence of the 5'UTR element which is derived from a 5'UTR of a TOP gene terminates at its 3'-end with a nucleotide located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (e.g. A(U/T)G) of the gene or mRNA it is derived from.
- the 5'UTR element does not comprise any part of the protein coding region.
- the only protein coding part of the inventive artificial nucleic acid molecule is provided by the open reading frame.
- the open reading frame is preferably derived - as said above - from a gene that is different to the gene the 5'UTR element is derived from.
- the 5'UTR element does not comprise a start codon, such as the nucleotide sequence A(U/T)G.
- the artificial nucleic acid molecule will not comprise any upstream AUGs (or upstream ATGs in case it is a DNA molecule).
- the AUG or ATG, respectively, of the open reading frame is the only start codon of the artificial nucleic acid molecule.
- the 5'UTR element does not comprise an open reading frame.
- the artificial nucleic acid molecule will not comprise any upstream open reading frames.
- the nucleic acid sequence which is derived from the 5'UTR of a TOP gene is derived from a eukaryotic TOP gene, preferably a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a mammalian TOP gene, such as a human or mouse TOP gene.
- the artificial nucleic acid molecule according to the present invention comprises a 5'UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene or which is derived from a variant of the 5'UTR of a TOP gene, wherein the TOP gene is a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a mammalian TOP gene, such as a human or mouse TOP gene and which optionally does not comprise the nucleotide sequence A(U/T)C and optionally does not comprise an open reading frame; at least one open reading frame (ORF); wherein optionally the 5'UTR element does not comprise a TOP motif and wherein optionally the 5'UTR element starts at its 5'-end with a nucleotide located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 downstream of the 5'terminal oligopyrimidine tract (TOP) of the 5'UT
- the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1 - 1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422, from the homologs of SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422, from a variant thereof, or a corresponding RNA sequence.
- the term "homologs of SEQ ID NOs. 1 - 1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422" refers to sequences of other species, e.g.
- SEQ ID NO. 1 relates to a sequence comprising the 5'UTR of Homo sapiens alpha 2 macroglobuiin (A2M). A homolog of SEQ ID NO.
- A2M alpha 2 macroglobuiin
- the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a nucleic acid sequence extending from nucleotide position 5 (i.e. the nucleotide that is located at position 5 in the sequence) to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g. the nucleotide position immediately 5' to the ATG sequence, of a nucleic acid sequence selected from SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 or SEQ ID NO. 1422, from the homologs of SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO.
- the 5' UTR element is derived from a nucleic acid sequence extending from the nucleotide position immediately 3' to the 5'TOP to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g. the nucleotide position immediately 5' to the ATG sequence, of a nucleic acid sequence selected from SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 or SEQ ID NO. 1422, from the homologs of SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO.
- the 5'UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence extending from nucleotide position 5 to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g.
- the at least one 5'UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence extending from nucleotide position 5 to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g.
- nucleotide position immediately 5' to the ATG sequence of a nucleic acid sequence selected from SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 or SEQ ID NO. 1422, or a corresponding RNA sequence, wherein, preferably, the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc. of the full-length 5'UTR the fragment is derived from.
- the 5'UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence extending from the nucleotide position immediately 3' to the 5'TOP to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g. the nucleotide position immediately 5' to the ATG sequence, of a nucleic acid sequence selected from SEQ ID NOs.
- the at least one 5'UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence extending from the nucleotide position immediately 3' to the 5'TOP to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g.
- nucleotide position immediately 5' to the ATG sequence of a nucleic acid sequence selected from SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 or SEQ ID NO. 1422, or a corresponding RNA sequence, wherein, preferably, the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc. of the full-length 5'UTR the fragment is derived from.
- the above defined fragments and variants are functional fragments and variants as described herein.
- the artificial nucleic acid molecule according to the present invention may comprise more than one 5'UTR elements as described above.
- the artificial nucleic acid molecule according to the present invention may comprise one, two, three, four or more 5'UTR elements, wherein the individual 5'UTR elements may be the same or they may be different.
- the artificial nucleic acid molecule according to the present invention may comprise two essentially identical 5'UTR elements as described above, e.g. two 5'UTR elements comprising or consisting of a nucleic acid sequence which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422, from the homologs of SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422, from a variant thereof, or a corresponding RNA sequence or from functional variants thereof, functional fragments thereof, or functional variant fragments thereof as described above.
- two 5'UTR elements comprising or consisting of a nucleic acid sequence which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO
- the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a 5'UTR of a TOP gene encoding a ribosomal protein or from a variant of a 5'UTR of a TOP gene encoding a ribosomal protein.
- Particularly preferred 5'UTR elements comprise or consist of a nucleic acid sequence which are derived from a 5' UTR of a TOP gene coding for a ribosomal protein se- lected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS1 1 , RPS12, RPS13, RPS14, RPS1 5, RPS15A, RPS1 6, RPS1 7, RPS1 8, RPS19, RPS20, RPS21 , RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL1 1 , RPL12, RPL13, RPL13A, RPL14, RPL15, RPL1 7, RPL18, RPL
- nucleic acid sequences which are derived from a 5' UTR of TOP genes vertebrate coding for ribosomal proteins, such as mammalian ribosomal proteins e.g. human or mouse ribosomal proteins.
- the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a 5'UTR of a nucleic acid sequence according to any of SEQ ID NOs: 1 70, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291 , 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301 , 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 131 1 , 1312, 1313, 1314, 1315, 131 6, 131 7, 1318, 1319, 1320, 1 321 , 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331 , 1332, 1333, 1334, 1 335, 1336, 1337, 1338, 1339, 1340, 1341 , 1342, 1343, 1344, 1346, 1347, 1348
- the sequence extending from position 5 to the nucleotide immediately 5' to the ATG corresponds to the 5'UTR of said sequences.
- the 5'UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5'UTR of a nucleic acid sequence according to any of SEQ ID NOs.: 1 70, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291 ,
- the at least one 5'UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5'UTR of a nucleic acid sequence according to SEQ ID NOs: 1 70, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291 , 1292,
- the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc. of the full-length 5'UTR, preferably lacking the 5'TOP motif.
- the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more.
- the fragment is a functional fragment as described herein.
- the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a 5'UTR of a TOP gene encoding a ribosomai Large protein (RPL) or from a variant of a 5'UTR of a TOP gene encoding a ribosomai Large protein (RPL).
- RPL ribosomai Large protein
- the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a 5'UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and 1422, a corresponding RNA sequence, a homolog thereof, or a variant thereof as described herein, preferably lacking the 5'TOP motif.
- the 5'UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5'UTR of a nucleic acid sequence according to any of SEQ ID NOs.
- the at least one 5'UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5'UTR of a nucleic acid sequence according to SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and 1422 or a corresponding RNA sequence, wherein, preferably, the fragment is as described above, i.e.
- the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more.
- the fragment is a functional fragment as described herein.
- the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomal protein Large 21 gene (RPL21 ), an ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 , cardiac muscle (ATP5A1 ) gene, an hydroxysteroid (1 7-beta) dehydrogenase 4 gene (HSD1 7B4), an androgen-induced 1 gene (Aid ), cytochrome c oxidase subunit Vic gene (COX6C), or a N-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1 ) or from a variant thereof, preferably from a vertebrate ribosomal protein Large 32 gene (RPL32), a vertebrate ribosomal protein Large 32
- the 5'UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No.
- the at least one 5'UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1368, or SEQ ID NOs 1412- 1420, wherein, preferably, the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc.
- the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more.
- the fragment is a functional fragment as described herein.
- the at least one 5'UTR element exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more.
- the 5'UTR may have a length of less than about 200, preferably less than 150, more preferably less than 100 nucleotides.
- the 5'UTR may have a length of less than about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 nucleotides
- the 5'UTR element may have a length of about 20-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71- 80, 81-85, 86-90, 91-95, 96-100, 101-105, 106-110, 111-115, 116-120, 121-125, 126- 130, 131-135, 136-140, 141-145,
- the 5'UTR element may have a length of about 20, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171,
- the 5'UTR element may have a length from about 20, 30, 40 or more to less than about 200 nucleotides, more preferably from about 20, 30, 40 or more to less than about 150 nucleotides, most preferably from about 20, 30, 40 or more to less than about 100 nucleotides.
- Preferred 5'UTR elements are derived from a 5' UTR of a TOP gene selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, R
- the artificial nucleic acid molecule comprises a 5'UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a vertebrate TOP gene, such as a mammalian, e.g.
- a human TOP gene selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS1 1 , RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS1 7, RPS18, RPS19, RPS20, RPS21 , RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL1 1 , RPL12, RPL13, RPL13A, RPL14, RPL15, RPL1 7, RPL18, RPL18A, RPL19, RPL21 , RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28
- the artificial nucleic acid molecule according to the present invention further comprises c. at least one 3'UTR element which comprises or consists of a nucleic acid sequence derived from the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene.
- '3'UTR element' refers to a nucleic acid sequence which comprises or consists of a nucleic acid sequence that is derived from a 3'UTR or from a variant of a 3'UTR.
- a 3'UTR element in the sense of the present invention may represent the 3'UTR of an mRNA, e.g., in the event that the artificial nucleic acid molecule is an mRNA, or it may represent a sequence in a nucleic acid construct, such as a vector construct, that when transcribed represents the 3'UTR of the transcription product, such as the mRNA.
- a 3'UTR element may be the 3'UTR of an mRNA, preferably of an artificial mRNA, or it may be the transcription template for a 3'UTR of an mRNA.
- a 3'UTR element preferably is a nucleic acid sequence which corresponds to the 3'UTR of an mRNA, preferably to the 3'UTR of an artificial mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct.
- the 3'UTR element fulfils the function of a 3'UTR or encodes a sequence which fulfils the function of a 3'UTR.
- '3UTR element' furthermore refers to a fragment or part of a 3'UTR of an artificial nucleic acid sequence, such as an artificial mRNA, or which codes for a part or fragment of a 3'UTR of an artificial nucleic acid molecule.
- the 3'UTR element in the sense of the present invention may be comprised in the 3'UTR of an artificial nucleic acid sequence, such as an artificial mRNA, or which codes for a 3'UTR of an artificial nucleic acid molecule.
- the 3'UTR element and the at least one open reading frame are heterologous.
- the artificial nucleic acid molecule may consist of at least two sequence parts that are derivable from two different genes, the 5'UTR element which is derivable from a TOP gene and the open reading frame and the 3'UTR which may be derivable from the gene encoding the desired protein product.
- the artificial nucleic molecule consist of three sequence parts that are derivable from three different genes: the 5'UTR element which is derivable from a TOP gene, the open reading frame which is derivable from the gene encoding the desired gene product and the 3'UTR element which may be derivable from a gene that relates to an mRNA with an enhanced half-life, for example a 3'UTR element as defined and described below.
- the at least one 3'UTR element is functionally linked to the ORF. This means preferably that the 3'UTR element is associated with the ORF such that it may exert a function, such as a stabilizing function on the expression of the ORF or a stabilizing function on the artificial nucleic acid molecule.
- the ORF and the 3'UTR element are associated in 5'- 3' direction.
- the artificial nucleic acid molecule comprises the structure 5'-ORF-(optional)linker-3'UTR element-3', wherein the linker may be present or absent.
- the linker may be one or more nucleotides, such as a stretch of 1 -50 or 1 -20 nucleotides, e.g., comprising or consisting of one or more restriction enzyme recognition sites (restriction sites).
- the at least one 5'UTR element and the at least one 3'UTR element are functionally linked to the ORF.
- the 5'UTR element and the 3'UTR element are associated with the ORF such that they may exert a function, preferably in an additive, more preferably in a synergistic manner, such as a stabilizing function on the expression of the ORF, a protein production increasing function for the protein encoded by the ORF, or a stabilizing function on the artificial nucleic acid molecule.
- the 5'UTR element, the ORF, and the 3'UTR element are associated in 5'->3' direction.
- the artificial nucleic acid molecule comprises the structure 5'-5'UTR element- (optional)linker-ORF-(optional)linker-3'UTR element-3', wherein the linker may be present or absent.
- the linker may be one or more nucleotides, such as a stretch of 1 -50 or 1 -20 nucleotides, e.g., comprising or consisting of one or more restriction enzyme recognition sites (restriction sites).
- the 5'UTR element and the 3'UTR element are heterologous, e.g. preferably the 5'UTR and the 3'UTR are derived from different genes of the same or of different species.
- the 3'UTR is not derived from the TOP gene the 5'UTR is derived from.
- the 3'UTR element is chosen such that it exerts at least an additive, preferably a synergistic function with the 5'UTR element on the protein production from the ORF of the artificial nucleic acid molecule.
- the protein production is increased in at least an additive, preferably a synergistic way by the 3'UTR element and the 5'UTR element.
- the protein amount of the protein encoded by the ORF such as a reporter protein, e.g. luciferase, at a certain time point after initiation of expression of the ORF, e.g.
- the additive preferably the synergistic effect may, for example, be determined by the following assay.
- Samples are taken at specific time points after initiation of expression, for example, after 6 hours, 24 hours, 48 hours, and 72 hours and the amount of protein produced by expression of the ORF contained in the artificial nucleic acid molecules is measured, for example, by an ELISA assay or a luciferase test, depending on the type of protein encoded by the ORF.
- the predicted amount of protein at a certain time point after initiation of expression obtained by construct E1 E2 if the effects of the 3'UTR element and the 5'UTR element were purely additive (PPA) may be calculated as follows:
- E0 is the amount of protein obtained for the construct E0 (lacking UTRs)
- E1 is the amount of protein obtained for the construct E1
- E2 is the protein amount obtained for the construct E2
- x is the time point after initiation of expression.
- E1 E2 X PPA X and synergistic in the sense of the present invention if E1 E2 X > PPA X , wherein E1 E2 X is the amount of protein obtained from construct E1 E2 at time point x.
- E1 E2 is at least 1 .0, preferably at least 1 .1 , more preferably at least 1 .3, more preferably at least 1 .5, even more preferably at least 1.75 times PPA at a given time point post initiation of expression, such as 24 hours, 48 hours or 72 hours post initiation of expression.
- the present invention provides an artificial nucleic acid molecule comprising (a.) at least one 5'UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene or which is derived from a variant of the 5'UTR of a TOP gene; (b.) at least one open reading frame (ORF); and (c.) at least one 3'UTR element, wherein the 3'UTR element and the 5'UTR element act at least additively, preferably synergistically to increase protein production from the ORF, preferably wherein El E2 > PPA, preferably E1 E2 is at least 1 .0 times PPA, preferably El E2 is at least 1 .1 times PPA, more preferably E1 E2 is at least 1.3 times PPA, even more preferably El E2 is at least 1 .5 times PPA at a given time point post initiation of expression of the ORF, for example 24 hours, preferably 48 hours post initiation of expression
- the 3'UTR element and the 5'UTR element have at least an additive, preferably a synergistic effect on the total protein production from the artificial nucleic acid molecule in a certain time span, such as within 24 hours, 48 hours, or 72 hours post initiation of expression.
- the additive or the synergistic effect may be determined as described above, with the difference that the area under the curve (AUC) for the amount of protein over time predicted for E1 E2 if the effects were purely additive is compared to the actual AUC measured for E1 E2.
- the 3'UTR element comprises or consists of a nucleic acid sequence which is derived from the 3'UTR of a stable mRNA or from a variant of the 3'UTR of a stable mRNA.
- the 3'UTR element comprises or consists of a sequence which is derived from a gene providing a stable mRNA or from a variant of a 3'UTR of a gene providing a stable mRNA.
- stable mRNA preferably refers to mRNAs which exhibit a longer half-life in mammalian cells than the average half- life of mRNA molecules in mammalian cells.
- a stable mRNA in the sense of the present application refers to an mRNA which exhibits a half-life of more than 5 hours, preferably more than 8 hours, in a mammalian cell, such as in a mammalian cell line, e.g. in HELA cells, or in primary cells, e.g. in HDF cells, preferably determined by using a transcription inhibitor such as actinomycin D.
- a mammalian cell such as in a mammalian cell line, e.g. in HELA cells, or in primary cells, e.g. in HDF cells, preferably determined by using a transcription inhibitor such as actinomycin D.
- the half-life of an mRNA in mammalian cells, such as HELA or HDF cells may be determined by culturing the cells in presence of a transcription inhibitor, e.g.
- actinomycin D 5,6-dichloro-1-P-D-ribofuranosylbenzimidazole (DRB), or oc-amanitin
- DRB 5,6-dichloro-1-P-D-ribofuranosylbenzimidazole
- oc-amanitin harvesting the cells at different time points after inhibition of transcription, and determining the amount of the mRNA present in the cell samples by methods well known to the person skilled in the art, e.g. by quantitative RT-PCR.
- the half-life of a particular mRNA may be calculated based on the amounts of the particular mRNA measured at the different time points post inhibition of transcription.
- pulse-chase methods e.g. using radioactively labelled nucleotides, or constructs comprising inducible promoters may be used for determining the half-life of an mRNA in mammalian cells.
- the enhanced stability of a stable mRNA in the sense of the present invention is affected by its 3'UTR.
- the 3'UTR element comprises or consists of a nucleic acid sequence which is derived from the 3'UTR of a stable mRNA which exhibits a half-life of more than 5 hours, preferably more than 8 hours, in a mammalian cell, such as in a mammalian cell line, e.g. in HeLa cells, or in mammalian primary cells, e.g. in HDF cells, preferably determined by using a transcription inhibitor such as actinomycin D, wherein the enhanced stability of said stable mRNA is effected by its 3'UTR.
- a 3'UTR for enhancing stability may be tested as described herein, e.g. by using a reporter open reading frame such as a luciferase encoding open reading frame.
- an artificial construct encoding the test stable mRNA may be generated, wherein the 3'UTR of the stable mRNA is replaced with a reference 3'UTR, such as a 3'UTR of a short lived mRNA, e.g. a Myc 3'UTR.
- the stability of the wild type stable mRNA and the 3'UTR modified mRNA may be determined as described above. In the event the 3'UTR modified mRNA exhibits a shorter half-life than the wild type stable mRNA, it may be concluded that a stability enhancing effect is exerted by the 3'UTR of the stable mRNA.
- the 3'UTR element comprises or consists of a nucleic acid sequence which is derived from a 3'UTR of a gene selected from the group consisting of an albumin gene, an a-globin gene, a ⁇ -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (1) gene, or from a variant of a 3'UTR of a gene selected from the group consisting of an albumin gene, an a-globin gene, a ⁇ -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (1) gene.
- the 3'UTR element comprises or consists of a nucleic acid sequence which is derived from a 3'UTR of an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene, most preferably a human albumin gene.
- the 3'UTR element comprises or consists of a nucleic acid sequence which is derived from a 3'UTR of an ⁇ -globin gene, preferably a vertebrate ⁇ -globin gene, more preferably a mammalian ⁇ -globin gene, most preferably a human a- globin gene.
- the 3'UTR element may comprise or consist of the center, a- complex-binding portion of the 3'UTR of an ⁇ -globin gene, such as of a human a-globin gene.
- the at least one 3'UTR element comprises or consists of a nucleic acid sequence which is derived from the 3'UTR of a vertebrate albumin gene, a vertebrate ⁇ -globin gene, a vertebrate ⁇ -globin gene, a vertebrate tyrosine hydroxylase gene, a vertebrate lipoxygenase gene, and a vertebrate collagen alpha gene, such as a vertebrate collagen alpha 1 (1) gene, or from a variant thereof, preferably from the 3'UTR of a mammalian albumin gene, a mammalian ⁇ -globin gene, a mammalian ⁇ -globin gene, a mammalian tyrosine hydroxylase gene, a mammalian lipoxygenase gene, and a mammalian collagen alpha gene, such as a mammalian collagen alpha 1 (I) gene, or from a variant thereof, more preferably from the 3'UTR of a human albumin
- the 3'UTR element is not derived from the 3'UTR of a Xenopus albumin gene.
- the 3'UTR element does not comprise a poly(A) limiting element B (PLEB) of a 3'UTR from a Xenopus albumin gene.
- the 3'UTR element does not consist of a PLEB of a 3'UTR from a Xenopus albumin gene.
- the 3'UTR element and the at least one open reading frame are heterologous, e.g. preferably the 3'UTR element and the ORF are derived from different genes of the same or of different species.
- the ORF does not encode an ⁇ -globin protein if the 3'UTR element is derived from an cc-globin gene.
- the ORF does not encode a ⁇ - globin protein if the 3'UTR element is derived from a ⁇ -globin gene.
- the ORF does not encode an albumin protein if the 3'UTR element is derived from an albumin gene.
- the ORF does not encode a tyrosine hydroxylase protein if the 3'UTR element is derived from a tyrosine hydroxylase gene.
- the ORF does not encode a lipoxygenase protein if the 3'UTR element is derived from a lipoxygenase gene.
- the ORF does not encode a collagen alpha protein if the 3'UTR element is derived from a collagene alpha gene.
- the ORF does not code for a protein selected from the group consisting of albumin proteins, growth hormones, e.g.
- the open reading frame does not code for a reporter protein, e.g., selected from the group consisting of globin proteins, in particular beta-globin,, luciferase protein, GFP proteins, e.g. EGFP, or variants thereof, for example, variants exhibiting at least 70% sequence identity to a globin protein, a luciferase protein, or a GFP protein.
- a reporter protein e.g., selected from the group consisting of globin proteins, in particular beta-globin,, luciferase protein, GFP proteins, e.g. EGFP, or variants thereof, for example, variants exhibiting at least 70% sequence identity to a globin protein, a luciferase protein, or a GFP protein.
- nucleic acid sequence which is derived from the 3'UTR of a noted gene' preferably refers to a nucleic acid sequence which is based on the 3'UTR sequence of a noted gene or on a part thereof, such as on the 3'UTR of an albumin gene, an a-globin gene, a ⁇ - globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagen alpha gene, such as a collagen alpha 1 (1) gene, preferably of an albumin gene or on a part thereof.
- This term includes sequences corresponding to the entire 3'UTR sequence, i.e.
- the full length 3'UTR sequence of a gene and sequences corresponding to a fragment of the 3'UTR sequence of a gene, such as an albumin gene, ⁇ -globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (1) gene, preferably of an albumin gene.
- a gene such as an albumin gene, ⁇ -globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (1) gene, preferably of an albumin gene.
- a fragment in this context preferably consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length 3'UTR, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length 3'UTR.
- Such a fragment in the sense of the present invention, is preferably a functional fragment as described herein.
- '3'UTR of a section gene' preferably refers to the 3'UTR of a naturally occurring gene, such as of a naturally occurring albumin gene, oc-globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (1) gene, preferably of a naturally occurring albumin gene.
- a naturally occurring gene such as of a naturally occurring albumin gene, oc-globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (1) gene, preferably of a naturally occurring albumin gene.
- the terms 'variant of the 3'UTR of a section gene' and 'variant thereof in the context of a 3'UTR refers to a variant of the 3'UTR of a naturally occurring gene, such as a naturally occurring albumin gene, a naturally occurring a-globin gene, a naturally occurring ⁇ -globin gene, a naturally occurring tyrosine hydroxylase gene, a naturally occurring lipoxygenase gene, or a naturally occurring collagen alpha gene, such as a naturally occurring collagen alpha 1 (1) gene, preferably to a variant of the 3'UTR of a vertebrate albumin gene, a vertebrate a-globin gene, a vertebrate ⁇ -globin gene, a vertebrate tyrosine hydroxylase gene, a vertebrate lipoxygenase gene, and a vertebrate collagen alpha gene, such as a vertebrate collagen alpha 1 (1) gene, preferably to a variant of the 3'UTR of a mammalian albumin
- Such variant may be a modified 3'UTR of a gene.
- a variant 3'UTR may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the naturally occurring 3'UTR from which the variant is derived.
- a variant of a 3'UTR is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the naturally occurring 3'UTR the variant is derived from.
- the variant is a functional variant as described herein.
- nucleic acid sequence which is derived from a variant of the 3'UTR of a noted gene' preferably refers to a nucleic acid sequence which is based on a variant of the 3'UTR sequence of a gene, such as on a variant of the 3'UTR of an albumin gene, an a-globin gene, a ⁇ -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagen alpha gene, such as a collagen alpha 1 (I) gene, or on a part thereof as described above.
- This term includes sequences corresponding to the entire sequence of the variant of the 3'UTR of a gene, i.e.
- a fragment in this context preferably consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length variant 3'UTR, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length variant 3'UTR.
- Such a fragment of a variant in the sense of the present invention, is preferably a functional fragment of a variant as described herein.
- 'functional variant', 'functional fragment', and 'functional fragment of a variant' mean that the fragment of the 5'UTR or the 3'UTR, the variant of the 5'UTR or the 3'UTR, or the fragment of a variant of the 5'UTR or the 3'UTR of a gene fulfils at least one, preferably more than one, function of the naturally occurring 5'UTR or 3'UTR of the gene of which the variant, the fragment, or the fragment of a variant is derived.
- Such function may be, for example, stabilizing mRNA and/or stabilizing and/or prolonging protein production from an mRNA and/or increasing protein production from an mRNA, preferably in a mammalian cell, such as in a human cell. It is particularly preferred that the variant, the fragment, and the variant fragment in the context of the present invention fulfil the function of stabilizing an mRNA, preferably in a mammalian cell, such as a human cell, compared to an mRNA comprising a reference 5'UTR and/or a reference 3'UTR or lacking a 5'UTR and/or a 3'UTR, and/or the function of stabilizing and/or prolonging protein production from an mRNA, preferably in a mammalian cell, such as in a human cell, compared to an mRNA comprising a reference 5'UTR and/or a reference 3'UTR or lacking a 5'UTR and/or a 3'UTR, and/or the function of increasing protein production from an mRNA
- a reference 3'UTR may be, for example, a 3'UTR naturally occurring in combination with the ORF.
- a functional variant, a functional fragment, or a functional variant fragment of a 5'UTR or of a 3'UTR of a gene preferably does not have a substantially diminishing effect on the efficiency of translation of the mRNA which comprises such variant of a 5'UTR and/or such variant of a 3'UTR compared to the wild type 5'UTR and/or 3'UTR from which the variant is derived.
- a particularly preferred function of a "functional fragment", a “functional variant” or a “functional fragment of a variant” of the 3'UTR of a gene such as an albumin gene, a-globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (1) gene, in the context of the present invention is the stabilization and/or prolongation of protein production by expression of an mRNA carrying the functional fragment, functional variant or functional fragment of a variant as described above.
- a particularly preferred function of a "functional fragment", a “functional variant” or a “functional fragment of a variant” of the 5'UTR in the context of the present invention is the protein production increasing function.
- the efficiency of the one or more functions exerted by the functional variant, the functional fragment, or the functional variant fragment is at least 40%, more preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% of the mRNA and/or protein production stabilizing efficiency and/or the protein production increasing efficiency exhibited by the naturally occurring 5'UTR and/or 3'UTR of which the variant, the fragment or the variant fragment is derived.
- a fragment or part of the 3'UTR of a gene such as an albumin gene, a-globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (I) gene, or of a variant thereof preferably exhibits a length of at least about 40 nucleotides, preferably of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.
- such fragment of the 3'UTR of a gene or of a variant of the 3'UTR of a gene is a functional fragment as described above.
- a fragment or part of the 5'UTR of a TOP gene or of a variant thereof preferably exhibits a length of at least about 20 nucleotides, preferably of at least about 30 nucleotides, more preferably of at least about 50 nucleotides.
- such fragment of the 5'UTR of a TOP gene or of a variant of the 5'UTR of a TOP gene is a functional fragment as described above.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention comprises or consists of a "functional fragment", a "functional variant” or a "functional fragment of a variant” of the 3'UTR of a gene, such as of an albumin gene, cc-globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (I) gene, or of a variant thereof.
- a gene such as of an albumin gene, cc-globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (I) gene, or of a variant thereof.
- the at least one 5'UTR element of the artificial nucleic acid molecule according to the present invention comprises or consists of a "functional fragment", a “functional variant” or a “functional fragment of a variant” of the 5'UTR of a TOP gene.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention increases the stability of the artificial nucleic acid molecule, e.g. increases the stability of an mRNA according to the present invention, compared to a respective mRNA (reference mRNA) lacking a 3'UTR element or comprising a reference 3'UTR element, such as a 3'UTR naturally occurring in combination with the ORF.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention increases the stability of protein production from the artificial nucleic acid molecule according to the present invention, e.g.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention prolongs protein production from the artificial nucleic acid molecule according to the present invention, e.g. from an mRNA according to the present invention, compared to a respective mRNA lacking a 3'UTR element or comprising a reference 3'UTR element, such as a 3'UTR naturally occurring in combination with the ORF.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention increases the protein production from the artificial nucleic acid molecule according to the present invention, e.g. from an mRNA according to the present invention, compared to a respective mRNA lacking a 3'UTR element or comprising a reference 3'UTR element, such as a 3'UTR naturally occurring in combination with the ORF.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention does not negatively influence translational efficiency of an mRNA compared to the translational efficiency of a respective mRNA lacking a 3'UTR element or comprising a reference 3'UTR element, such as a 3'UTR naturally occurring in combination with the ORF.
- the term 'respective mRNA' in this context means that - apart from the different 3'UTR - the reference mRNA is comparable, preferably identical, to the mRNA comprising the 3'UTR element.
- the at least one 5'UTR element of the artificial nucleic acid molecule according to the present invention increases the stability of the artificial nucleic acid molecule, e.g. increases the stability of an mRNA according to the present invention, compared to a respective mRNA (reference mRNA) lacking a 5'UTR element or comprising a reference 5'UTR element, such as a 5'UTR naturally occurring in combination with the ORF.
- the at least one 5'UTR element of the artificial nucleic acid molecule according to the present invention increases protein production from the artificial nucleic acid molecule according to the present invention, e.g.
- mRNA from an mRNA according to the present invention, compared to a respective mRNA lacking a 5'UTR element or comprising a reference 5'UTR element, such as a 5'UTR naturally occurring in combination with the ORF.
- the term 'respective mRNA' in this context means that - apart from the different 5'UTR - the reference mRNA is comparable, preferably identical, to the mRNA comprising the inventive 5'UTR element.
- the at least one 5'UTR element and the at least one 3'UTR element act synergistically to increase protein production from the artificial nucleic acid molecule according to the present invention, e.g. from an mRNA according to the present invention, as described above.
- 'stabilizing and/or prolonging protein production from an mRNA' preferably means that the protein production from the mRNA is stabilized and/or prolonged compared to the protein production from a reference mRNA, e.g. comprising a reference 3'UTR element or lacking a 3'UTR element.
- 'Stabilized protein expression' in this context preferably means that there is more uniform protein production from the artificial nucleic acid molecule according to the present invention over a predetermined period of time, such as over 24 hours, more preferably over 48 hours, even more preferably over 72 hours, when compared to a reference nucleic acid molecule, for example, an mRNA comprising a reference 3'UTR element or lacking a 3'UTR element.
- a reference nucleic acid molecule for example, an mRNA comprising a reference 3'UTR element or lacking a 3'UTR element.
- the level of protein production e.g. in a mammalian system, from the artificial nucleic acid molecule comprising a 3'UTR element according to the present invention, e.g.
- the amount of a protein (encoded by the ORF) observed 6 hours after initiation of expression, e.g. 6 hours post transfection of the artificial nucleic acid molecule according to the present invention into a cell, such as a mammalian cell may be comparable to the amount of protein observed 48 hours after initiation of expression, e.g. 48 hours post transfection.
- the ratio of the amount of protein encoded by the ORF, such as of a reporter protein, e.g., luciferase, observed at 48 hours post initiation of expression e.g.
- 48 hours post transfection to the amount of protein observed 6 hours after initiation of expression, e.g. 6 hours post transfection, is preferably above 0.4, preferably above 0.5, more preferably above 0.6, even more preferably above 0.7, e.g. between about 0.4 and about 4, preferably between about 0.65 and about 3, more preferably between about 0.7 and about 2 for a nucleic acid molecule according to the present invention.
- a nucleic acid molecule e.g. an mRNA comprising a reference 3'UTR element or lacking a 3'UTR element
- said ratio may be, e.g. between about 0.05 and about 0.3.
- the present invention provides an artificial nucleic acid molecule comprising an ORF and a 3'UTR element as described above, wherein the ratio of the (reporter) protein amount observed 48 hours after initiation of expression to the (reporter) protein amount observed 6 hours after initiation of expression, preferably in a mammalian expression system, such as in mammalian cells, is preferably above 0.4, preferably above 0.5, more preferably above 0.6, even more preferably above 0.7, e.g. between about 0.4 and about 4, preferably between about 0.65 and about 3, more preferably between about 0.7 and about 2.
- the protein level observed at a certain time point after initiation of expression, e.g. after transfection, of the artificial nucleic acid molecule according to the present invention, e.g. after transfection of an mRNA according to the present invention, for example, 24, 48, or 72 hours post transfection, or the total protein produced in a time span of, e.g. 24, 48 or 72 hours, is preferably higher than the protein level observed at the same time point after initiation of expression, e.g.
- a reference nucleic acid molecule such as a reference mRNA comprising a reference 5' and/or a reference 3'UTR or lacking a 5'UTR element and/or 3'UTR element.
- a reference nucleic acid molecule such as a reference mRNA comprising a reference 5' and/or a reference 3'UTR or lacking a 5'UTR element and/or 3'UTR element.
- the increase in protein production effected by the 5'UTR element compared to a reference nucleic acid molecule lacking such 5'UTR element at a given time point post initiation of expression is at least 1 .5-fold, more preferably at least 2- fold, more preferably at least 3-fold, even more preferably at least 4-fold, most preferably at least 5-fold of the protein production observed for a reference nucleic acid molecule lacking the 5'UTR element.
- Said increase in stability of the artificial nucleic acid molecule, said increase in stability of protein production, said prolongation of protein production and/or said increase in protein production is preferably determined by comparison with a respective reference nucleic acid molecule lacking a 5'UTR element and/or a 3'UTR element, e.g. an mRNA lacking a 5'UTR element and/or a 3'UTR element, or a reference nucleic acid molecule comprising a reference 5'UTR element and/or a reference 3'UTR element, such as a 3'UTR and/or a 5'UTR naturally occurring with the ORF or a 5'UTR and/or a 3'UTR of a reference gene.
- a respective reference nucleic acid molecule lacking a 5'UTR element and/or a 3'UTR element
- a reference nucleic acid molecule comprising a reference 5'UTR element and/or a reference 3'UTR element, such as a 3'UTR and/or a
- the mRNA and/or protein production stabilizing effect and efficiency and/or the protein production increasing effect and efficiency of the variants, fragments and/or variant fragments of the 3'UTR of an albumin gene as well as the mRNA and/or protein production stabilizing effect and efficiency and/or the protein production increasing effect and efficiency of the at least one 3'UTR element, the at least one 5'UTR element, or the at least one 3'UTR element and the at least one 5'UTR element of the artificial nucleic acid molecule according to the present invention may be determined by any method suitable for this purpose known to the skilled person.
- artificial mRNA molecules may be generated comprising a coding sequence for a reporter protein, such as luciferase, and no 3'UTR and/or no 5'UTR, a 5'UTR element derived from a TOP gene and/or a 3'UTR elem,ent derived from a gene as described above, a 5'UTR element derived from a reference gene and/or a 3'UTR derived from a reference gene (i.e., a reference 3'UTR element or a reference 5'UTR element, such as a 5'UTR or a 3'UTR naturally occurring with the ORF), as 3'UTR a variant of a 3'UTR of a gene as described above, as 3'UTR a fragment of a 3'UTR of a gene as described above, or as 3'UTR a fragment of a variant of a 3'UTR of a gene as described above, as 5'UTR a variant of a 5'UTR of a gene as described
- Such mRNAs may be generated, for example, by in vitro transcription of respective vectors such as plasmid vectors, e.g. comprising a T7 promoter and a sequence encoding the respective mRNA sequences.
- the generated mRNA molecules may be transfected into cells by any transfection method suitable for transfecting mRNA, for example they may be electroporated into mammalian cells, such as HELA or HDF cells, and samples may be analyzed certain time points after transfection, for example, 6 hours, 24 hours, 48 hours, and 72 hours post transfection. Said samples may be analyzed for mRNA quantities and/or protein quantities by methods well known to the skilled person.
- the quantities of reporter mRNA present in the cells at the sample time points may be determined by quantitative PCR methods.
- the quantities of reporter protein encoded by the respective mRNAs may be determined, e.g., by ELISA assays or reporter assays such as luciferase assays depending on the reporter protein used.
- the effect of stabilizing protein expression and/or prolonging protein expression may be, for example, analyzed by determining the ratio of the protein level observed 48 hours post transfection and the protein level observed 6 hours post transfection. The closer said value is to 1 , the more stable the protein expression is within this time period. Said value may also be above 1 if the protein level is higher at the later time point.
- Such measurements may of course also be performed at 72 or more hours and the ratio of the protein level observed 72 hours post transfection and the protein level observed 6 hours post transfection may be determined to determine stability of protein expression.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99%, most preferably of 100% to a nucleic acid sequence selected from SEQ ID No. 1369-1377, 1391 , 1392, and 1393 and wherein the variants of the sequences according to SEQ ID No. 1369-1377, 1391 , 1 392 and 1393 are preferably functional variants as described above.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention may also comprise or consist of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99%, most preferably of 100% to the nucleic acid sequence according to SEQ ID No. 1369-1377, 1391 , 1392, or 1393 wherein the fragment is preferably a functional fragment or a functional variant fragment as described above.
- the fragment is as described above, i.e.
- Such fragment preferably exhibits a length of at least about 40 nucleotides, preferably of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.
- such fragment may exhibit a nucleic acid sequence according to SEQ ID Nos. 1378-1390, such as
- CAGCCTACCA TGAGAATAAG AGAAAGAAAA TGAAGATCAA AAGCTTATTC ATCTGTTTTT CTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAAC ATAAATTTCT (SEQ ID No. 1381 )
- ATAAATTTCT TTAATCATTT TGCCTCTTTT CTCTGTGCTT CAATTAATAA (SEQ ID No. 1385) ATCTGTTTTT CTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAAC ATAAATTTCT
- CAGCCTACCA TGAGAATAAG AGAAAGAAAA TGAAGATCAA AAGCTTATTC ATCTGTTTTT CTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAAC ATAAATTTCT TTAATCATTT
- TGCCTCTTTT CTCTGTGCTT CAATTAATAA A (SEQ ID No . 1387) TGAAGATCAA AAGCTTATTC ATCTGTTTTT CTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAAC ATAAATTTCT TTAATCATTT TGCCTCTTTT CTCTGTGCTT CAATTAATAA
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention may comprise or consist of a nucleic acid fragment as described above.
- the thymidine nucleotides comprised in the fragments according to SEQ ID Nos. 1378-1390 may be replaced by uridine nucleotides.
- said variants, fragments or variant fragments are functional variants, functional fragments, or functional variant fragments as described above, exhibiting at least one function of the nucleic acid sequence according to SEQ ID Nos. 1369-1377, 1391 , 1392, or 1393 such as stabilization of the artificial nucleic acid molecule according to the invention, stabilizing and/or prolonging protein expression from the artificial nucleic acid molecule according to the invention, and/or increasing protein production, preferably with an efficiency of at least 40%, more preferably of at least 50%, more preferably of at least 60%, even more preferably of at least 70%, even more preferably of at least 80%, most preferably of at least 90% of the stabilizing efficiency and/or protein production increasing efficiency exhibited by the nucleic acid sequence according to SEQ ID Nos.
- variants, fragments or variant fragments are functional variants, functional fragments, or functional variant fragments exhibit the function of acting synergistically with the 5'UTR element to increase protein production from the artificial nucleic acid molecule.
- the at least one 3'UTR element of the artificial nucleic acid molecule according to the present invention exhibits a length of at least about 40 nucleotides, preferably of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.
- the 3'UTR may exhibit a length of about 50 to about 300 nucleotides, preferably of about 100 to about 250 nucleotides, more preferably of about 150 to about 200 nucleotides.
- the artificial nucleic acid molecule according to the present invention may comprise more than one 3'UTR elements as described above.
- the artificial nucleic acid molecule according to the present invention may comprise one, two, three, four or more 3'UTR elements, wherein the individual 3'UTR elements may be the same or they may be different.
- the artificial nucleic acid molecule according to the present invention may comprise two essentially identical 3'UTR elements as described above, e.g.
- two 3'UTR elements comprising or consisting of a nucleic acid sequence which is derived from the 3'UTR of an albumin gene or from a variant of the 3'UTR of an albumin gene, such as a nucleic acid sequence according to SEQ ID No. 1369 or 1 376, functional variants thereof, functional fragments thereof, or functional variant fragments thereof as described above.
- an artificial nucleic acid molecule comprising a 5'UTR element comprising or consisting of a nucleic acid sequence derived from a TOP gene as described above may represent or may provide an mRNA molecule exhibiting strongly enhanced protein production from said artificial nucleic acid molecule.
- the artificial nucleic acid molecule according to the present invention may be RNA, such as mRNA, DNA, such as a DNA vector, or may be a modified RNA or DNA molecule. It may be provided as a double-stranded molecule having a sense strand and an anti-sense strand, for example, as a DNA molecule having a sense strand and an anti-sense strand.
- the artificial nucleic acid molecule according to the present invention may further comprise a 5'-cap.
- the optional 5'-cap is preferably attached to the 5'-side of the 5'UTR element.
- the artificial nucleic acid sequence comprises a 5'UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene encoding a ribosomal protein as described above, for example, encoding a ribosomal Large protein, or from a variant thereof, and a 3'UTR element which comprises or consists of a nucleic acid sequence which is derived from the 3'UTR of an albumin gene or a variant thereof as described above.
- the artificial nucleic acid sequence comprises a 5'UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomal protein Large 21 gene (RPL21 ), an ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 , cardiac muscle (ATP5A1 ) gene, an hydroxysteroid (1 7-beta) dehydrogenase 4 gene (HSD1 7B4), an androgen-induced 1 gene (AIG1 ), cytochrome c oxidase subunit Vic gene (COX6C), or a N-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1 ) or from a variant thereof, preferably from a vertebrate ribosomal protein Large 32 gene (RPL32), a
- the artificial nucleic acid molecule according to the present invention comprises a 5'UTR element which comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No.
- a 5'UTR element which comprises or consists of a nucleic acid sequence which has an identity of at least about 90% to the nucleic acid sequence according to SEQ ID No. 1368 or a corresponding RNA sequence and a 3'UTR element which comprises or consist of a nucleic acid sequence which has an identity of at least about 90% to the nucleic acid sequence according to SEQ ID No. 1369, 1376, 1377, 1391 , or 1392.
- the artificial nucleic acid molecule according to the present invention further comprises a poly(A) sequence and/or a polyadenylation signal.
- the optional poly(A) sequence is located 3' to the ORF or the at least one 3'UTR element, preferably is connected to the 3'-end of the ORF or the 3'UTR element.
- the connection may be direct or indirect, for example, via a stretch of 2, 4, 6, 8, 10, 20 etc. nucleotides, such as via a linker of 1 -50, preferably of 1 -20 nucleotides, e.g. comprising or consisting of one or more restriction sites.
- the optional polyadenylation signal is located within the 3'UTR element.
- consensus sequence may be recognised by most animal and bacterial cell-systems, for example by the polyadenylation-factors, such as cleavage/polyadenylation specificity factor (CPSF) cooperating with CstF, PAP, PAB2, CFI and/or CFII.
- CPSF cleavage/polyadenylation specificity factor
- the polyadenylation signal preferably the consensus sequence NNUANA
- the polyadenylation signal is located less than about 50 nucleotides, more preferably less than about 30 nucleotides, most preferably less than about 25 nucleotides, for example 21 nucleotides, upstream of the 3'-end of the 3'UTR element.
- the inventive artificial nucleic acid molecule may be a DNA molecule comprising a 3'UTR element as described above and a polyadenylation signal, which may result in polyadenylation of an RNA upon transcription of this DNA molecule. Accordingly, a resulting RNA may comprise a combination of the 3'UTR element followed by a poly(A) sequence.
- transcription of an artificial nucleic acid molecule according to the invention e.g. transcription of an artificial nucleic acid molecule comprising a 5'UTR element, an open reading frame, a 3'UTR element and a polyadenylation-signal, may result in an mRNA molecule comprising a 5'UTR element, an open reading frame, a 3'UTR element and a poly(A) sequence.
- the invention also provides an artificial nucleic acid molecule which is an mRNA molecule comprising a, 5'UTR element, an open reading frame, an optional 3'UTR element as described above and a poly(A) sequence.
- the invention provides an artificial nucleic acid molecule which is an artificial DNA molecule comprising a 5'UTR element as described above, an open reading frame and optionally a nucleic acid sequence according to any one of SEQ ID Nos. 1369- 1377, 1391 , and 1392 or a sequence having an identity of at least about 40% or more to a nucleic acid sequence according to any one of SEQ ID Nos. 1369-1377, 1391 , and 1392 or a fragment thereof.
- the invention provides an artificial nucleic acid molecule which is an artificial RNA molecule comprising a 5'UTR element as described above, an open reading frame and optionally an RNA sequence corresponding to a sequence according to any one of SEQ ID Nos. 1369-1377, 1391 , and 1392 or a sequence having an identity of at least about 40 % or more to any one of SEQ ID Nos. 1369-1377, 1391 , and 1392, or a fragment thereof.
- the invention provides an artificial nucleic acid molecule which may be a template for an RNA molecule, preferably for an mRNA molecule, which is stabilised and optimized with respect to translation efficiency.
- the artificial nucleic acid molecule may be a DNA or RNA which may be used for production of an mRNA.
- the obtainable mRNA may, in turn, be translated for production of a desired peptide or protein encoded by the open reading frame.
- the artificial nucleic acid molecule is a DNA, it may, for example, be used as a double-stranded storage form for continued and repetitive in vitro or in vivo production of mRNA.
- the artificial nucleic acid molecule according to the present invention further comprises a poly(A) sequence.
- the length of the poly(A) sequence may vary.
- the poly(A) sequence may have a length of about 20 adenine nucleotides up to about 300 adenine nucleotides, preferably of about 40 to about 200 adenine nucleotides, more preferably from about 50 to about 100 adenine nucleotides, such as about 60, 70, 80, 90 or 100 adenine nucleotides.
- the artificial nucleic acid molecule according to the present invention may comprise a nucleic acid sequence corresponding to the DNA sequence
- Such artificial RNA molecule may also be obtainable in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA progenitor.
- the artificial nucleic acid molecule according to the present invention is an RNA molecule, preferably an mRNA molecule comprising in 5'-to- 3'-direction a 5'UTR element as described above, an open reading frame, a 3'UTR element as described above and a poly(A) sequence.
- the open reading frame does not code for human albumin, provided that the 3'UTR element is identical to the 3'UTR of human albumin. In some further embodiments, it is preferred that the open reading frame does not code for human albumin according to GenBank Accession number NM_000477.5 provided that the 3'UTR element is identical to the 3'UTR of human albumin. In some further embodiments, it is preferred that the open reading frame does not code for human albumin or variants thereof provided that the 3'UTR element is a sequence which is identical to SEQ ID No. 1369.
- the open reading frame does not code for a reporter protein, e.g., selected from the group consisting of globin proteins, luciferase proteins, GFP proteins or variants thereof, for example, variants exhibiting at least 70% sequence identity to a globin protein, a luciferase protein, or a GFP protein.
- a reporter protein e.g., selected from the group consisting of globin proteins, luciferase proteins, GFP proteins or variants thereof, for example, variants exhibiting at least 70% sequence identity to a globin protein, a luciferase protein, or a GFP protein.
- the 3'UTR element does not consist of a histone stem-loop, preferably does not comprise a histone stem-loop.
- the artificial nucleic acid molecule according to the present invention does not comprise a histone stem-loop.
- the 3'UTR element of the artificial nucleic acid molecule or the artificial nucleic acid molecule according to the present invention may comprise a histone stem-loop in addition to the nucleic acid sequence derived form the 3'UTR of an albumin gene.
- Such artificial nucleic acid molecule may comprise in 5'-to-3'-direction a 5'UTR element, an ORF, a 3'UTR element, preferably comprising a polyadenylation signal, an optional histone stem-loop and an optional poly(A) sequence. It may also comprise in 5'-to-3'- direction a 5'UTR element as described above, an ORF, a 3'UTR element, e.g. comprising a polyadenylation signal, a poly(A) sequence and an optional histone stem-loop.
- such a histone stem-loop is typically derived from a histone gene and comprises an intramolecular base pairing of two neighbored entirely or partially reverse complementary sequences, thereby forming a stem-loop.
- a stem-loop can occur in single-stranded DNA or, more commonly, in RNA.
- the structure is also known as a hairpin or hairpin loop and usually consists of a stem and a (terminal) loop within a consecutive sequence, wherein the stem is formed by two neighbored entirely or partially reverse complementary sequences separated by a short sequence as sort of spacer, which builds the loop of the stem-loop structure.
- the two neighbored entirely or partially reverse complementary sequences may be defined as e.g. stem-loop elements steml and stem2.
- the stem loop is formed when these two neighbored entirely or partially reverse complementary sequences, e.g. stem-loop elements steml and stem2, form base-pairs with each other, leading to a double stranded nucleic acid sequence comprising an unpaired loop at its terminal ending formed by the short sequence located between stem-loop elements steml and stem2 on the consecutive sequence.
- the unpaired loop thereby typically represents a region of the nucleic acid which is not capable of base pairing with either of these stem-loop elements.
- the resulting lollipop-shaped structure is a key building block of many RNA secondary structures.
- a stem-loop structure is thus dependent on the stability of the resulting stem and loop regions, wherein the first prerequisite is typically the presence of a sequence that can fold back on itself to form a paired double strand.
- the stability of paired stem-loop elements is determined by the length, the number of mismatches or bulges it contains (a small number of mismatches is typically tolerable, especially in a long double strand), and the base composition of the paired region.
- optimal loop length is 3-10 bases, more preferably 3 to 8, 3 to 7, 3 to 6 or even more preferably 4 to 5 bases, and most preferably 4 bases.
- the artificial nucleic acid molecule according to the present invention comprises (a.) at least one 5'UTR element as described herein, (b.) at least one open reading frame, and at least one histone-stem loop which may, for example, comprise or consist of a sequence having a sequence identity of at least about 75%, preferably of at least about 80%, preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95% to the sequence according to SEQ ID NO.
- the artificial nucleic acid molecule comprises further elements such as a 5'-cap, a poly(C) sequence and/or an IRES-motif.
- a 5'-cap may be added post- transcriptionally to the 5'end of an RNA.
- the inventive artificial nucleic acid molecule particularly if the nucleic acid is in the form of an mRNA or codes for an mRNA, may be modified by a sequence of at least 10 cytidines, preferably at least 20 cytidines, more preferably at least 30 cytidines (so-called "poly(C) sequence").
- the inventive nucleic acid molecule may contain, especially if the nucleic acid is in the form of an (m)RNA or codes for an mRNA, a poly(C) sequence of typically about 10 to 200 cytidine nucleotides, preferably about 10 to 100 cytidine nucleotides, more preferably about 10 to 70 cytidine nucleotides or even more preferably about 20 to 50 or even 20 to 30 cytidine nucleotides.
- IRES internal ribosome entry side
- IRES-motif may separate several open reading frames, for example if the artificial nucleic acid molecule encodes for two or more peptides or proteins.
- An IRES-sequence may be particularly helpful if the mRNA is a bi- or multicistronic RNA.
- the artificial nucleic acid molecule may comprise additional 5'-elements such as a promoter containing-sequence.
- the promoter may drive and or regulate transcription of the artificial nucleic acid molecule according to the present invention, for example of an artificial DNA-molecule according to the present invention.
- the invention provides artificial nucleic acid molecules, prefer- ably m NA molecules, comprising in 5'-to-3'-direction at least one of the following structures
- the artificial nucleic acid molecule preferably the open reading frame
- the inventive artificial nucleic acid molecule may be thermodynamically stabilized by modifying the G (guanosine)/C (cytidine) content of the molecule.
- the G/C content of the open reading frame of an artificial nucleic acid molecule according to the present invention may be increased compared to the G/C content of the open reading frame of a corresponding wild type sequence, preferably by using the degeneration of the genetic code.
- the encoded amino acid sequence of the nucleic acid molecule is preferably not modified by the G/C modification compared to the coded amino acid sequence of the particular wild type sequence.
- the codons of a coding sequence or a whole nucleic acid molecule, e.g. an mRNA, may therefore be varied compared to the wild type coding sequence, such that they include an increased amount of G/C nucleotides while the translated amino acid sequence is maintained.
- the most favourable codons for the stability can be determined (so-called alternative codon usage).
- nucleic acid sequence e.g. the open reading frame
- codons which contain exclusively G or C nucleotides
- no modification of the codon is necessary.
- the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modifica- tion, since no A or U/T is present.
- codons which contain A and/or U T nucleotides may be modified by substitution of other codons which code for the same amino acids but contain no A and/or U/T.
- codons for Pro can be modified from CC(U/T) or CCA to CCC or CCG;
- the codons for Arg can be modified from CG(U/T) or CGA or AGA or AGG to CGC or CGG; the codons for Ala can be modified from GC(U/T) or GCA to GCC or GCG;
- the codons for Gly can be modified from GG(U/T) or GGA to GGC or GGG.
- a or (U/T) nucleotides cannot be eliminated from the codons, it is however possible to decrease the A and (U/T) content by using codons which contain a lower content of A and/or (U/T) nucleotides. Examples of these are:
- the codons for Phe can be modified from (U T)(U T)(U/T) to (U T) (UAT)C;
- the codons for Leu can be modified from (U/T) (U/T) A, (U/T) (U T)G, C(U T) (U/T) or C(U T)A to C(UJT)C or C(UJT)G;
- the codons for Ser can be modified from (U/T)C(U/T) or (U T)CA or AG(U/T) to (UAT)CC, (U DCG or AGC;
- the codon for Tyr can be modified from (U T)A(U T) to (UAT)AC;
- the codon for Cys can be modified from (U/T)G(U/T) to (U/T)GC;
- the codon for His can be modified from CA(U/T) to CAC;
- the codon for Gin can be modified from CAA to CAG;
- the codons for lie can be modified from A(UAT)(U T) or A(U T)A to A(U T)C;
- the codons for Thr can be modified from AC(U/T) or ACA to ACC or ACG;
- the codon for Asn can be modified from AA(U/T) to AAC;
- the codon for Lys can be modified from AAA to AAG;
- the codons for Val can be modified from G(U/T)(U/T) or G(U/T)A to G(U T)C or G(U/T)G; the codon for Asp can be modified from GA(U/T) to GAC;
- the codon for Glu can be modified from GAA to GAG;
- the stop codon (U T)AA can be modified to (U T)AG or (U T)GA.
- the codons for Met (A(U/T)G) and Trp ((U/T)GG) on the other hand, there is no possibility of sequence modification without altering the encoded amino acid sequence.
- substitutions listed above can be used either individually or in all possible combinations to increase the G/C content of the open reading frame of the inventive nucleic acid sequence as defined herein, compared to its particular wild type open reading frame (i.e. the original sequence).
- all codons for Thr occurring in the wild type sequence can be modified to ACC (or ACG).
- the G/C content of the open reading frame of the inventive artificial nucleic acid molecule as defined herein is increased by at least 7%, more preferably by at least 1 5%, particularly preferably by at least 20%, compared to the G/C content of the wild type coding region.
- at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70 %, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the open reading frame of the inventive artificial nucleic acid molecule or a fragment, variant or derivative thereof are substituted, thereby increasing the G/C content of said open reading frame.
- the open reading frame is preferably at least partially codon-optimized. Codon-optimization is based on the finding that the translation efficiency may be determined by a different frequency in the occurrence of transfer RNAs (tRNAs) in cells. Thus, if so-called "rare codons" are present in the coding region of the inventive artificial nucleic acid molecule as defined herein, to an increased extent, the translation of the corresponding modified nucleic acid sequence is less efficient than in the case where codons coding for relatively "frequent" tRNAs are present.
- tRNAs transfer RNAs
- the open reading frame of the inventive nucleic acid sequence is preferably modified compared to the corresponding wild type coding region such that at least one codon of the wild type sequence which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is comparably frequent in the cell and carries the same amino acid as the relatively rare tRNA.
- the open reading frame of the inventive artificial nucleic acid molecule as defined herein is modified such that codons for which frequently occurring tRNAs are available may replace codons which correspond to rare tRNAs.
- all codons of the wild type open reading frame which code for a rare tRNA may be exchanged for a codon which codes for a tRNA which is more frequent in the cell and which carries the same amino acid as the rare tRNA.
- Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely is known to a person skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001 , 1 1 (6): 660-666.
- the open reading frame is codon-optimized, preferably with respect to the system in which the nucleic acid molecule according to the present invention is to be ex- pressed, preferably with respect to the system in which the nucleic acid molecule according to the present invention is to be translated.
- the codon usage of the open reading frame is codon-optimized according to mammalian codon usage, more preferably according to human codon usage.
- the open reading frame is codon-optimized and G/C-content modified. For further improving degradation resistance, e.g.
- the artificial nucleic acid molecule may further comprise modifications, such as backbone modifications, sugar modifications and/or base modifications, e.g., lipid-modifications or the like.
- modifications such as backbone modifications, sugar modifications and/or base modifications, e.g., lipid-modifications or the like.
- the tran- scription and/or the translation of the artificial nucleic acid molecule according to the present invention is not significantly impaired by said modifications.
- Nucleotide analogues/modifications that may be used in the context of the present invention may be selected, for example, from 2-amino-6-chloropurineriboside-5'-triphosphate, 2- aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'- triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5- aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'- triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5- methylcytidine-5 '-triphosphate, 5-methyluridine-5 '-triphosphate, 6-azacytidine-5'- triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurineriboside-5'-triphosphate,
- nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'- triphosphate, 5 -bromocytidine-5' -triphosphate, and pseudouridine-5'-triphosphate.
- lipid-modified artificial nucleic acid molecules may typically comprise at least one linker which is covalently linked with the artificial nucleic acid molecule, and at least one lipid which is covalently linked with this linker.
- a lipid-modified artificial nu- cleic acid molecule may comprise at least one artificial nucleic acid molecule as defined herein and at least one, preferably bifunctional lipid which is covalently linked, preferably without a linker, with that artificial nucleic acid molecule.
- a lipid-modified artificial nucleic acid molecule may comprise an artificial nucleic acid molecule as defined herein, at least one linker which is covalently linked with that artificial nucleic acid molecule, at least one lipid which is covalently linked with this linker, and additionally at least one, preferably bifunctional lipid which is covalently linked, preferably without a linker, with the artificial nucleic acid molecule.
- the present invention provides a vector comprising
- 5'-untranslated region element which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene or which is derived from a variant of the 5'UTR of a TOP gene;
- ORF open reading frame
- At least one 3'UTR element which comprises or consists of a nucleic acid sequence derived from the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene.
- the at least one 5'UTR element, the optional at least one 3'UTR element and the at least one ORF are as described herein for the artificial nucleic acid molecule according to the present invention.
- the cloning site may be any sequence that is suitable for introducing an open reading frame or a sequence comprising an open reading frame, such as one or more restriction sites.
- the vector comprising a cloning site is preferably suitable for inserting an open reading frame into the vector 3' to the 5'UTR element, preferably directly 3' to the 5'UTR element.
- the vector comprising a cloning site is preferably suitable for inserting an open reading frame into the vector, preferably for inserting an open reading frame between the 5'UTR element and the optional 3'UTR element, preferably 5' to the optional 3'UTR element and 3' to the 5'UTR element.
- the cloning site or the ORF is located 5' to the 3'UTR element, preferably in close proximity to the 5'-end of the 3'UTR element.
- the cloning site or the ORF may be directly connected to the 5'-end of the 3'UTR element or they may be connected via a stretch of nucleotides, such as by a stretch of 2, 4, 6, 8, 10, 20 etc.
- the cloning site or the ORF is located 3' to the 5'UTR element, preferably in close proximity to the 3'-end of the 5'UTR element.
- the cloning site or the ORF may be directly connected to the 3'-end of the 5'UTR element or they may be connected via a stretch of nucleotides, such as by a stretch of 2, 4, 6, 8, 10, 20 etc. nucleotides as described above for the artificial nucleic acid molecule according to the present invention.
- the vector according to the present invention is suitable for producing the artificial nucleic acid molecule according to the present invention, preferably for producing an artificial mRNA according to the present invention, for example, by optionally inserting an open reading frame or a sequence comprising an open reading frame into the vector and transcribing the vector.
- the vector comprises elements needed for transcription, such as a promoter, e.g. an RNA polymerase promoter.
- the vector is suitable for transcription using eukaryotic, prokaryotic, viral or phage transcription systems, such as eukaryotic cells, prokaryotic cells, or eukaryotic, prokaryotic, viral or phage in vitro transcription systems.
- the vector may comprise a promoter sequence, which is recognized by a polymerase, such as by an RNA polymerase, e.g. by a eukaryotic, prokaryotic, viral, or phage RNA polymerase.
- a polymerase such as by an RNA polymerase, e.g. by a eukaryotic, prokaryotic, viral, or phage RNA polymerase.
- the vector comprises a phage RNA polymerase promoter such as an SP6 or T7, preferably a T7 promoter.
- the vector is suitable for in vitro transcription using a phage based in vitro transcription system, such as a T7 RNA polymerase based in vitro transcription system.
- the vector may further comprise a poly(A) sequence and/or a polyadenylation signal as described above for the artificial nucleic acid molecule according to the present invention.
- the vector may be an RNA vector or a DNA vector.
- the vector is a DNA vector.
- the vector may be any vector known to the skilled person, such as a viral vector or a plasmid vector.
- the vector is a plasmid vector, preferably a DNA plasmid vector.
- the vector according to the present invention comprises the artificial nucleic acid molecule according to the present invention.
- a vector according to the present invention comprises a sequence according to SEQ ID NOs. 1 -1363, 1395, 1421 , 1422, 1368, or 1412-1420, or a sequence having an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%; even more preferably of at least about 99%; even more preferably of 100% sequence identity to a sequence according to any one of SEQ ID NOs. 1 -1363, 1395, 1421 , 1422, 1368, or 1412- 1420, or a fragment thereof, preferably a functional fragment thereof, or a corresponding RNA sequence.
- the vector is a circular molecule.
- the vector is a double-stranded molecule, such as a double stranded DNA molecule.
- Such circular, preferably double stranded DNA molecule may be used conveniently as a storage form for the inventive artificial nucleic acid molecule.
- it may be used for transfection of cells, for example, cultured cells.
- it may be used for in vitro transcription for obtaining an artificial RNA molecule according to the invention.
- the vector, preferably the circular vector is linearizable, for example, by restriction enzyme digestion.
- the vector comprises a cleavage site, such as a restriction site, preferably a unique cleavage site, located immediately 3' to the ORF, or - if present - immediately 3' to the 3'UTR element, or - if present - immediately 3' to the poly(A) sequence or polyadenylation signal, or - if present - located 3' to the poly(C) sequence, or - if present - located 3' to the histone stem-loop".
- a cleavage site such as a restriction site, preferably a unique cleavage site, located immediately 3' to the ORF, or - if present - immediately 3' to the 3'UTR element, or - if present - immediately 3' to the poly(A) sequence or polyadenylation signal, or - if present - located 3' to the poly(C) sequence, or - if present - located 3' to the histone stem-loop.
- the product obtained by linearizing the vector terminates at the 3'end with the stop codon, or - if present - the 3'-end of the 3'UTR element, or - if present - with the 3'-end of the poly(A) sequence or with the 3'-end of the polyadenylation signal, or - if present - with the 3'-end of the poly(C) sequence, or - if present - with the 3'-end of the histone stem-loop, plus optionally some nucleotides remaining from the restriction site after cleavage.
- the present invention relates to a cell comprising the artificial nucleic acid molecule according to the present invention or the vector according to present invention.
- the cell may be any cell, such as a bacterial cell, insect cell, plant cell, vertebrate cell, e.g. a mammalian cell. Such cell may be, e.g., used for replication of the vector of the present invention, for example, in a bacterial cell.
- the cell may be used for transcribing the artificial nucleic acid molecule or the vector according to the present invention and/or translating the open reading frame of the artificial nucleic acid molecule or the vector according to the present invention.
- the cell may be used for recombinant protein production.
- the cells according to the present invention are, for example, obtainable by standard nucleic acid transfer methods, such as standard transfection methods.
- the artificial nucleic acid molecule or the vector according to the present invention may be transferred into the cell by electroporation, lipofection, e.g. based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or based on cationic polymers, such as DEAE-dextran or polyethylenimine etc.
- the cell is a mammalian cell, such as a cell of human subject, a domestic animal, a laboratory animal, such as a mouse or rat cell.
- the cell is a human cell.
- the cell may be a cell of an established cell line, such as a CHO, BHK, 293T, COS-7, HELA, HEK etc. cell, or the cell may be a primary cell, e.g. a HDF cell, preferably a cell isolated from an organism.
- the cell is an isolated cell of a mammalian subject, preferably of a human subject.
- the cell may be an immune cell, such as a dendritic cell, a cancer or tumor cell, or any somatic cell etc., preferably of a mammalian subject, preferably of a human subject.
- the present invention provides a pharmaceutical composition comprising the artificial nucleic acid molecule according to the present invention, the vector according the present invention, or the cell according to the present invention.
- the pharmaceutical composition according to the invention may be used, e.g., as a vaccine, for example, for genetic vaccination.
- the O F may, e.g., encode an antigen to be administered to a patient for vaccination.
- the pharmaceutical composition according to the present invention is a vaccine.
- the pharmaceutical composition according to the present invention may be used, e.g., for gene therapy.
- the pharmaceutical composition further comprises one or more pharmaceuti- cally acceptable excipients, vehicles, fillers and/or diluents.
- a pharmaceutically acceptable vehicle typically includes a liquid or non-liquid basis for the inventive pharmaceutical composition.
- the pharmaceutical composition is provided in liquid form.
- the vehicle is based on water, such as pyrogen-free water, isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions.
- the buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e.
- the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of mammalian cells due to osmosis or other concentration effects.
- Reference media are e.g. liquids occurring in "in vivd' methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Ringer-Lactate solution is particularly preferred as a liquid basis.
- compatible solid or liquid fillers or diluents or encapsulating compounds suitable for administration to a patient may be used as well for the inventive pharmaceutical composition.
- the term "compatible” as used herein preferably means that these components of the inventive pharmaceutical composition are capable of being mixed with the inventive nucleic acid, vector or cells as defined herein in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive pharmaceutical composition under typical use conditions.
- the pharmaceutical composition according to the present invention may optionally further comprise one or more additional pharmaceutically active components.
- a pharmaceutically active component in this context is a compound that exhibits a therapeutic effect to heal, ameliorate or prevent a particular indication or disease.
- Such compounds include, without implying any limitation, peptides or proteins, nucleic acids, (therapeutically active) low molecular weight organic or inorganic compounds (molecular weight less than 5000, preferably less than 1000), sugars, antigens or antibodies, therapeutic agents already known in the prior art, antigenic cells, antigenic cellular fragments, cellular fractions, cell wall components (e.g. polysaccharides), modified, attenuated or de-activated (e.g. chemically or by irradiation) pathogens (virus, bacteria etc.).
- the inventive pharmaceutical composition may comprise a carrier for the artificial nucleic acid molecule or the vector.
- Such a carrier may be suitable for mediating dissolution in physiological acceptable liquids, transport and cellular uptake of the pharmaceutical active artificial nucleic acid molecule or the vector.
- a carrier may be a component which may be suitable for depot and delivery of an artificial nucleic acid molecule or vector according to the invention.
- Such components may be, for example, cationic or polycationic carriers or compounds which may serve as transfection or com- plexation agent.
- transfection or complexation agents are cationic or polycationic compounds, including protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes sim- plex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), p
- cationic or polycationic compounds or carriers may be cationic or polycationic peptides or proteins, which preferably comprise or are additionally modified to comprise at least one -SH moiety.
- a cationic or polycationic carrier is selected from cationic peptides having the following sum formula (I):
- the cationic or polycationic peptide or protein when defined according to formula ⁇ (Arg)i;(Lys) m ;(His) n ;(Orn) 0 ;(Xaa) x ⁇ (formula (I)) as shown above and which comprise or are additionally modified to comprise at least one -SH moeity, may be, without being restricted thereto, selected from subformula (la):
- cationic or polycationic compounds which can be used as transfection or complexation agent may include cationic polysaccharides, for example chitosan, poly- brene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
- cationic polysaccharides for example chitosan, poly- brene
- cationic polymers e.g. polyethyleneimine (PEI)
- PEI polyethyleneimine
- cationic lipids e.g.
- DOTMA [1 - (2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphati- dylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O- ditetradecanoyl-N ⁇ a rimethylammonioacetyl)diethanolamine chloride, CLIP1 : rac-[(2,3- dioctadecyloxypropyl)(2-hydroxyethyl)]-
- modified polyaminoacids such as ⁇ -aminoacid- polymers or reversed polyamides, etc.
- modified polyethylenes such as PVP (poly(N-ethyl- 4-vinylpyridinium bromide)), etc.
- modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
- modified Amidoamines such as pAMAM (poly(amidoamine)), etc.
- modified polybetaaminoester (PBAE) such as diamine end modi- fied 1 ,4 butanediol diacrylate-co-5-amino-l -pentanol polymers, etc.
- dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
- polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine,
- inventive artificial nucleic acid molecule or the inventive vector is complexed at least partially with a cationic or polycationic compound, preferably cationic proteins or peptides.
- a cationic or polycationic compound preferably cationic proteins or peptides.
- the ratio of complexed nucleic acid to: free nucleic acid is selected from a range, of about 5:1 (w/w) to about 1 :10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1 :8 (w/w), even more preferably from a range of about 3:1 (w/w) to about 1 :5 (w/w) or 1 :3 (w/w), and most preferably the ratio of complexed nucleic acid to free nucleic acid is selected from a ratio of about 1 :1 (w/w).
- the pharmaceutical composition according to the present invention may optionally further comprise one or more adjuvants, for example, adjuvants for stimulating the innate immune system or for enhancing cellular uptake of the artificial nucleic acid molecule or vector.
- an adjuvant may be understood as any compound, which is suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response.
- the inventive pharmaceutical composition when administered, preferably elicits an innate immune response due to the adjuvant, optionally contained therein.
- such an adjuvant may be an adjuvant supporting the induction of an innate immune response in a mammal.
- Such an adjuvant may be, for example, an immunostimulatory nucleic acid, i.e. a nucleic acid that may bind to a Toll-like-receptor or the like, preferably an immunostimulatory RNA.
- Such adjuvants preferably such immunostimulatory nucleic acids, may induce an innate, i.e. unspecific, immune response which may support a specific, i.e. adaptive, immune response to the peptide or protein, i.e. the antigen, encoded by the artificial nucleic acid molecule of the pharmaceutical composition, preferably the vaccine.
- the inventive pharmaceutical composition may also additionally comprise any further compound, which is known to be immunostimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13.
- any further compound which is known to be immunostimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13.
- emulsifiers such as, for example, Tween ®
- wetting agents such as, for example, sodium lauryl sulfate
- colouring agents such as, for example, sodium lauryl sulfate
- taste-imparting agents pharmaceutical carriers
- tablet- forming agents such as, for example, stabilizers; antioxidants; preservatives etc.
- the pharmaceutical composition according to the present invention preferably comprises a "safe and effective amount" of the components of the pharmaceutical composition, particularly of the inventive nucleic acid sequence, the vector and/or the cells as defined herein.
- a "safe and effective amount” means an amount sufficient to significantly induce a positive modification of a disease or disorder as defined herein.
- a "safe and effective amount” preferably avoids serious side-effects and permits a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.
- the present invention provides the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention for use as a medicament, for example, as vaccine (in genetic vaccination) or in gene therapy.
- the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention are particularly suitable for any medical application which makes use of the therapeutic action or effect of peptides, polypeptides or proteins, or where supplementation of a particular peptide or protein is needed.
- the present invention provides the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention for use in the treatment or prevention of diseases or disorders amenable to treatment by the therapeutic action or effect of peptides, polypeptides or proteins or amenable to treatment by supplementation of a particular peptide, polypeptide or protein.
- the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may be used for the treatment or prevention of genetic diseases, autoimmune diseases, cancerous or tumour-related diseases, infectious diseases, chronic diseases or the like, e.g., by genetic vaccination or gene therapy.
- such therapeutic treatments which benefit from a stable, prolonged and/or increased presence of therapeutic peptides, polypeptides or proteins in a subject to be treated are especially suitable as medical application in the context of the present invention, since the 5'UTR element optionally in combination with the 3'UTR element provides for increased protein expression from the ORF and the 3'UTR element provides for a stable and prolonged expression of the ORF of the inventive nucleic acid molecule.
- a particularly suitable medical application for the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention is vaccination, for example against infections or tumours.
- the present invention provides the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention for vaccination of a subject, preferably a mammalian subject, more preferably a human subject.
- Preferred vaccination treatments are vaccination against infectious diseases, such as bacterial, protozoal or viral infections, and anti-tumour-vaccination. Such vaccination treatments may be prophylactic or therapeutic.
- the ORF may be selected.
- the open reading frame may code for a protein that has to be supplied to a patient suffering from total lack or at least partial loss of function of a protein, such as a patient suffering from a genetic disease.
- the open reading frame may be chosen from an ORF coding for a peptide or protein which beneficially influences a disease or the condition of a subject.
- the open reading frame may code for a peptide or protein which effects down-regulation of a pathological overproduction of a natural peptide or protein or elimination of cells expressing pathologically a protein or peptide. Such lack, loss of function or overproduction may, e.g., occur in the context of tumour and neoplasia, autoimmune diseases, allergies, infections, chronic diseases or the like.
- the open reading frame may code for an antigen or immunogen, e.g. for an epitope of a pathogen or for a tumour antigen.
- the artificial nucleic acid molecule or the vector according to the present invention comprises an ORF encoding an amino acid sequence comprising or consisting of an antigen or immunogen, e.g. an epitope of a pathogen or a tumour-associated antigen, a 5'UTR element as described above, and optional further components, such as a 3'UTR element and/or a poly(A) sequence etc. as described above.
- an antigen or immunogen e.g. an epitope of a pathogen or a tumour-associated antigen, a 5'UTR element as described above, and optional further components, such as a 3'UTR element and/or a poly(A) sequence etc. as described above.
- the artificial nucleic acid molecule according to the present invention is RNA, preferably mRNA, since DNA harbours the risk of eliciting an anti-DNA immune response and tends to insert into genomic DNA.
- a viral delivery vehicle such as an adenoviral delivery vehicle
- the artificial nucleic acid molecule or the vector is a DNA molecule.
- the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
- parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracra- nial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or infusion techniques.
- the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the phar- maceutical composition according to the present invention is administered parenterally, e.g. by parenteral injection, more preferably by subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, sublingual injection or via infusion techniques. Particularly preferred is intradermal and intramus- cular injection.
- Sterile injectable forms of the inventive pharmaceutical composition may be aqueous or oleaginous suspension.
- suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
- the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
- the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may also be administered topically, espe- dally when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs.
- the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may be formulated in a suitable ointment suspended or dissolved in one or more carriers.
- the use as a medicament comprises the step of transfection of mammalian cells, preferably in vitro transfection of mammalian cells, more preferably in vitro transfection of isolated cells of a subject to be treated by the medicament. If the use comprises the in vitro transfection of isolated cells, the use as a medicament may further comprise the (re)administration of the transfected cells to the patient.
- the use of the inventive artificial nucleic acid molecules or the vector as a medicament may further comprise the step of selection of successfully transfected isolated cells. Thus, it may be beneficial if the vector further comprises a selection marker.
- the use as a medicament may comprise in vitro transfection of isolated cells and purification of an expression- product, i.e. the encoded peptide or protein from these cells. This purified peptide or protein may subsequently be administered to a subject in need thereof.
- the present invention also provides a method for treating or preventing a disease or disorder as described above comprising administering the artificial nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention to a subject in need thereof.
- the present invention provides a method for treating or preventing a disease or disorder comprising transfection of a cell with an artificial nucleic acid molecule according to the present invention or with the vector according to the present invention.
- Said transfection may be performed in vitro or in vivo.
- transfection of a cell is performed in vitro and the transfected cell is administered to a subject in need thereof, preferably to a human patient.
- the cell which is to be transfected in vitro is an isolated cell of the subject, preferably of the human patient.
- the present invention provides a method of treatment comprising the steps of isolating a cell from a subject, preferably from a human patient, transfecting the isolated cell with the artificial nucleic acid molecule according to the present invention or the vector according to the present invention, and administering the transfected cell to the subject, preferably the human patient.
- the method of treating or preventing a disorder according to the present invention is preferably a vaccination method and/or a gene therapy method as described above.
- the 5'UTR element and the optional 3'UTR element are capable of increasing protein production from an artificial nucleic acid molecule, such as an mRNA or vector, comprising the 5'UTR element and an ORF.
- the present invention relates to a method for increasing protein production from an artificial nucleic acid molecule
- a method for increasing protein production from an artificial nucleic acid molecule comprising the step of associating the artificial nucleic acid molecule, preferably the ORF contained within the artificial nucleic acid molecule, with (i) at least one 5'-untranslated region element (5'UTR element) which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene or which is derived from a variant of the 5'UTR of a TOP gene as described above and (ii) optionally at least one 3'UTR element which comprises or consists of a nucleic acid sequence derived from the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene,
- the term "associating the artificial nucleic acid molecule or the vector with a 5 'UTR element and an optional 3'UTR element" in the context of the present invention preferably means functionally associating or functionally combining the artificial nucleic acid molecule, such as the mRNA or the vector, with the 5'UTR element and the optional 3'UTR element.
- the artificial nucleic acid molecule preferably the ORF contained within the artificial nucleic acid molecule, the 5'UTR element and the optional 3'UTR element as described above are associated or coupled such that the function of the 5'UTR element and the optional 3'UTR element, e.g., protein production increasing function, is exerted.
- the 5'UTR element and the optional 3'UTR element are integrated into the artificial nucleic acid molecule, preferably into the m NA molecule or the vector, such that the open reading frame is positioned 3' to the 5'UTR element, preferably between the 5'UTR element and the optional 3'UTR element.
- the present invention provides the use of at least one 5 '-untranslated region element (5'UTR element) which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene or which is derived from a variant of the 5'UTR of a TOP gene as described above and optionally at least one 3'UTR element which comprises or consists of a nucleic acid sequence derived from the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene as described above for increasing protein production from an artificial nucleic acid molecule, such as an mRNA or a vector.
- 5'UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene or which is
- the use according to the present invention preferably comprises associating the artificial nucleic acid molecule with the 5'UTR element and the optional 3'UTR element as described above.
- the method for increasing protein production from an artificial nucleic acid molecule and the above use may also comprise associating the artificial nucleic acid molecules with one or more further elements, such as a polyadenylation signal, a poly(A) sequence, a poly(C) sequence and/or a histone stem loop as described above.
- the compounds and ingredients of the inventive pharmaceutical composition may also be manufactured and traded separately of each other.
- kit or kit of parts comprising an artificial nucleic acid molecule according to the invention, a vector according the invention, a cell according to the invention, and/or a pharmaceutical composition according to the invention.
- kit or kit of parts may, additionally, comprise instructions for use, cells for transfection, an adjuvant, a means for administration of the pharmaceutical composition, a pharmaceutically acceptable carrier and/or an pharmaceutically acceptable solution for dissolution or dilution of the artificial nucleic acid molecule, the vector, the cells or the pharmaceutical composition.
- Figures, Sequences and Examples are intended to illustrate the invention further. They are not intended to limit the subject-matter of the invention thereto.
- Fig. 1 shows the nucleotide sequence of a Photinus pyralis luciferase encoding nucleic acid molecule PpLuc(GC) - A64N64. This artificial construct does not comprise a 5'UTR element or a 3'UTR element in the sense of the present invention. The coding region for PpLuc(GC) is depicted in italics.
- Fig. 2 shows the nucleotide sequence of PpLuc(GC) - albumin7 - A64N64.
- the coding region for PpLuc(GC) is depicted in italics.
- the albumin 3'UTR is underlined.
- Fig. 3 shows the nucleotide sequence of RPL32 - PpLuc(GC) - A64N64.
- the coding region for PpLuc(GC) is depicted in italics.
- the RPL32 5'UTR is underlined.
- Fig. 4 shows the nucleotide sequence of RPL32 - PpLuc(GC) - albumin7 - A64N64.
- the 5'UTR of human ribosomal protein Large 32 gene lacking the 5' terminal oligopyrimidine tract (RPL32) according to SEQ ID NO. 1368 and the albumin7 3'UTR element according to SEQ ID NO. 1376 were inserted 5' and 3' of the ORF in the construct shown in Figure 1 , respectively.
- Fig. 5 is a graphical representation of the effect of the TOP 5'UTR element which is derived from the 5'UTR of the TOP gene RPL23 according to SEQ ID NO. 1368, the albumin 3' UTR element according to SEQ ID NO. 1376 and the combination of the TOP 5'UTR element and the albumin 3'UTR element on luciferase expression from mRNA.
- a variety of mRNAs were transfected into human dermal fibroblasts (HDF) by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- the albumin 3'UTR element extends luciferase expression, while the TOP 5'UTR element increases luciferase levels compared to mRNA lacking 5'- and 3'UTR elements. Strikingly, the combination of TOP 5'UTR element and albumin 3'UTR element further strongly increases the luciferase level, much above the level observed with either of the individual elements, thus acting synergistically. Data are graphed as mean RLU ⁇ SD (relative light units ⁇ standard deviation) for triplicate transfections. RLU are summarized in Example 5.1 . shows the nucleotide sequence of RPL35 - PpLuc(GC) - albumin7 - A64N64.
- the 5'UTR of human ribosomal protein Large 35 gene lacking the 5' terminal oligopyrimidine tract (RPL35) according to SEQ ID NO. 1412 and the albumin7 3'UTR element according to SEQ ID NO. 1376 were inserted 5' and 3' of the ORF in the construct shown in Figure 1 , respectively. shows the nucleotide sequence of RPL21 - PpLuc(GC) - albumin7 - A64N64.
- HSD1 7B4 shows the nucleotide sequence of HSD1 7B4 - PpLuc(GC) - albumin7 - A64N64.
- the 5'UTR of human hydroxysteroid (17-beta) dehydrogenase 4 gene lacking the 5' terminal oligopyrimidine tract (HSD1 7B4) according to SEQ ID NO. 1415 and the albumin7 3'UTR element according to SEQ ID NO. 1376 were inserted 5' and 3' of the ORF in the construct shown in Figure 1 , respectively. shows the nucleotide sequence of A1G1 - PpLuc(GC) - albumin7 - A64N64.
- the 5'UTR of human androgen-induced 1 gene lacking the 5' terminal oligopyrimidine tract (AIG1 ) according to SEQ ID NO. 141 6 and the albumin7 3'UTR element according to SEQ ID NO. 1376 were inserted 5' and 3' of the ORF in the construct shown in Figure 1 , respectively.
- Fig. 1 1 shows the nucleotide sequence of COX6C - PpLuc(GC) - albumin7 - A64N64.
- the 5'UTR of human cytochrome c oxidase subunit Vic gene lacking the 5' terminal oligopyrimidine tract (COX6C) according to SEQ ID NO. 141 7 and the albumin7 3'UTR element according to SEQ ID NO. 1376 were inserted 5' and 3' of the ORF in the construct shown in Figure 1 , respectively.
- Fig. 12 shows the nucleotide sequence of ASAH1 - PpLuc(GC) - albumin7 - A64N64.
- the 5'UTR of human N-acylsphingosine amidohydrolase (acid cera- midase) 1 lacking the 5' terminal oligopyrimidine tract (ASAH1 ) according to SEQ ID NO. 1418 and the albumin7 3'UTR according to SEQ ID NO. 1376 were inserted 5' and 3' of the ORF in the construct shown in Figure 1 , respectively.
- Fig. 13 shows the nucleotide sequence of mRPL21 - PpLuc(GC) - albumin7 - A64N64.
- Fig. 14 shows the nucleotide sequence of mRPL35A - PpLuc(GC) - albumin7 - A64N64.
- the 5'UTR of murine ribosomal protein Large 35a gene lacking the 5' terminal oligopyrimidine tract (mRPL35A) according to SEQ ID NO. 1420 and the albumin7 3'UTR element according to SEQ ID NO. 1376 were inserted 5' and 3' of the ORF in the construct shown in Figure 1 , respectively.
- Fig. 15 shows the nucleotide sequence of RPL35 - PpLuc(GC) - A64N64.
- Fig. 16 shows the nucleotide sequence of RPL21 - PpLuc(GC) - A64N64.
- 5'UTR of human ribosomal protein Large 21 gene lacking the 5' terminal oli- gopyrimidine tract (RPL21 ) according to SEQ ID NO. 1413 was inserted 5' of the ORF in the construct shown in Figure 1 . shows the nucleotide sequence of atp5a1 - PpLuc(GC) - A64N64.
- the 5'UTR of human ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 gene lacking the 5' terminal oligopyrimidine tract (atp5a1 ) according to SEQ ID NO. 1414 was inserted 5' of the ORF in the construct shown in Figure 1 .
- FIG. 14 shows the nucleotide sequence of HSD1 7B4 - PpLuc(GC) - A64N64.
- the 5'UTR of human hydroxysteroid (1 7-beta) dehydrogenase 4 gene lacking the 5' terminal oligopyrimidine tract (HSD1 7B4) according to SEQ ID NO. 1415 was inserted 5' of the ORF in the construct shown in Figure 1 .
- the 5'UTR of human androgen-induced 1 gene lacking the 5' terminal oligopyrimidine tract (AIG1 ) according to SEQ ID NO. 1416 was inserted 5' of the ORF in the construct shown in Figure 1 .
- FIG. 1 shows the nucleotide sequence of COX6C - PpLuc(GC) - A64N64.
- the 5'UTR of human cytochrome c oxidase subunit Vic gene lacking the 5' terminal oligopyrimidine tract (COX6C) according to SEQ ID NO. 141 7 was inserted 5' of the ORF in the construct shown in Figure 1 .
- FIG. 1418 was inserted 5' of the ORF in the construct shown in Figure 1 .
- Figure 1 is a graphical representation of the effect of different TOP 5'UTR elements on luciferase expression from mRNA.
- a variety of mRNAs were transfected into human dermal fibroblasts (HDF) by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- TOP 5'UTR elements strongly increase luciferase levels compared to mRNA lacking a 5'UTR element.
- mRNAs comprising 5'UTR elements derived from the 5'UTRs of the TOP genes ASAH1 , COX6C, AIG1 , HSD17B4, atp5a1 , RPL21 , RPL35 and RPL32 were transfected into human dermal fibroblasts (HDF) by Iipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection. The TOP 5'UTR elements increases luciferase levels compared to mRNA lacki ng a 5'UTR element. Data are graphed as mean RLU + SEM (relative light units + standard error) for triplicate transfections. RLU are summarized in Example 5.2.
- Fig. 23 is a graphical representation of the effect of the RPL35 TOP 5'UTR element, the albumin 3'UTR element and the combi nation of RPL35 TOP 5' UTR element and al bum i n 3 'UTR element on luciferase expression from mRNA.
- a variety of mRNAs were transfected into human dermal fibroblasts (HDF) by Iipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- the albumin 3'UTR element extends luciferase expression, while the RPL35 TOP 5'UTR element increases luciferase levels compared to mRNA lacking 5'- and 3 'UTR elements.
- Fig. 24 is a graphical representation of the effect of the RPL21 TOP 5'UTR element, the albumin 3'UTR element and the combination of RPL21 TOP 5'UTR element and albumin 3'UTR element on luciferase expression from mRNA.
- a variety of mRNAs were transfected into human dermal fibroblasts (HDF) by Iipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- the albumin 3'UTR element extends luciferase expression, while the RPL21 TOP 5'UTR element increases luciferase levels compared to mRNA lacking 5'- and 3'UTR elements.
- Example Fig. 25 is a graphical representation of the effect of the atp5a1 TOP 5'UTR element, the albumin 3'UTR element and the combi nation of atp5a1 TOP 5 ' UTR element and album i n 3 ' UTR element on luciferase expression from mRNA.
- mRNAs were transfected into human dermal fibroblasts (HDF) by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- the albumin 3'UTR element extends luciferase expression, while the atp5a1 TOP 5'UTR element increases luciferase levels compared to mRNA lacking 5'- and 3'UTR elements. Stri kingly, the combination of atp5a1 TOP 5'UTR element and albumin 3'UTR element further strongly increases the luciferase level, much above the level observed with either of the individual elements, thus acting synergistically. Data are graphed as mean RLU ⁇ SEM (relative light units ⁇ standard error) for triplicate transfections. Synergy is summarized in Example 5.3.
- Fig. 26 is a graphical representation of the effect of the HSD17B4 TOP 5'UTR element, the albumin 3'UTR element and the combi nation of HSD 1 7B4 TOP 5' UTR element and a lbu m i n 3 'UTR element on luciferase expression from mRNA.
- a variety of mRNAs were transfected into human dermal fibroblasts (HDF) by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- the albumin 3'UTR element extends luciferase expression, while the HSD1 7B4 TOP 5'UTR element increases luciferase levels compared to mRNA lacking 5'- and 3'UTR elements.
- Fig. 27 is a graphical representation of the effect of the AIG1 TOP 5'UTR element, the albumin 3'UTR element and the combi nation of AIG 1 TOP 5 'UTR element and albumi n 3 ' UTR element on luciferase expression from mRNA.
- a variety of mRNAs were transfected into human dermal fibroblasts (HDF) by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- the albumin 3'UTR element extends luciferase expression, while the AIG1 TOP 5'UTR element increases luciferase levels compared to mRNA lacki ng 5'- and 3'UTR elements.
- Fig. 28 is a graphical representation of the effect of the COX6C TOP 5'UTR element, the albumin 3'UTR element and the combi nation of COX6C TOP 5 ' UTR element and album i n 3 ' UTR element on luciferase expression from mRNA.
- a variety of mRNAs were transfected into human dermal fibroblasts (HDF) by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- the albumin 3'UTR element extends luciferase expression, while the COX6C TOP 5'UTR element increases luciferase levels compared to mRNA lacking 5'- and 3'UTR elements.
- Fig. 29 is a graphical representation of the effect of the ASAH1 TOP 5'UTR element, the albumin 3'UTR element and the combi nation of ASAH 1 TOP 5 ' UTR element and album i n 3 ' UTR element on luciferase expression from mRNA.
- a variety of mRNAs were transfected into human dermal fibroblasts (HDF) by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- the albumin 3'UTR element extends luciferase expression, while the ASAH1 TOP 5'UTR element increases luciferase levels compared to mRNA lacking 5'- and 3'UTR elements.
- Fig. 30 is a graphical representation of the effect of the TOP 5'UTR element from mouse genes on luciferase expression from mRNA.
- mRNAs containing either a mouse or a human TOP 5'UTR element were transfected into human dermal fibroblasts (HDF) by lipofection. Luciferase levels were measured at 24, 48, and 72 hours after transfection.
- Mouse TOP 5'UTR elements strongly increase luciferase levels compared to mRNA lacking a 5'-element, similarly as the human TOP 5'UTR element.
- Data are graphed as mean RLU + SEM (relative light units ⁇ standard error) for triplicate transfections. RLU are summarized in
- SEQ ID NO. 1416 5'UTR of human androgen-induced 1 (AIG1 ) lacking the 5' terminal oligopyrimidine tract
- a vector for in vitro transcription was constructed containing a T7 promoter followed by a GC-enriched sequence coding for Photinus pyralis luciferase (PpLuc(GQ) and an A64 poly(A) sequence.
- the poly(A) sequence was followed by a restriction site used for linearization of the vector before in vitro transcription.
- mRNA obtained from this vector accordingly by in vitro transcription is designated as skillPpLuc(GC) - A64N64".
- This vector was modified to include untranslated sequences 5' or 3' of the open reading frame (5'UTR or 3'UTR, respectively).
- vectors comprising the following mRNA encoding sequences have been generated (the mRNA coding sequences are depicted in Figures 1 to 4 and 6 to 21 ):
- the DNA-tempIate according to Example 1 was linearized and transcribed in vitro using T7- Polymerase.
- the DNA-template was then digested by DNase-treatment.
- mRNA transcripts contained a 5'-CAP structure obtained by adding an excess of N7-Methyl-Guanosine-5'- Triphosphate-5'-Guanosine to the transcription reaction. mRNA thus obtained was purified and resuspended in water.
- HDF Human dermal fibroblasts
- lysis buffer 25 mM Tris, pH 7.5 (HCI), 2 mM EDTA, 10% glycerol, 1 % Triton X-100, 2 mM DTT, 1 mM PMSF. Lysates were stored at -20°C until luciferase activity was measured.
- HDF were seeded in 96 well plates three days before transfection at a density of 10 4 cells per well.
- cells were washed in opti-MEM.
- Cells were lipofected with 25 ng of PpLuc-encoding mRNA per well com- plexed with Lipofectamine2000.
- mRNA coding for Renilla reniformis luciferase (RrLuc) was transfected together with PpLuc mRNA to control for transfection efficiency (2.5 ng of RrLuc mRNA per well).
- opti- MEM was exchanged for medium. 24, 48, 72 hours after transfection, medium was aspirated and cells were lysed in 100 ⁇ of lysis buffer (Passive Lysis Buffer, Promega). Lysates were stored at -80°C until luciferase activity was measured.
- Luciferase activity was measured as relative light units (RLU) in a BioTek SynergyHT plate reader.
- PpLuc activity was measured at 15 seconds measuring time using 50 ⁇ of lysate and 200 ⁇ of luciferin buffer (75 ⁇ luciferin, 25 mM Glycylglycin, pH 7.8 (NaOH), 15 mM MgS04, 2 mM ATP).
- RrLuc activity was measured at 15 seconds measuring time using 50 ⁇ of lysate and 200 ⁇ of coelenterazin buffer (40 ⁇ coelenterazin in phosphate buffered saline adjusted to 500 mM NaCl).
- luciferase activity was measured as relative light units (RLU) in a Hidex Cha- meleon plate reader.
- PpLuc activity was measured at 2 seconds measuring time using 20 ⁇ of lysate and 50 ⁇ of luciferin buffer (Beetle-Juice, PJK GmbH).
- RrLuc activity was measured at 2 seconds measuring time using 20 ⁇ of lysate and 50 ⁇ of coelenterazin buffer (Renilla-Juice, PJK GmbH).
- mRNAs with different UTRs were synthesized: mRNAs either lacked both TOP 5'UTR element and albumin 3'UTR element, or contained either a TOP 5'UTR element (RPL32) or an albumin 3'UTR element (albumin7), or both TOP 5'UTR element and albumin 3'UTR element.
- Luciferase encoding mRNAs or control mRNA were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured at 24, 48, and 72 hours after transfection. The PpLuc signal was corrected for transfection efficiency by the signal of cotransfected RrLuc (see following Table 1 and Figure 5).
- Luciferase was clearly expressed from mRNA having neither TOP 5'UTR nor albumin 3'UTR (PpLuc(GC)-A64N64).
- the albumin 3'UTR element extended luciferase expression, while the TOP 5'UTR element increased luciferase levels compared to mRNA lacking 5'- and 3'UTR elements. Strikingly however, the combination of TOP 5'UTR element and albumin 3'UTR element further strongly increased the luciferase level, much above the level observed with either of the individual elements.
- the magnitude of the rise in luciferase level due to combining TOP 5'UTR element and albumin 3'UTR element in the same mRNA demonstrates that they are acting synergistically.
- the synergy between TOP 5'UTR element and albumin 3'UTR element was quantified by dividing the signal from mRNA combining both elements by the sum of the signal from mRNA lacking both elements plus the rise in signal effected by the TOP 5'UTR element plus the rise in signal effected by the albumin 3'UTR element. This calculation was performed for the three time points individually and for total protein expressed from 0 to 72 hours calculated from the area under the curve (AUC) (see following Table 2).
- the synergy thus calculated specifies how much higher the luciferase level from mRNA combining TOP 5'UTR element and albumin 3'UTR element is than would be expected if the effects of TOP 5'UTR element and albumin 3'UTR element were purely additive.
- the luciferase level from mRNA combining TOP 5'UTR element and albumin 3'UTR element was up to two times higher than if their effects were purely additive. This result confirms that the combination of TOP 5'UTR element and albumin 3'UTR element effects a markedly synergistic increase in protein expression.
- TOP 5'UTR elements increase protein expression from mRNA.
- mRNAs comprising different TOP 5'UTR elements were synthesized.
- mRNAs contained the albumin7 3'UTR element.
- Luciferase encoding mRNAs were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured at 24, 48, and 72 hours after transfection (see following Table 3 and Figure 22).
- mRNAs comprising different UTR elements were synthesized: mRNAs either lacked both TOP 5'UTR element and albumin 3'UTR element, or contained an albumin 3'UTR element, or contained one of different TOP 5'UTR elements, or contained both one of different TOP 5'UTR elements and an albumin 3'UTR element.
- Luciferase encoding mRNAs were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured at 24, 48, and 72 hours after transfection (see Figures 23 to 30).
- Luciferase was clearly expressed from mRNA having neither a TOP 5'UTR element nor an albumin 3'UTR element.
- the albumin 3'UTR element extended luciferase expression, while TOP 5'UTR elements increased luciferase levels compared to mRNA lacking 5' and 3'UTRs. Strikingly however, the combinations of TOP 5'UTR elements and albumin 3'UTR element further strongly increased the luciferase level, much above the level observed with either of the individual elements.
- the magnitude of the rise in luciferase level due to combining TOP 5'UTR element and albumin 3'UTR element in the same mRNA demonstrates that they are acting synergisti- cally.
- the synergy between TOP 5'UTR element and albumin 3'UTR element was quantified by dividing the signal from mRNA combining both elements by the sum of the signal from mRNA lacking both elements plus the rise in signal effected by the TOP 5'UTR element plus the rise in signal effected by the albumin 3'UTR element. This calculation was performed for total protein expressed from 0 to 72 hours calculated from the area under the curve (AUC) (see following Table 4). Table 4:
- the synergy thus calculated specifies how much higher the luciferase level from mRNA combining TOP 5'UTR elements and albumin 3'UTR element is than would be expected if the effects of TOP 5'UTR element and albumin 3'UTR element were purely additive.
- the luciferase level from mRNA combining TOP 5'UTR element and albumin 3'UTR element was up to three times higher than if their effects were purely additive. This result confirms that the combination of TOP 5'UTR element and albumin 3'UTR element effects a markedly synergistic increase in protein expression.
- TOP 5'UTR elements from mouse genes increase protein expression from mRNA.
- mRNAs with two different mouse TOP 5'UTR elements were synthesized.
- Luciferase encoding mRNAs were transfected into human dermal fibroblasts (HDF).
- HDF human dermal fibroblasts
- Luciferase levels were measured at 24, 48, and 72 hours after transfection (see following Table 5 and Figure 30). Table 5:
- A2M alpha-2-macroglobulin
- gctccttctttctgcaacatg (Seq ID No: 1)
- ACADM acyl-CoA dehydrogenase
- arylsulfatase E Chondrodysplasia punctata 1
- ARSE arylsulfatase E : cttcctcttcttgatcggggattcaggaaggagcccaggagcagaggaagtagagagagagacaac atg (Seq ID No: 3 )
- BTK Bruton agammaglobulinemia tyrosine kinase
- CDK4 Homo sapiens cyclin-dependent kinase 4
- cytochrome P450 family 17, subfamily A, polypeptide 1 (CYP17A1) : agctcttctactccactgctgtctatcttgcctgccggcacccagccaccatg (Seq ID No: 7)
- GLC galactosylceramidase
- GJB1 Homo sapiens gap junction protein, beta 1, 32kDa (GJB1) :
- GPI glucose-6-phosphate isomerase
- HSDHA HFDHA : ctgtcctcttcagctcaagatg (Seq ID No: 16)
- SGCG 35kDa dystrophin-associated glycoprotein
- LPL lipoprotein lipase
- NPC1 Homo sapiens Niemann-Pick disease, type CI
- PEX12 Homo sapiens peroxisomal biogenesis factor 12
- PEX6 Homo sapiens peroxisomal biogenesis factor 6
- PFKM phosphofructokinase, muscle
- aldehyde dehydrogenase 3 family member A2 (ALDH3A2) : ccgcctcccactccccagcgcccccggaccgtgcagttctctgcaggaccaggccatg
- BLMH Homo sapiens bleomycin hydrolase
- CSK Homo sapiens cathepsin K
- GM2A Homo sapiens GM2 ganglioside activator
- HSD17B4 Homo sapiens hydroxysteroid (17-beta) dehydrogenase 4 (HSD17B4) : ccgcctcctcctgtcccgcagtcggcgtccagcggctctgcttgttcgtgtgtgtcgttgcagg ccttattcatg (Seq ID No: 34)
- NCF2 neutrophil cytosolic factor 2
- arylsulfatase A :
- EPN Homo sapiens elastin
- HBA2 Homo sapiens hemoglobin, alpha 2
- HBA2 cactcttctggtccccaca- gactcagagagaacccaccatg
- HEXB Homo sapiens hexosaminidase B (beta polypeptide)
- MAN2B1 Homo sapiens mannosidase, alpha, class 2B, member 1 (MAN2B1) :
- RAG2 Homo sapiens recombination activating gene 2
- CD53 Homo sapiens CD53 molecule
- Fc fragment of IgG, low affinity Ilia, receptor (CD16a) (FCGR3A) : tggtccctttagggctccggatatctttggtgacttgtccactccag- tgtggcatcatg (Seq ID No: 46)
- IL1B Homo sapiens interleukin 1, beta
- CD4 molecule CD4 :
- serpin peptidase inhibitor clade A (al- pha-1 antiproteinase, antitrypsin) , member 5 (SERPINA5) :
- VTN vitronectin
- aldehyde dehydrogenase 9 family member Al (ALDH9A1) : ccgcccctcccgcggcccccgcccctcccgcggcccgtcagcctctgccgcggagctgcgtccgcca ctcatg (Seq ID No: 51)
- ANXAl Homo sapiens annexin Al
- ATP1A1 alpha 1 polypeptide
- ATP1A2 alpha 2 polypeptide
- CACNB3 Homo sapiens calcium channel, voltage-dependent, beta 3 subunit
- cytochrome P450 family 51, subfamily A, polypeptide 1 (CYP51A1) :
- HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase
- LTA4H Homo sapiens leukotriene A4 hydrolase
- NPY1R neuropeptide Y receptor Yl
- PDHB Homo sapiens pyruvate dehydrogenase (lipoamide) beta (PDHB) :
- UDP-glucose pyrophosphorylase 2 (UGP2)
- ATP1B1 beta 1 polypeptide
- Homo sapiens wntless homolog (Drosophila) (WLS) :
- MCTP1 transmembrane 1
- cagcctcttttgccggtattcagtgaagaaagcaagtctaaatatgcagttctctcactggagtga aagatgttttgttcatttctaatcaactatg (Seq ID No: 74)
- SMC4 Homo sapiens structural maintenance of chromosomes 4 :
- GLE1 Homo sapiens GLEl RNA export mediator homolog (yeast) (GLE1) :
- TAM6 Homo sapiens tripartite motif containing 6
- gagtctttcggcctgggtggaggacgcggctgcttcaagtccttggctctgatccaggccacagat tccaggattctacaggcaggaaacatcttagaaatcagggttgggcaggcaggagccaggagagta gctacaatg (Seq ID No: 77)
- EMI2A Homo sapiens ecotropic viral integration site 2A
- HNRNPL Hemo sapiens heterogeneous nuclear ribonucleoprotein L
- HNRNPL heterogeneous nuclear ribonucleoprotein L
- MTIF2 mitochondrial translational initiation factor 2
- NFKBIZ nuclear factor of kappa light polypeptide gene enhanc er in B-cells inhibitor
- ERBB3 Homo sapiens v-erb-b2 erythroblastic leukemia viral oncogene homol og 3 (avian) (ERBB3) :
- PDPN podoplanin
- RRMl Homo sapiens ribonucleotide reductase Ml
- Homo sapiens solute carrier family 2 (facilitated glucose transporter) , member 4 (SLC2A4) :
- TBXAS1 Homo sapiens thromboxane A synthase 1 (platelet) (TBXAS1) :
- TKT Homo sapiens transketolase
- TNFRSF1A tumor necrosis factor receptor superfamily, member 1A
- TUBB2A Homo sapiens tubulin, beta 2A class Ila
- ADSS adenylosuccinate synthase
- ANPEP Homo sapiens alanyl (membrane) aminopeptidase
- BTF3 Homo sapiens basic transcription factor 3
- Homo sapiens complement component 1, q subcomponent binding protei n (C1QBP) : ttgtcctttgcatctgcacgtgttcgcagtcgtttccgcgatg
- CAV3 Homo sapiens caveolin 3
- CD68 Homo sapiens CD68 molecule
- gagcctctcctcaaagcctggctcccacggaaaatatgctcagtgcagccgcgtgcatgaatgaaaacgccgccgggcgcttctagtcggacaaaatg (Seq ID No: 102)
- LIM domain binding 2 LIM domain binding 2
- Homo sapiens chloride channel, nucleotide-sensitive, 1A (CLNS1A) : ctgcctcttccagggcgggcggtgtggtgcacgcattgctgtgctccaactccctcagggcctgtg ttgccgcactctgctgctatg (Seq ID No: 106)
- CRMP1 collapsin response mediator protein 1
- DGKA diacylglycerol kinase, alpha 80kDa
- DARS Homo sapiens aspartyl-tRNA synthetase
- DHYSL2 dihydropyrimidinase-like 2
- DRG2 developmentally regulated GTP binding protein 2
- EEF1A1 Homo sapiens eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) :
- EEF1G Homo sapiens eukaryotic translation elongation factor 1 gamma
- EIF4B eukaryotic translation initiation factor 4B
- Homo sapiens eukaryotic translation initiation factor 4 gamma, 2 (EIF4G2) : tattcttttgaagattcttcgttgtcaagccgccaaagtg
- EMP1 epithelial membrane protein 1
- FBL Homo sapiens fibrillarin
- EXTL2 Homo sapiens exostoses (multiple) -like 2
- Homo sapiens solute carrier family 37 (glucoses-phosphate transporter) , member 4 (SLC37A4) : ccgcctctgttcaggacactgggtccccttggagcctcccaggcttaatgattgtccagaaggcg gctataaagggagcctgggaggctgggtggaggagggagcagaaaaacccaactcagcagatctg ggaactgtgtgagagcggcaagcaggaactgtggtcagaggctgtgcgtcttggctggtagggcctgc tcttaccatg (Seq ID No: 123)
- GDI2 Homo sapiens GDP dissociation inhibitor 2
- GMDS GDP-mannose 4, 6-dehydratase
- HDAC2 histone deacetylase 2
- CD79A binding protein 1 IGBP1
- IGBP1 gttcctctctccccaagatg
- EIF3E Homo sapiens eukaryotic translation initiation factor 3, subunit E (EIF3E) : actcccttttctttggcaagatg (Seq ID No: 130)
- ACAM activated leukocyte cell adhesion molecule
- AOAH Homo sapiens acyloxyacyl hydrolase
- ADP-ribosylation factor 1 ADP-ribosylation factor 1
- ADP-ribosylation factor 6 ADP-ribosylation factor 6
- RHOA Homo sapiens ras homolog family member A
- Homo sapiens ras homolog family member G (RHOG) :
- ATP synthase H+ transporting, mitochondrial Fl compl ex, 0 subunit (ATP50) : ctctcttcccactcgggtttgacctacagccgcccgggagaa- gatg (Seq ID No: 137)
- B lymphoid tyrosine kinase BLK
- BCG1 Homo sapiens B-cell translocation gene 1, anti-proliferative
- CAMLG Homo sapiens calcium modulating ligand
- CAV1 Homo sapiens caveolin 1, caveolae protein, 22kDa
- CD1D Homo sapiens CDld molecule
- CD22 Homo sapiens CD22 molecule
- CD37 Homo sapiens CD37 molecule
- CD38 Homo sapiens CD38 molecule
- CD48 Homo sapiens CD48 molecule
- chromogranin B secretogranin 1 (CHGB) :
- cytochrome c oxidase subunit IV isoform 1 (COX4I1) : ctacccttttccgctccacggtgacctccgtgcggccgggtgcgggcggagtcttcctcgatcccg tggtgctccgcggcgcggccttgctcttccggtcgcgggacaccgggtgtagagggcggtcgcg gcgggcagtggcggcagaatg (Seq ID No: 152)
- ATF2 Homo sapiens activating transcription factor 2
- CSNK1A1 Homo sapiens casein kinase 1, alpha 1
- DCTD Homo sapiens dCMP deaminase
- DDB1 Homo sapiens damage-specific DNA binding protein 1, 127kDa (DDB1) : ctgtcttttcgcttgtgtccctctttctagtgtcgcgctcgagtcccgacgggccgctccaagcct cgacatg (Seq ID No: 158)
- DES Homo sapiens desmin
- DHPS deoxyhypusine synthase
- ENRA Homo sapiens endothelin receptor type A
- EEF1A2 Homo sapiens eukaryotic translation elongation factor 1 alpha 2
- EEF2 Homo sapiens eukaryotic translation elongation factor 2 (EEF2) : cgttctcttccgccgtcgtcgccgccatcctcggcgcgactcgcttctttcggttctacctgggag aatccaccgccatccgccaccatg (Seq ID No: 165)
- Homo sapiens eukaryotic translation initiation factor 4A2 Homo sapiens eukaryotic translation initiation factor 4A2
- Homo sapiens enolase 2 (gamma, neuronal) (EN02) :
- FMOD Homo sapiens fibromodulin
- FTH1 Homo sapiens ferritin, heavy polypeptide 1 (FTH1) :
- GPDH Homo sapiens glyceraldehyde-3-phosphate dehydrogenase (GAPDH) : cgctctctgctcctcctgttcgacagtcagccgcatcttcttttgcgtcgccagccgagccacatc gctcagacaccatg (Seq ID No: 174)
- GAS glycyl-tRNA synthetase
- GAT2 glutamic-oxaloacetic transaminase 2
- GAT2 mitochondrial (aspartate aminotransferase 2)
- GTF2F1 Homo sapiens general transcription factor IIF, polypeptide 1, 74kD a (GTF2F1) :
- GYS1 Homo sapiens glycogen synthase 1 (muscle) (GYS1) :
- Homo sapiens major histocompatibility complex class I, C (HLA-C) : cattctccccagaggccgagatg (Seq ID No: 179)
- Homo sapiens major histocompatibility complex class II, DP beta 1 (HLA-DPB1) :
- H GCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1 (soluble)
- HPCA Homo sapiens hippocalcin
- Homo sapiens hydroxysteroid (17-beta) dehydrogenase 2 (HSD17B2) : ctcccttcttgactctctgttcacagaactcaggctgcctccagccagcctttgcccgctagactc actggccctgagcacttgaaggtgcagcaagtcactgagaatg (Seq ID No: 183)
- Homo sapiens heat shock 60kDa protein 1 (chaperonin) (HSPD1) :
- ICM3 intercellular adhesion molecule 3
- IRF2 interferon regulatory factor 2
- ITIH2 inter-alpha-trypsin inhibitor heavy chain 2
- KPNBl Homo sapiens karyopherin (importin) beta 1 (KPNBl) :
- KPNA3 Homo sapiens karyopherin alpha 3 (importin alpha 4) (KPNA3) :
- LAMB1 Homo sapiens laminin, beta 1 (LAMB1) :
- RPSA ribosomal protein SA
- L-plastin lymphocyte cytosolic protein 1
- LCP1 lymphocyte cytosolic protein 1
- LGALS1 Homo sapiens lectin, galactoside-binding, soluble, 1
- LGALS1 Homo sapiens lectin, galactoside-binding, soluble, 1
- MAN2A1 Homo sapiens mannosidase, alpha, class 2A, member 1 (MAN2A1) :
- MBP myelin basic protein
- M1R melanocortin 1 receptor
- MEF2C myocyte enhancer factor 2C
- GAT1 Homo sapiens mannosyl (al- pha-1, 3-) -glycoprotein beta-1, 2-N-acetylglucosaminyltransferase ( GAT1) :
- mitogen-activated protein kinase kinase kinase 11 (MAP3K11) :
- NCBPl nuclear cap binding protein subunit 1, 80kDa
- tggcctctcggttccgcggcgcaccggagggcagcatg (Seq ID No: 206)
- NNN Homo sapiens necdin homolog
- NADH dehydrogenase ubiquinone
- Fe-S protein 4 18kDa (NADH-coenzyme Q reductase) (NDUFS4) :
- NFKB2 Homo sapiens nuclear factor of kappa light polypeptide gene enhanc er in B-cells 2 (p49/pl00) (NFKB2) :
- NME2 Homo sapiens non-metastatic cells 2, protein (NM23B) expressed in (NME2) :
- nucleophosmin nucleolar phosphoprotein B23, numatrin
- N5E Homo sapiens 5 ' -nucleotidase, ecto (CD73) (NT5E) :
- PEBP1 Homo sapiens phosphatidylethanolamine binding protein 1 (PEBP1) : gcgtcttcccgagccagtgtgctgagctctccgcgtcgcctctgtcgcgcctggcctaccgc ggcactcccggctgcacgctctgcttggcctcgccatg (Seq ID No: 214)
- PTD phosphogluconate dehydrogenase
- PGM1 Homo sapiens phosphoglucomutase 1
- Homo sapiens solute carrier family 25 (mitochon- drial carrier; phosphate carrier) , member 3 (SLC25A3) :
- PIMl pim-1 oncogene
- PLM2 Homo sapiens pyruvate kinase, muscle
- PLD2 Homo sapiens phospholipase D2
- PGP2 colonic epithelium-enriched
- PPAT phosphoribosyl pyrophosphate amidotransferase
- Homo sapiens protein phosphatase 1, catalytic subunit, gamma isozy me PPP1CC:
- Homo sapiens protein phosphatase 1, regulatory subunit 8 PPP1R8
- PPP1R8 protein phosphatase 1, regulatory subunit 8
- Homo sapiens protein phosphatase 6, catalytic subunit PPP6C: cggcctccgccgctgccgccgctgctacagccgccgccgctgttgccgcggcttgttatt cttaaatg (Seq ID No: 229)
- mitogen-activated protein kinase 6 MAPK6
- PRPSAP1 Homo sapiens phosphoribosyl pyrophosphate synthetase-associated pr otein 1 (PRPSAP1) :
Abstract
Description
Claims
Priority Applications (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES13713359.1T ES2660129T3 (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5'UTR-TOP |
US14/388,224 US10080809B2 (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5′TOP UTR |
RU2014142881A RU2660565C2 (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising 5'top utr |
JP2015502143A JP6301906B2 (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecule containing 5 'TOPUTR |
KR1020147030143A KR20140139101A (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5'top utr |
BR112014023898A BR112014023898A2 (en) | 2012-03-27 | 2013-03-27 | artificial nucleic acid molecules comprising 5''utr top |
CN201380016968.7A CN104321432B (en) | 2012-03-27 | 2013-03-27 | Include the artificial nucleic acid molecule of 5 ' TOP UTR |
SG11201405545XA SG11201405545XA (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5'top utr |
MX2014011625A MX358706B (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5'top utr. |
EP13713359.1A EP2831240B1 (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5'top utr |
CA2866945A CA2866945C (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5'top utr |
AU2013242405A AU2013242405B2 (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5'TOP UTR |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP2012001334 | 2012-03-27 | ||
EPPCT/EP2012/001334 | 2012-03-27 | ||
EPPCT/EP2012/002448 | 2012-06-08 | ||
EP2012002448 | 2012-06-08 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2013143700A2 true WO2013143700A2 (en) | 2013-10-03 |
WO2013143700A3 WO2013143700A3 (en) | 2013-12-27 |
Family
ID=48044723
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2013/000938 WO2013143700A2 (en) | 2012-03-27 | 2013-03-27 | Artificial nucleic acid molecules comprising a 5'top utr |
Country Status (12)
Country | Link |
---|---|
US (1) | US10080809B2 (en) |
JP (1) | JP6301906B2 (en) |
KR (1) | KR20140139101A (en) |
CN (2) | CN104321432B (en) |
AU (1) | AU2013242405B2 (en) |
BR (1) | BR112014023898A2 (en) |
CA (1) | CA2866945C (en) |
ES (1) | ES2660129T3 (en) |
MX (1) | MX358706B (en) |
RU (1) | RU2660565C2 (en) |
SG (2) | SG11201405545XA (en) |
WO (1) | WO2013143700A2 (en) |
Cited By (132)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8822663B2 (en) | 2010-08-06 | 2014-09-02 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
WO2014152211A1 (en) | 2013-03-14 | 2014-09-25 | Moderna Therapeutics, Inc. | Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions |
WO2015024667A1 (en) * | 2013-08-21 | 2015-02-26 | Curevac Gmbh | Method for increasing expression of rna-encoded proteins |
US8980864B2 (en) | 2013-03-15 | 2015-03-17 | Moderna Therapeutics, Inc. | Compositions and methods of altering cholesterol levels |
US8999380B2 (en) | 2012-04-02 | 2015-04-07 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
WO2015101414A2 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Artificial nucleic acid molecules |
WO2015101415A1 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Artificial nucleic acid molecules |
US9107886B2 (en) | 2012-04-02 | 2015-08-18 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding basic helix-loop-helix family member E41 |
WO2015149944A2 (en) | 2014-04-01 | 2015-10-08 | Curevac Gmbh | Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant |
US9186372B2 (en) | 2011-12-16 | 2015-11-17 | Moderna Therapeutics, Inc. | Split dose administration |
US9283287B2 (en) | 2012-04-02 | 2016-03-15 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of nuclear proteins |
US9334328B2 (en) | 2010-10-01 | 2016-05-10 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
WO2016091391A1 (en) * | 2014-12-12 | 2016-06-16 | Curevac Ag | Artificial nucleic acid molecules for improved protein expression |
WO2016097065A1 (en) * | 2014-12-16 | 2016-06-23 | Curevac Ag | Ebolavirus and marburgvirus vaccines |
WO2016107877A1 (en) * | 2014-12-30 | 2016-07-07 | Curevac Ag | Artificial nucleic acid molecules |
US9428535B2 (en) | 2011-10-03 | 2016-08-30 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
WO2016165831A1 (en) | 2015-04-17 | 2016-10-20 | Curevac Ag | Lyophilization of rna |
WO2016170176A1 (en) | 2015-04-22 | 2016-10-27 | Curevac Ag | Rna containing composition for treatment of tumor diseases |
WO2016180430A1 (en) | 2015-05-08 | 2016-11-17 | Curevac Ag | Method for producing rna |
WO2016184822A1 (en) | 2015-05-15 | 2016-11-24 | Curevac Ag | Prime-boost regimens involving administration of at least one mrna construct |
US9533047B2 (en) | 2011-03-31 | 2017-01-03 | Modernatx, Inc. | Delivery and formulation of engineered nucleic acids |
WO2017015630A2 (en) * | 2015-07-23 | 2017-01-26 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
WO2017021546A1 (en) * | 2015-08-05 | 2017-02-09 | Curevac Ag | Epidermal mrna vaccine |
US9572897B2 (en) | 2012-04-02 | 2017-02-21 | Modernatx, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
US9597380B2 (en) | 2012-11-26 | 2017-03-21 | Modernatx, Inc. | Terminally modified RNA |
WO2017049245A2 (en) | 2015-09-17 | 2017-03-23 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
WO2017059902A1 (en) * | 2015-10-07 | 2017-04-13 | Biontech Rna Pharmaceuticals Gmbh | 3' utr sequences for stabilization of rna |
WO2017066782A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Hydrophobic mrna cap analogs |
WO2017066793A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Mrna cap analogs and methods of mrna capping |
WO2017066791A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Sugar substituted mrna cap analogs |
WO2017066789A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Mrna cap analogs with modified sugar |
WO2017066781A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Mrna cap analogs with modified phosphate linkage |
WO2017112865A1 (en) | 2015-12-22 | 2017-06-29 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of agents |
WO2017140905A1 (en) * | 2016-02-17 | 2017-08-24 | Curevac Ag | Zika virus vaccine |
WO2017182634A1 (en) | 2016-04-22 | 2017-10-26 | Curevac Ag | Rna encoding a tumor antigen |
WO2017186928A1 (en) | 2016-04-29 | 2017-11-02 | Curevac Ag | Rna encoding an antibody |
WO2017191264A1 (en) | 2016-05-04 | 2017-11-09 | Curevac Ag | Nucleic acid molecules and uses thereof |
WO2017191274A2 (en) | 2016-05-04 | 2017-11-09 | Curevac Ag | Rna encoding a therapeutic protein |
WO2017212009A1 (en) | 2016-06-09 | 2017-12-14 | Curevac Ag | Hybrid carriers for nucleic acid cargo |
WO2017212007A1 (en) | 2016-06-09 | 2017-12-14 | Curevac Ag | Cationic carriers for nucleic acid delivery |
WO2017212006A1 (en) | 2016-06-09 | 2017-12-14 | Curevac Ag | Hybrid carriers for nucleic acid cargo |
WO2017218704A1 (en) | 2016-06-14 | 2017-12-21 | Modernatx, Inc. | Stabilized formulations of lipid nanoparticles |
WO2018033254A2 (en) | 2016-08-19 | 2018-02-22 | Curevac Ag | Rna for cancer therapy |
CN107849574A (en) * | 2015-06-30 | 2018-03-27 | 埃泽瑞斯公司 | Increase the UTR of the translation efficiency of RNA molecule |
WO2018078053A1 (en) | 2016-10-26 | 2018-05-03 | Curevac Ag | Lipid nanoparticle mrna vaccines |
WO2018089540A1 (en) | 2016-11-08 | 2018-05-17 | Modernatx, Inc. | Stabilized formulations of lipid nanoparticles |
WO2018104540A1 (en) | 2016-12-08 | 2018-06-14 | Curevac Ag | Rnas for wound healing |
WO2018104538A1 (en) | 2016-12-08 | 2018-06-14 | Curevac Ag | Rna for treatment or prophylaxis of a liver disease |
WO2018115527A2 (en) | 2016-12-23 | 2018-06-28 | Curevac Ag | Mers coronavirus vaccine |
WO2018115507A2 (en) | 2016-12-23 | 2018-06-28 | Curevac Ag | Henipavirus vaccine |
WO2018115525A1 (en) | 2016-12-23 | 2018-06-28 | Curevac Ag | Lassa virus vaccine |
WO2018170336A1 (en) | 2017-03-15 | 2018-09-20 | Modernatx, Inc. | Lipid nanoparticle formulation |
WO2018170306A1 (en) | 2017-03-15 | 2018-09-20 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
WO2018167320A1 (en) | 2017-03-17 | 2018-09-20 | Curevac Ag | Rna vaccine and immune checkpoint inhibitors for combined anticancer therapy |
EP3415629A1 (en) * | 2013-12-30 | 2018-12-19 | CureVac AG | Artificial nucleic acid molecules |
WO2018232120A1 (en) | 2017-06-14 | 2018-12-20 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of agents |
EP3424524A2 (en) | 2017-07-04 | 2019-01-09 | CureVac AG | Cancer rna-vaccine |
WO2019036638A1 (en) | 2017-08-18 | 2019-02-21 | Modernatx, Inc. | Methods of preparing modified rna |
WO2019038332A1 (en) | 2017-08-22 | 2019-02-28 | Curevac Ag | Bunyavirales vaccine |
EP3450561A1 (en) | 2013-08-21 | 2019-03-06 | CureVac AG | Method for increasing expression of rna-encoded proteins |
WO2019046809A1 (en) | 2017-08-31 | 2019-03-07 | Modernatx, Inc. | Methods of making lipid nanoparticles |
EP3461498A1 (en) | 2013-08-21 | 2019-04-03 | CureVac AG | Rabies vaccine |
WO2019077001A1 (en) * | 2017-10-19 | 2019-04-25 | Curevac Ag | Novel artificial nucleic acid molecules |
WO2019092153A1 (en) | 2017-11-08 | 2019-05-16 | Curevac Ag | Rna sequence adaptation |
EP3495486A1 (en) | 2013-12-30 | 2019-06-12 | CureVac AG | Artificial nucleic acid molecules |
US10323076B2 (en) | 2013-10-03 | 2019-06-18 | Modernatx, Inc. | Polynucleotides encoding low density lipoprotein receptor |
WO2019115635A1 (en) | 2017-12-13 | 2019-06-20 | Curevac Ag | Flavivirus vaccine |
WO2019193183A2 (en) | 2018-04-05 | 2019-10-10 | Curevac Ag | Novel yellow fever nucleic acid molecules for vaccination |
EP3586871A2 (en) | 2013-08-21 | 2020-01-01 | CureVac AG | Respiratory syncytial virus (rsv) vaccine |
WO2020002525A1 (en) | 2018-06-27 | 2020-01-02 | Curevac Ag | Novel lassa virus rna molecules and compositions for vaccination |
WO2020061367A1 (en) | 2018-09-19 | 2020-03-26 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
WO2020061457A1 (en) | 2018-09-20 | 2020-03-26 | Modernatx, Inc. | Preparation of lipid nanoparticles and methods of administration thereof |
WO2020128031A2 (en) | 2018-12-21 | 2020-06-25 | Curevac Ag | Rna for malaria vaccines |
WO2020160430A1 (en) | 2019-01-31 | 2020-08-06 | Modernatx, Inc. | Vortex mixers and associated methods, systems, and apparatuses thereof |
WO2020160397A1 (en) | 2019-01-31 | 2020-08-06 | Modernatx, Inc. | Methods of preparing lipid nanoparticles |
WO2020161342A1 (en) | 2019-02-08 | 2020-08-13 | Curevac Ag | Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases |
EP3701963A1 (en) | 2015-12-22 | 2020-09-02 | CureVac AG | Method for producing rna molecule compositions |
US10815291B2 (en) | 2013-09-30 | 2020-10-27 | Modernatx, Inc. | Polynucleotides encoding immune modulating polypeptides |
WO2020254535A1 (en) | 2019-06-18 | 2020-12-24 | Curevac Ag | Rotavirus mrna vaccine |
WO2021028439A1 (en) | 2019-08-14 | 2021-02-18 | Curevac Ag | Rna combinations and compositions with decreased immunostimulatory properties |
WO2021123332A1 (en) | 2019-12-20 | 2021-06-24 | Curevac Ag | Lipid nanoparticles for delivery of nucleic acids |
JP2021519595A (en) * | 2018-04-17 | 2021-08-12 | キュアバック アーゲー | New RSV RNA molecule and vaccination composition |
WO2021156267A1 (en) | 2020-02-04 | 2021-08-12 | Curevac Ag | Coronavirus vaccine |
EP3865579A1 (en) * | 2020-02-12 | 2021-08-18 | Pantherna Therapeutics GmbH | Recombinant nucleic acid construct and use thereof |
WO2021160758A3 (en) * | 2020-02-11 | 2021-09-23 | Pantherna Therapeutics Gmbh | Recombinant nucleic acid construct and use thereof |
WO2021204179A1 (en) | 2020-04-09 | 2021-10-14 | Suzhou Abogen Biosciences Co., Ltd. | Nucleic acid vaccines for coronavirus |
WO2021204175A1 (en) | 2020-04-09 | 2021-10-14 | Suzhou Abogen Biosciences Co., Ltd. | Lipid nanoparticle composition |
WO2021239880A1 (en) | 2020-05-29 | 2021-12-02 | Curevac Ag | Nucleic acid based combination vaccines |
US20210403925A1 (en) * | 2017-03-24 | 2021-12-30 | Curevac Ag | Nucleic acids encoding crispr-associated proteins and uses thereof |
WO2022002040A1 (en) | 2020-06-30 | 2022-01-06 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2022023559A1 (en) | 2020-07-31 | 2022-02-03 | Curevac Ag | Nucleic acid encoded antibody mixtures |
US11254951B2 (en) | 2014-12-30 | 2022-02-22 | Curevac Ag | Artificial nucleic acid molecules |
WO2022037652A1 (en) | 2020-08-20 | 2022-02-24 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2022043551A2 (en) | 2020-08-31 | 2022-03-03 | Curevac Ag | Multivalent nucleic acid based coronavirus vaccines |
WO2022137133A1 (en) | 2020-12-22 | 2022-06-30 | Curevac Ag | Rna vaccine against sars-cov-2 variants |
WO2022135993A2 (en) | 2020-12-22 | 2022-06-30 | Curevac Ag | Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration |
WO2022152141A2 (en) | 2021-01-14 | 2022-07-21 | Suzhou Abogen Biosciences Co., Ltd. | Polymer conjugated lipid compounds and lipid nanoparticle compositions |
WO2022152109A2 (en) | 2021-01-14 | 2022-07-21 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
EP4035659A1 (en) | 2016-11-29 | 2022-08-03 | PureTech LYT, Inc. | Exosomes for delivery of therapeutic agents |
WO2022162027A2 (en) | 2021-01-27 | 2022-08-04 | Curevac Ag | Method of reducing the immunostimulatory properties of in vitro transcribed rna |
WO2022200575A1 (en) | 2021-03-26 | 2022-09-29 | Glaxosmithkline Biologicals Sa | Immunogenic compositions |
WO2022207862A2 (en) | 2021-03-31 | 2022-10-06 | Curevac Ag | Syringes containing pharmaceutical compositions comprising rna |
WO2022233880A1 (en) | 2021-05-03 | 2022-11-10 | Curevac Ag | Improved nucleic acid sequence for cell type specific expression |
WO2022247755A1 (en) | 2021-05-24 | 2022-12-01 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2022256597A1 (en) | 2021-06-04 | 2022-12-08 | Translate Bio, Inc. | Assay for quantitative assessment of mrna capping efficiency |
EP4108774A1 (en) | 2015-08-28 | 2022-12-28 | CureVac AG | Artificial nucleic acid molecules |
WO2023006999A2 (en) | 2021-07-30 | 2023-02-02 | CureVac SE | Mrnas for treatment or prophylaxis of liver diseases |
WO2023025404A1 (en) | 2021-08-24 | 2023-03-02 | BioNTech SE | In vitro transcription technologies |
WO2023031394A1 (en) | 2021-09-03 | 2023-03-09 | CureVac SE | Novel lipid nanoparticles for delivery of nucleic acids |
WO2023031392A2 (en) | 2021-09-03 | 2023-03-09 | CureVac SE | Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine |
WO2023044343A1 (en) | 2021-09-14 | 2023-03-23 | Renagade Therapeutics Management Inc. | Acyclic lipids and methods of use thereof |
WO2023044333A1 (en) | 2021-09-14 | 2023-03-23 | Renagade Therapeutics Management Inc. | Cyclic lipids and methods of use thereof |
EP4162950A1 (en) | 2021-10-08 | 2023-04-12 | Suzhou Abogen Biosciences Co., Ltd. | Nucleic acid vaccines for coronavirus |
WO2023056914A1 (en) | 2021-10-08 | 2023-04-13 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2023056917A1 (en) | 2021-10-08 | 2023-04-13 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
US11667910B2 (en) | 2015-05-29 | 2023-06-06 | CureVac Manufacturing GmbH | Method for producing and purifying RNA, comprising at least one step of tangential flow filtration |
WO2023133525A1 (en) * | 2022-01-07 | 2023-07-13 | Precision Biosciences, Inc. | Optimized polynucleotides for protein expression |
WO2023144193A1 (en) | 2022-01-25 | 2023-08-03 | CureVac SE | Mrnas for treatment of hereditary tyrosinemia type i |
WO2023144330A1 (en) | 2022-01-28 | 2023-08-03 | CureVac SE | Nucleic acid encoded transcription factor inhibitors |
US11739125B2 (en) | 2013-08-21 | 2023-08-29 | Cure Vac SE | Respiratory syncytial virus (RSV) vaccine |
EP4233898A2 (en) | 2016-05-04 | 2023-08-30 | CureVac SE | Influenza mrna vaccines |
EP4239080A2 (en) | 2015-07-01 | 2023-09-06 | CureVac Manufacturing GmbH | Method for analysis of an rna molecule |
WO2023196931A1 (en) | 2022-04-07 | 2023-10-12 | Renagade Therapeutics Management Inc. | Cyclic lipids and lipid nanoparticles (lnp) for the delivery of nucleic acids or peptides for use in vaccinating against infectious agents |
US11786590B2 (en) | 2015-11-09 | 2023-10-17 | CureVac SE | Rotavirus vaccines |
WO2023227608A1 (en) | 2022-05-25 | 2023-11-30 | Glaxosmithkline Biologicals Sa | Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide |
EP4289965A2 (en) | 2016-02-12 | 2023-12-13 | CureVac SE | Method for analyzing rna |
US11872280B2 (en) | 2020-12-22 | 2024-01-16 | CureVac SE | RNA vaccine against SARS-CoV-2 variants |
WO2024037578A1 (en) | 2022-08-18 | 2024-02-22 | Suzhou Abogen Biosciences Co., Ltd. | Composition of lipid nanoparticles |
US11920174B2 (en) | 2016-03-03 | 2024-03-05 | CureVac SE | RNA analysis by total hydrolysis and quantification of released nucleosides |
WO2024068545A1 (en) | 2022-09-26 | 2024-04-04 | Glaxosmithkline Biologicals Sa | Influenza virus vaccines |
EP4353257A2 (en) | 2015-04-13 | 2024-04-17 | CureVac Manufacturing GmbH | Method for producing rna compositions |
Families Citing this family (52)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012019630A1 (en) | 2010-08-13 | 2012-02-16 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded protein |
WO2013120498A1 (en) | 2012-02-15 | 2013-08-22 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded allergenic antigen or an autoimmune self-antigen |
WO2013120500A1 (en) | 2012-02-15 | 2013-08-22 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded tumour antigen |
WO2013120497A1 (en) | 2012-02-15 | 2013-08-22 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein |
WO2013120499A1 (en) | 2012-02-15 | 2013-08-22 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly (a) sequence or a polyadenylation signal for increasing the expression of an encoded pathogenic antigen |
WO2013143700A2 (en) | 2012-03-27 | 2013-10-03 | Curevac Gmbh | Artificial nucleic acid molecules comprising a 5'top utr |
ES2742473T3 (en) | 2012-03-27 | 2020-02-14 | Curevac Ag | Artificial nucleic acid molecules for enhanced protein or peptide expression |
AU2013242403B2 (en) | 2012-03-27 | 2018-10-18 | Curevac Ag | Artificial nucleic acid molecules |
WO2014127917A1 (en) | 2013-02-22 | 2014-08-28 | Curevac Gmbh | Combination of vaccination and inhibition of the pd-1 pathway |
AU2014310935B2 (en) | 2013-08-21 | 2019-11-21 | CureVac SE | Combination vaccine |
RU2712743C2 (en) | 2013-08-21 | 2020-01-30 | Куревак Аг | Rabies vaccine |
WO2015062738A1 (en) | 2013-11-01 | 2015-05-07 | Curevac Gmbh | Modified rna with decreased immunostimulatory properties |
SG11201604198YA (en) | 2013-12-30 | 2016-07-28 | Curevac Ag | Methods for rna analysis |
ES2754239T3 (en) | 2014-03-12 | 2020-04-16 | Curevac Ag | Combination of vaccination and OX40 agonists |
EP3521456B1 (en) | 2014-06-10 | 2023-01-04 | CureVac Manufacturing GmbH | Methods and means for enhancing rna production |
PT3766916T (en) | 2014-06-25 | 2022-11-28 | Acuitas Therapeutics Inc | Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids |
CN107889503A (en) | 2015-04-30 | 2018-04-06 | 库瑞瓦格股份公司 | Poly- (N) polymerase of immobilization |
WO2016184575A1 (en) | 2015-05-20 | 2016-11-24 | Curevac Ag | Dry powder composition comprising long-chain rna |
WO2016184576A2 (en) | 2015-05-20 | 2016-11-24 | Curevac Ag | Dry powder composition comprising long-chain rna |
EP4098743A1 (en) | 2015-05-29 | 2022-12-07 | CureVac AG | Method for adding cap structures to rna using immobilized enzymes |
US10221127B2 (en) | 2015-06-29 | 2019-03-05 | Acuitas Therapeutics, Inc. | Lipids and lipid nanoparticle formulations for delivery of nucleic acids |
WO2017009376A1 (en) | 2015-07-13 | 2017-01-19 | Curevac Ag | Method of producing rna from circular dna and corresponding template dna |
EP3359670B2 (en) | 2015-10-05 | 2024-02-14 | ModernaTX, Inc. | Methods for therapeutic administration of messenger ribonucleic acid drugs |
US11225682B2 (en) | 2015-10-12 | 2022-01-18 | Curevac Ag | Automated method for isolation, selection and/or detection of microorganisms or cells comprised in a solution |
IL286515B2 (en) | 2015-10-28 | 2024-02-01 | Acuitas Therapeutics Inc | Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids |
EP3383361B1 (en) * | 2015-12-03 | 2021-08-11 | DNA Essence GmbH | Oligonucleotides in food, beverage, cosmetic and medicinal formulations |
EP3394280A1 (en) | 2015-12-23 | 2018-10-31 | CureVac AG | Method of rna in vitro transcription using a buffer containing a dicarboxylic acid or tricarboxylic acid or a salt thereof |
US10626413B2 (en) | 2016-01-15 | 2020-04-21 | Enyu Ding | Nucleic acid vector |
WO2018096179A1 (en) | 2016-11-28 | 2018-05-31 | Curevac Ag | Method for purifying rna |
CN108456669B (en) * | 2017-02-20 | 2023-08-08 | 上海凯赛生物技术股份有限公司 | Ribosome binding site, recombinant expression plasmid, transformant and application thereof |
WO2018191657A1 (en) | 2017-04-13 | 2018-10-18 | Acuitas Therapeutics, Inc. | Lipids for delivery of active agents |
WO2018200943A1 (en) | 2017-04-28 | 2018-11-01 | Acuitas Therapeutics, Inc. | Novel carbonyl lipids and lipid nanoparticle formulations for delivery of nucleic acids |
EP3642345A1 (en) * | 2017-06-20 | 2020-04-29 | The U.S.A. As Represented By The Secretary, Department Of Health And Human Services | Codon-optimized human npc1 genes for the treatment of niemann-pick type c1 deficiency and related conditions |
WO2019036008A1 (en) | 2017-08-16 | 2019-02-21 | Acuitas Therapeutics, Inc. | Lipids for use in lipid nanoparticle formulations |
WO2019036030A1 (en) | 2017-08-17 | 2019-02-21 | Acuitas Therapeutics, Inc. | Lipids for use in lipid nanoparticle formulations |
US11542225B2 (en) | 2017-08-17 | 2023-01-03 | Acuitas Therapeutics, Inc. | Lipids for use in lipid nanoparticle formulations |
WO2019122371A1 (en) | 2017-12-21 | 2019-06-27 | Curevac Ag | Linear double stranded dna coupled to a single support or a tag and methods for producing said linear double stranded dna |
CN109971786B (en) * | 2019-04-19 | 2022-09-23 | 上海市东方医院(同济大学附属东方医院) | Nucleopore protein Nup54, vector thereof and application of recombinant adenovirus |
CN112190591B (en) * | 2019-07-08 | 2021-12-17 | 中国农业科学院兰州兽医研究所 | Use of ribosomal protein RPL13 inhibitor for the preparation of a medicament for inhibiting IRES-dependent translation of viral replication |
CN111041099B (en) * | 2019-07-22 | 2021-09-17 | 江苏医药职业学院 | Application of reagent for detecting expression level of G protein-coupled receptor 137B and kit |
CN114787180A (en) | 2019-10-17 | 2022-07-22 | 斯特里迪比奥公司 | Adeno-associated virus vectors for the treatment of niemann-pick disease type C |
CN112980877A (en) * | 2019-12-12 | 2021-06-18 | 中国科学院大连化学物理研究所 | Method for improving exogenous gene expression efficiency |
US11241493B2 (en) | 2020-02-04 | 2022-02-08 | Curevac Ag | Coronavirus vaccine |
US11576966B2 (en) | 2020-02-04 | 2023-02-14 | CureVac SE | Coronavirus vaccine |
CN111218458B (en) * | 2020-02-27 | 2020-11-20 | 珠海丽凡达生物技术有限公司 | mRNAs encoding SARS-CoV-2 virus antigen and vaccine and preparation method of vaccine |
CN111118018B (en) * | 2020-03-05 | 2021-06-01 | 泰州博莱得利生物科技有限公司 | Cat serum albumin recombinant protein and expression method thereof in pichia pastoris |
CN112662673B (en) * | 2021-01-08 | 2022-06-28 | 石河子大学 | Human KLF7 gene promoter as well as construction method and application thereof |
CN113174427B (en) * | 2021-04-29 | 2023-02-28 | 遵义医科大学 | Method for detecting ERGIC3 mRNA based on DNA molecule |
CN113801213B (en) * | 2021-06-23 | 2022-04-08 | 广东省农业科学院水稻研究所 | Meloidogyne graminifolia transcription factor MgBTF3 and application thereof in disease control |
KR20230083893A (en) * | 2021-12-03 | 2023-06-12 | 고려대학교 산학협력단 | 5'-UTR with improved translation efficiency, a synthetic nucleic acid molecule comprising the same, and a vaccine or therapeutic composition comprising the same |
CN114645029A (en) * | 2022-03-11 | 2022-06-21 | 上海交通大学 | Separation of artificially synthesized MpgS protein polypeptide and MpgP protein polypeptide and application thereof |
CN117517657B (en) * | 2024-01-08 | 2024-04-09 | 中国农业科学院北京畜牧兽医研究所 | Application of LNX1 gene or protein in regulation of avian innate immune response |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002098443A2 (en) | 2001-06-05 | 2002-12-12 | Curevac Gmbh | Stabilised mrna with an increased g/c content and optimised codon for use in gene therapy |
Family Cites Families (50)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5908779A (en) * | 1993-12-01 | 1999-06-01 | University Of Connecticut | Targeted RNA degradation using nuclear antisense RNA |
WO1998042856A1 (en) | 1997-03-21 | 1998-10-01 | Enzo Therapeutics, Inc., A Fully Owned Subsidiary Of Enzo Biochem, Inc. | Vectors and viral vectors, and packaging cell lines for propagating same |
US6423885B1 (en) | 1999-08-13 | 2002-07-23 | Commonwealth Scientific And Industrial Research Organization (Csiro) | Methods for obtaining modified phenotypes in plant cells |
US8021875B2 (en) * | 2001-08-27 | 2011-09-20 | Roche Madison Inc. | Methods for expression of transgenes |
US7985553B2 (en) | 2001-10-29 | 2011-07-26 | Nathaniel Heintz | Method for isolating cell type-specific mRNAs |
DE10162480A1 (en) | 2001-12-19 | 2003-08-07 | Ingmar Hoerr | The application of mRNA for use as a therapeutic agent against tumor diseases |
DE10229872A1 (en) | 2002-07-03 | 2004-01-29 | Curevac Gmbh | Immune stimulation through chemically modified RNA |
GB0216414D0 (en) * | 2002-07-15 | 2002-08-21 | Novartis Ag | Organic compounds |
US9068234B2 (en) | 2003-01-21 | 2015-06-30 | Ptc Therapeutics, Inc. | Methods and agents for screening for compounds capable of modulating gene expression |
EP2500437B1 (en) * | 2003-01-21 | 2016-11-30 | PTC Therapeutics, Inc. | Methods for identifying compounds that modulate untranslated region-dependent gene expression and methods of using same |
DE10335833A1 (en) | 2003-08-05 | 2005-03-03 | Curevac Gmbh | Transfection of blood cells with mRNA for immune stimulation and gene therapy |
NZ546554A (en) | 2003-10-10 | 2009-04-30 | Powderject Vaccines Inc | Nucleic acid constructs |
DE102004035227A1 (en) | 2004-07-21 | 2006-02-16 | Curevac Gmbh | mRNA mixture for vaccination against tumor diseases |
DE102004042546A1 (en) | 2004-09-02 | 2006-03-09 | Curevac Gmbh | Combination therapy for immune stimulation |
DE102005023170A1 (en) | 2005-05-19 | 2006-11-23 | Curevac Gmbh | Optimized formulation for mRNA |
ES2937245T3 (en) * | 2005-08-23 | 2023-03-27 | Univ Pennsylvania | RNA containing modified nucleosides and methods of using the same |
JP2010507361A (en) | 2006-07-31 | 2010-03-11 | キュアバック ゲーエムベーハー | Specifically, a nucleic acid represented by the general formula (I): GlXmGn or the general formula (II): ClXmCn as an immunostimulant / adjuvant |
DE102006035618A1 (en) | 2006-07-31 | 2008-02-07 | Curevac Gmbh | New nucleic acid useful as immuno-stimulating adjuvant for manufacture of a composition for treatment of cancer diseases e.g. colon carcinomas and infectious diseases e.g. influenza and malaria |
DE102006061015A1 (en) | 2006-12-22 | 2008-06-26 | Curevac Gmbh | Process for the purification of RNA on a preparative scale by HPLC |
DE102007001370A1 (en) | 2007-01-09 | 2008-07-10 | Curevac Gmbh | RNA-encoded antibodies |
WO2009030254A1 (en) | 2007-09-04 | 2009-03-12 | Curevac Gmbh | Complexes of rna and cationic peptides for transfection and for immunostimulation |
WO2009046739A1 (en) | 2007-10-09 | 2009-04-16 | Curevac Gmbh | Composition for treating prostate cancer (pca) |
WO2009046738A1 (en) | 2007-10-09 | 2009-04-16 | Curevac Gmbh | Composition for treating lung cancer, particularly of non-small lung cancers (nsclc) |
PT2176408E (en) | 2008-01-31 | 2015-04-23 | Curevac Gmbh | Nucleic acids comprising formula (nugixmgnnv)a and derivatives thereof as an immunostimulating agents /adjuvants |
GB0815872D0 (en) | 2008-09-01 | 2008-10-08 | Pasteur Institut | Novel method and compositions |
WO2010037408A1 (en) | 2008-09-30 | 2010-04-08 | Curevac Gmbh | Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof |
US8343497B2 (en) | 2008-10-12 | 2013-01-01 | The Brigham And Women's Hospital, Inc. | Targeting of antigen presenting cells with immunonanotherapeutics |
ES2679043T3 (en) | 2009-05-15 | 2018-08-21 | Irx Therapeutics, Inc. | Vaccine immunotherapy |
US20110053829A1 (en) | 2009-09-03 | 2011-03-03 | Curevac Gmbh | Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids |
WO2011069529A1 (en) | 2009-12-09 | 2011-06-16 | Curevac Gmbh | Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids |
DK2449113T3 (en) | 2010-07-30 | 2016-01-11 | Curevac Ag | Complex formation of nucleic acids with the disulfide cross-linked cationic components for transfection and immunostimulation |
WO2012019630A1 (en) | 2010-08-13 | 2012-02-16 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded protein |
WO2012089225A1 (en) | 2010-12-29 | 2012-07-05 | Curevac Gmbh | Combination of vaccination and inhibition of mhc class i restricted antigen presentation |
WO2012116715A1 (en) | 2011-03-02 | 2012-09-07 | Curevac Gmbh | Vaccination in newborns and infants |
WO2012113413A1 (en) | 2011-02-21 | 2012-08-30 | Curevac Gmbh | Vaccine composition comprising complexed immunostimulatory nucleic acids and antigens packaged with disulfide-linked polyethyleneglycol/peptide conjugates |
WO2012116714A1 (en) | 2011-03-02 | 2012-09-07 | Curevac Gmbh | Vaccination in elderly patients |
WO2013113326A1 (en) | 2012-01-31 | 2013-08-08 | Curevac Gmbh | Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen |
WO2013113325A1 (en) | 2012-01-31 | 2013-08-08 | Curevac Gmbh | Negatively charged nucleic acid comprising complexes for immunostimulation |
WO2013120498A1 (en) | 2012-02-15 | 2013-08-22 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded allergenic antigen or an autoimmune self-antigen |
WO2013120500A1 (en) | 2012-02-15 | 2013-08-22 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded tumour antigen |
WO2013120499A1 (en) | 2012-02-15 | 2013-08-22 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly (a) sequence or a polyadenylation signal for increasing the expression of an encoded pathogenic antigen |
WO2013120497A1 (en) | 2012-02-15 | 2013-08-22 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein |
WO2013143700A2 (en) | 2012-03-27 | 2013-10-03 | Curevac Gmbh | Artificial nucleic acid molecules comprising a 5'top utr |
AU2013242403B2 (en) | 2012-03-27 | 2018-10-18 | Curevac Ag | Artificial nucleic acid molecules |
ES2742473T3 (en) | 2012-03-27 | 2020-02-14 | Curevac Ag | Artificial nucleic acid molecules for enhanced protein or peptide expression |
EP2854857B1 (en) | 2012-05-25 | 2018-11-28 | CureVac AG | Reversible immobilization and/or controlled release of nucleic acid containing nanoparticles by (biodegradable) polymer coatings |
LT2922554T (en) | 2012-11-26 | 2022-06-27 | Modernatx, Inc. | Terminally modified rna |
RU2712743C2 (en) | 2013-08-21 | 2020-01-30 | Куревак Аг | Rabies vaccine |
WO2015024668A2 (en) | 2013-08-21 | 2015-02-26 | Curevac Gmbh | Respiratory syncytial virus (rsv) vaccine |
RU2717986C2 (en) * | 2013-12-30 | 2020-03-27 | Куревак Аг | Artificial molecules of nucleic acid |
-
2013
- 2013-03-27 WO PCT/EP2013/000938 patent/WO2013143700A2/en active Application Filing
- 2013-03-27 JP JP2015502143A patent/JP6301906B2/en not_active Expired - Fee Related
- 2013-03-27 ES ES13713359.1T patent/ES2660129T3/en active Active
- 2013-03-27 CN CN201380016968.7A patent/CN104321432B/en not_active Expired - Fee Related
- 2013-03-27 US US14/388,224 patent/US10080809B2/en not_active Expired - Fee Related
- 2013-03-27 SG SG11201405545XA patent/SG11201405545XA/en unknown
- 2013-03-27 CN CN201810777834.1A patent/CN108929880A/en active Pending
- 2013-03-27 AU AU2013242405A patent/AU2013242405B2/en not_active Ceased
- 2013-03-27 CA CA2866945A patent/CA2866945C/en not_active Expired - Fee Related
- 2013-03-27 KR KR1020147030143A patent/KR20140139101A/en active IP Right Grant
- 2013-03-27 RU RU2014142881A patent/RU2660565C2/en active
- 2013-03-27 BR BR112014023898A patent/BR112014023898A2/en not_active Application Discontinuation
- 2013-03-27 SG SG10201607966UA patent/SG10201607966UA/en unknown
- 2013-03-27 MX MX2014011625A patent/MX358706B/en active IP Right Grant
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002098443A2 (en) | 2001-06-05 | 2002-12-12 | Curevac Gmbh | Stabilised mrna with an increased g/c content and optimised codon for use in gene therapy |
Non-Patent Citations (11)
Cited By (239)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9937233B2 (en) | 2010-08-06 | 2018-04-10 | Modernatx, Inc. | Engineered nucleic acids and methods of use thereof |
US9181319B2 (en) | 2010-08-06 | 2015-11-10 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US9447164B2 (en) | 2010-08-06 | 2016-09-20 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US8822663B2 (en) | 2010-08-06 | 2014-09-02 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US9334328B2 (en) | 2010-10-01 | 2016-05-10 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US9657295B2 (en) | 2010-10-01 | 2017-05-23 | Modernatx, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US10064959B2 (en) | 2010-10-01 | 2018-09-04 | Modernatx, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US9950068B2 (en) | 2011-03-31 | 2018-04-24 | Modernatx, Inc. | Delivery and formulation of engineered nucleic acids |
US9533047B2 (en) | 2011-03-31 | 2017-01-03 | Modernatx, Inc. | Delivery and formulation of engineered nucleic acids |
US10751386B2 (en) | 2011-09-12 | 2020-08-25 | Modernatx, Inc. | Engineered nucleic acids and methods of use thereof |
US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US10022425B2 (en) | 2011-09-12 | 2018-07-17 | Modernatx, Inc. | Engineered nucleic acids and methods of use thereof |
US9428535B2 (en) | 2011-10-03 | 2016-08-30 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US9295689B2 (en) | 2011-12-16 | 2016-03-29 | Moderna Therapeutics, Inc. | Formulation and delivery of PLGA microspheres |
US9271996B2 (en) | 2011-12-16 | 2016-03-01 | Moderna Therapeutics, Inc. | Formulation and delivery of PLGA microspheres |
US9186372B2 (en) | 2011-12-16 | 2015-11-17 | Moderna Therapeutics, Inc. | Split dose administration |
US9587003B2 (en) | 2012-04-02 | 2017-03-07 | Modernatx, Inc. | Modified polynucleotides for the production of oncology-related proteins and peptides |
US9114113B2 (en) | 2012-04-02 | 2015-08-25 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding citeD4 |
US9192651B2 (en) | 2012-04-02 | 2015-11-24 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of secreted proteins |
US9216205B2 (en) | 2012-04-02 | 2015-12-22 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding granulysin |
US9220755B2 (en) | 2012-04-02 | 2015-12-29 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders |
US9220792B2 (en) | 2012-04-02 | 2015-12-29 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding aquaporin-5 |
US9221891B2 (en) | 2012-04-02 | 2015-12-29 | Moderna Therapeutics, Inc. | In vivo production of proteins |
US9233141B2 (en) | 2012-04-02 | 2016-01-12 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders |
US9255129B2 (en) | 2012-04-02 | 2016-02-09 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1 |
US9254311B2 (en) | 2012-04-02 | 2016-02-09 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins |
US9675668B2 (en) | 2012-04-02 | 2017-06-13 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding hepatitis A virus cellular receptor 2 |
US9283287B2 (en) | 2012-04-02 | 2016-03-15 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of nuclear proteins |
US9149506B2 (en) | 2012-04-02 | 2015-10-06 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding septin-4 |
US9301993B2 (en) | 2012-04-02 | 2016-04-05 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding apoptosis inducing factor 1 |
US9303079B2 (en) | 2012-04-02 | 2016-04-05 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
US9061059B2 (en) | 2012-04-02 | 2015-06-23 | Moderna Therapeutics, Inc. | Modified polynucleotides for treating protein deficiency |
US9878056B2 (en) | 2012-04-02 | 2018-01-30 | Modernatx, Inc. | Modified polynucleotides for the production of cosmetic proteins and peptides |
US9782462B2 (en) | 2012-04-02 | 2017-10-10 | Modernatx, Inc. | Modified polynucleotides for the production of proteins associated with human disease |
US9814760B2 (en) | 2012-04-02 | 2017-11-14 | Modernatx, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
US9827332B2 (en) | 2012-04-02 | 2017-11-28 | Modernatx, Inc. | Modified polynucleotides for the production of proteins |
US9107886B2 (en) | 2012-04-02 | 2015-08-18 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding basic helix-loop-helix family member E41 |
US9828416B2 (en) | 2012-04-02 | 2017-11-28 | Modernatx, Inc. | Modified polynucleotides for the production of secreted proteins |
US9095552B2 (en) | 2012-04-02 | 2015-08-04 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1 |
US9089604B2 (en) | 2012-04-02 | 2015-07-28 | Moderna Therapeutics, Inc. | Modified polynucleotides for treating galactosylceramidase protein deficiency |
US10501512B2 (en) | 2012-04-02 | 2019-12-10 | Modernatx, Inc. | Modified polynucleotides |
US9572897B2 (en) | 2012-04-02 | 2017-02-21 | Modernatx, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
US9050297B2 (en) | 2012-04-02 | 2015-06-09 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator |
US8999380B2 (en) | 2012-04-02 | 2015-04-07 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
US9597380B2 (en) | 2012-11-26 | 2017-03-21 | Modernatx, Inc. | Terminally modified RNA |
WO2014152211A1 (en) | 2013-03-14 | 2014-09-25 | Moderna Therapeutics, Inc. | Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions |
US8980864B2 (en) | 2013-03-15 | 2015-03-17 | Moderna Therapeutics, Inc. | Compositions and methods of altering cholesterol levels |
US11739125B2 (en) | 2013-08-21 | 2023-08-29 | Cure Vac SE | Respiratory syncytial virus (RSV) vaccine |
US10799602B2 (en) | 2013-08-21 | 2020-10-13 | Curevac Ag | Method for increasing expression of RNA-encoded proteins |
EP3586871A2 (en) | 2013-08-21 | 2020-01-01 | CureVac AG | Respiratory syncytial virus (rsv) vaccine |
US10293060B2 (en) | 2013-08-21 | 2019-05-21 | Curevac Ag | Method for increasing expression of RNA-encoded proteins |
EP3461498A1 (en) | 2013-08-21 | 2019-04-03 | CureVac AG | Rabies vaccine |
EP4043032A1 (en) | 2013-08-21 | 2022-08-17 | CureVac AG | Rabies vaccine |
WO2015024667A1 (en) * | 2013-08-21 | 2015-02-26 | Curevac Gmbh | Method for increasing expression of rna-encoded proteins |
EP3450561A1 (en) | 2013-08-21 | 2019-03-06 | CureVac AG | Method for increasing expression of rna-encoded proteins |
US10815291B2 (en) | 2013-09-30 | 2020-10-27 | Modernatx, Inc. | Polynucleotides encoding immune modulating polypeptides |
US10323076B2 (en) | 2013-10-03 | 2019-06-18 | Modernatx, Inc. | Polynucleotides encoding low density lipoprotein receptor |
EP3842537A1 (en) | 2013-12-30 | 2021-06-30 | CureVac AG | Artificial nucleic acid molecules |
US11697816B2 (en) | 2013-12-30 | 2023-07-11 | CureVac SE | Artificial nucleic acid molecules |
KR102399799B1 (en) * | 2013-12-30 | 2022-05-18 | 큐어백 아게 | Artificial nucleic acid molecules |
WO2015101414A3 (en) * | 2013-12-30 | 2015-10-08 | Curevac Gmbh | Artificial nucleic acid molecules |
US10047375B2 (en) | 2013-12-30 | 2018-08-14 | Curevac Ag | Artificial nucleic acid molecules |
WO2015101414A2 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Artificial nucleic acid molecules |
CN105874072A (en) * | 2013-12-30 | 2016-08-17 | 库瑞瓦格股份公司 | Artificial nucleic acid molecules |
EP3495486A1 (en) | 2013-12-30 | 2019-06-12 | CureVac AG | Artificial nucleic acid molecules |
KR20160104062A (en) * | 2013-12-30 | 2016-09-02 | 큐어백 아게 | Artificial nucleic acid molecules |
CN111304231A (en) * | 2013-12-30 | 2020-06-19 | 库瑞瓦格股份公司 | Artificial nucleic acid molecules |
WO2015101415A1 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Artificial nucleic acid molecules |
JP2017502670A (en) * | 2013-12-30 | 2017-01-26 | キュアバック アーゲー | Artificial nucleic acid molecule |
JP6997251B2 (en) | 2013-12-30 | 2022-01-17 | キュアバック アーゲー | Artificial nucleic acid molecule |
EP3415629A1 (en) * | 2013-12-30 | 2018-12-19 | CureVac AG | Artificial nucleic acid molecules |
JP2020184995A (en) * | 2013-12-30 | 2020-11-19 | キュアバック アーゲー | Artificial nucleic acid molecules |
WO2015149944A2 (en) | 2014-04-01 | 2015-10-08 | Curevac Gmbh | Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant |
DE202015009961U1 (en) | 2014-12-12 | 2022-01-25 | Curevac Ag | Artificial nucleic acid molecules for improved protein expression |
DE202015009974U1 (en) | 2014-12-12 | 2022-02-17 | Curevac Ag | Artificial nucleic acid molecules for improved protein expression |
US11149278B2 (en) | 2014-12-12 | 2021-10-19 | Curevac Ag | Artificial nucleic acid molecules for improved protein expression |
DE202015010000U1 (en) | 2014-12-12 | 2023-07-03 | CureVac SE | Artificial nucleic acid molecules for improved protein expression |
EP4023755A1 (en) | 2014-12-12 | 2022-07-06 | CureVac AG | Artificial nucleic acid molecules for improved protein expression |
WO2016091391A1 (en) * | 2014-12-12 | 2016-06-16 | Curevac Ag | Artificial nucleic acid molecules for improved protein expression |
US11286492B2 (en) | 2014-12-12 | 2022-03-29 | Curevac Ag | Artificial nucleic acid molecules for improved protein expression |
US11345920B2 (en) | 2014-12-12 | 2022-05-31 | Curevac Ag | Artificial nucleic acid molecules for improved protein expression |
EP4241784A2 (en) | 2014-12-12 | 2023-09-13 | CureVac SE | Artificial nucleic acid molecules for improved protein expression |
EP3708668A1 (en) * | 2014-12-12 | 2020-09-16 | CureVac AG | Artificial nucleic acid molecules for improved protein expression |
EP3708668B1 (en) | 2014-12-12 | 2022-07-27 | CureVac AG | Artificial nucleic acid molecules for improved protein expression |
US11761009B2 (en) | 2014-12-12 | 2023-09-19 | CureVac SE | Artificial nucleic acid molecules for improved protein expression |
DE202015010001U1 (en) | 2014-12-12 | 2023-07-03 | CureVac SE | Artificial nucleic acid molecules for improved protein expression |
EP4241784A3 (en) * | 2014-12-12 | 2023-11-15 | CureVac SE | Artificial nucleic acid molecules for improved protein expression |
EP3230458B1 (en) * | 2014-12-12 | 2020-02-19 | CureVac AG | Artificial nucleic acid molecules for improved protein expression |
WO2016097065A1 (en) * | 2014-12-16 | 2016-06-23 | Curevac Ag | Ebolavirus and marburgvirus vaccines |
KR20170100660A (en) * | 2014-12-30 | 2017-09-04 | 큐어백 아게 | New artificial nucleic acid molecule |
CN107124889A (en) * | 2014-12-30 | 2017-09-01 | 库瑞瓦格股份公司 | Artificial nucleic acid molecule |
EP3494982A1 (en) | 2014-12-30 | 2019-06-12 | CureVac AG | Artificial nucleic acid molecules |
KR102580696B1 (en) | 2014-12-30 | 2023-09-19 | 큐어백 에스이 | Novel artificial nucleic acid molecules |
US11254951B2 (en) | 2014-12-30 | 2022-02-22 | Curevac Ag | Artificial nucleic acid molecules |
WO2016107877A1 (en) * | 2014-12-30 | 2016-07-07 | Curevac Ag | Artificial nucleic acid molecules |
JP2018501802A (en) * | 2014-12-30 | 2018-01-25 | キュアバック アーゲー | Artificial nucleic acid molecule |
JP2021168664A (en) * | 2014-12-30 | 2021-10-28 | キュアバック アーゲー | Artificial nucleic acid molecules |
RU2757675C2 (en) * | 2014-12-30 | 2021-10-20 | Куревак Аг | Molecules of new artificial nucleic acids |
EP4353257A2 (en) | 2015-04-13 | 2024-04-17 | CureVac Manufacturing GmbH | Method for producing rna compositions |
EP4026568A1 (en) | 2015-04-17 | 2022-07-13 | CureVac Real Estate GmbH | Lyophilization of rna |
WO2016165831A1 (en) | 2015-04-17 | 2016-10-20 | Curevac Ag | Lyophilization of rna |
EP3326641A1 (en) | 2015-04-22 | 2018-05-30 | CureVac AG | Rna containing composition for treatment of tumor diseases |
EP3173092A2 (en) | 2015-04-22 | 2017-05-31 | CureVac AG | Rna containing composition for treatment of tumor diseases |
WO2016170176A1 (en) | 2015-04-22 | 2016-10-27 | Curevac Ag | Rna containing composition for treatment of tumor diseases |
EP3603661A2 (en) | 2015-04-22 | 2020-02-05 | CureVac AG | Rna containing composition for treatment of tumor diseases |
WO2016180430A1 (en) | 2015-05-08 | 2016-11-17 | Curevac Ag | Method for producing rna |
US11661634B2 (en) | 2015-05-08 | 2023-05-30 | CureVac Manufacturing GmbH | Method for producing RNA |
WO2016184822A1 (en) | 2015-05-15 | 2016-11-24 | Curevac Ag | Prime-boost regimens involving administration of at least one mrna construct |
US11667910B2 (en) | 2015-05-29 | 2023-06-06 | CureVac Manufacturing GmbH | Method for producing and purifying RNA, comprising at least one step of tangential flow filtration |
US11760992B2 (en) | 2015-05-29 | 2023-09-19 | CureVac Manufacturing GmbH | Method for producing and purifying RNA, comprising at least one step of tangential flow filtration |
US11834651B2 (en) | 2015-05-29 | 2023-12-05 | CureVac Manufacturing GmbH | Method for producing and purifying RNA, comprising at least one step of tangential flow filtration |
CN107849574A (en) * | 2015-06-30 | 2018-03-27 | 埃泽瑞斯公司 | Increase the UTR of the translation efficiency of RNA molecule |
US11136585B2 (en) | 2015-06-30 | 2021-10-05 | Ethris Gmbh | UTRs increasing the translation efficiency of RNA molecules |
EP4239080A2 (en) | 2015-07-01 | 2023-09-06 | CureVac Manufacturing GmbH | Method for analysis of an rna molecule |
WO2017015630A2 (en) * | 2015-07-23 | 2017-01-26 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
WO2017015630A3 (en) * | 2015-07-23 | 2017-03-30 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
WO2017021546A1 (en) * | 2015-08-05 | 2017-02-09 | Curevac Ag | Epidermal mrna vaccine |
EP4108774A1 (en) | 2015-08-28 | 2022-12-28 | CureVac AG | Artificial nucleic acid molecules |
EP4286012A2 (en) | 2015-09-17 | 2023-12-06 | ModernaTX, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
EP3736261A1 (en) | 2015-09-17 | 2020-11-11 | ModernaTX, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
WO2017049245A2 (en) | 2015-09-17 | 2017-03-23 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
EP3868885A1 (en) * | 2015-10-07 | 2021-08-25 | BioNTech RNA Pharmaceuticals GmbH | 3' utr sequences for stabilization of rna |
KR102363368B1 (en) | 2015-10-07 | 2022-02-16 | 비온테크 에스이 | 3' UTR sequence for stabilization of RNA |
WO2017059902A1 (en) * | 2015-10-07 | 2017-04-13 | Biontech Rna Pharmaceuticals Gmbh | 3' utr sequences for stabilization of rna |
KR20180057647A (en) * | 2015-10-07 | 2018-05-30 | 비온테크 알엔에이 파마슈티컬스 게엠베하 | 3 ' UTR sequence for stabilization of RNA |
EP3636764A1 (en) * | 2015-10-07 | 2020-04-15 | BioNTech RNA Pharmaceuticals GmbH | 3' utr sequences for stabilization of rna |
JP2021048847A (en) * | 2015-10-07 | 2021-04-01 | バイオエヌテック エールエヌアー ファーマシューティカルズ ゲーエムベーハーBiontech Rna Pharmaceuticals Gmbh | 3'utr sequences for stabilization of rna |
US11492628B2 (en) | 2015-10-07 | 2022-11-08 | BioNTech SE | 3′-UTR sequences for stabilization of RNA |
JP7084565B2 (en) | 2015-10-07 | 2022-06-15 | バイオエヌテック エスエー | 3'UTR sequence for RNA stabilization |
WO2017060314A3 (en) * | 2015-10-07 | 2017-05-18 | Biontech Rna Pharmaceuticals Gmbh | 3' utr sequences for stabilization of rna |
WO2017066782A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Hydrophobic mrna cap analogs |
WO2017066793A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Mrna cap analogs and methods of mrna capping |
EP4086269A1 (en) | 2015-10-16 | 2022-11-09 | ModernaTX, Inc. | Mrna cap analogs with modified phosphate linkage |
WO2017066791A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Sugar substituted mrna cap analogs |
WO2017066781A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Mrna cap analogs with modified phosphate linkage |
WO2017066789A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Mrna cap analogs with modified sugar |
US11786590B2 (en) | 2015-11-09 | 2023-10-17 | CureVac SE | Rotavirus vaccines |
EP4036079A2 (en) | 2015-12-22 | 2022-08-03 | ModernaTX, Inc. | Compounds and compositions for intracellular delivery of agents |
EP3701963A1 (en) | 2015-12-22 | 2020-09-02 | CureVac AG | Method for producing rna molecule compositions |
WO2017112865A1 (en) | 2015-12-22 | 2017-06-29 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of agents |
EP4289965A2 (en) | 2016-02-12 | 2023-12-13 | CureVac SE | Method for analyzing rna |
US11723967B2 (en) | 2016-02-17 | 2023-08-15 | CureVac SE | Zika virus vaccine |
WO2017140905A1 (en) * | 2016-02-17 | 2017-08-24 | Curevac Ag | Zika virus vaccine |
US20210205434A1 (en) * | 2016-02-17 | 2021-07-08 | Curevac Ag | Zika virus vaccine |
US11920174B2 (en) | 2016-03-03 | 2024-03-05 | CureVac SE | RNA analysis by total hydrolysis and quantification of released nucleosides |
WO2017182634A1 (en) | 2016-04-22 | 2017-10-26 | Curevac Ag | Rna encoding a tumor antigen |
EP3777881A1 (en) | 2016-04-22 | 2021-02-17 | CureVac AG | Rna encoding a tumor antigen |
WO2017186928A1 (en) | 2016-04-29 | 2017-11-02 | Curevac Ag | Rna encoding an antibody |
EP4233898A2 (en) | 2016-05-04 | 2023-08-30 | CureVac SE | Influenza mrna vaccines |
US11078247B2 (en) | 2016-05-04 | 2021-08-03 | Curevac Ag | RNA encoding a therapeutic protein |
WO2017191274A2 (en) | 2016-05-04 | 2017-11-09 | Curevac Ag | Rna encoding a therapeutic protein |
WO2017191274A3 (en) * | 2016-05-04 | 2018-02-08 | Curevac Ag | Rna encoding a therapeutic protein |
WO2017191264A1 (en) | 2016-05-04 | 2017-11-09 | Curevac Ag | Nucleic acid molecules and uses thereof |
WO2017212007A1 (en) | 2016-06-09 | 2017-12-14 | Curevac Ag | Cationic carriers for nucleic acid delivery |
WO2017212006A1 (en) | 2016-06-09 | 2017-12-14 | Curevac Ag | Hybrid carriers for nucleic acid cargo |
WO2017212009A1 (en) | 2016-06-09 | 2017-12-14 | Curevac Ag | Hybrid carriers for nucleic acid cargo |
WO2017218704A1 (en) | 2016-06-14 | 2017-12-21 | Modernatx, Inc. | Stabilized formulations of lipid nanoparticles |
WO2018033254A2 (en) | 2016-08-19 | 2018-02-22 | Curevac Ag | Rna for cancer therapy |
WO2018078053A1 (en) | 2016-10-26 | 2018-05-03 | Curevac Ag | Lipid nanoparticle mrna vaccines |
WO2018089540A1 (en) | 2016-11-08 | 2018-05-17 | Modernatx, Inc. | Stabilized formulations of lipid nanoparticles |
EP4035659A1 (en) | 2016-11-29 | 2022-08-03 | PureTech LYT, Inc. | Exosomes for delivery of therapeutic agents |
EP3808380A1 (en) | 2016-12-08 | 2021-04-21 | CureVac AG | Rna for treatment or prophylaxis of a liver disease |
US11464836B2 (en) | 2016-12-08 | 2022-10-11 | Curevac Ag | RNA for treatment or prophylaxis of a liver disease |
WO2018104538A1 (en) | 2016-12-08 | 2018-06-14 | Curevac Ag | Rna for treatment or prophylaxis of a liver disease |
WO2018104540A1 (en) | 2016-12-08 | 2018-06-14 | Curevac Ag | Rnas for wound healing |
WO2018115525A1 (en) | 2016-12-23 | 2018-06-28 | Curevac Ag | Lassa virus vaccine |
WO2018115527A2 (en) | 2016-12-23 | 2018-06-28 | Curevac Ag | Mers coronavirus vaccine |
WO2018115507A2 (en) | 2016-12-23 | 2018-06-28 | Curevac Ag | Henipavirus vaccine |
EP4186888A1 (en) | 2017-03-15 | 2023-05-31 | ModernaTX, Inc. | Compound and compositions for intracellular delivery of therapeutic agents |
WO2018170336A1 (en) | 2017-03-15 | 2018-09-20 | Modernatx, Inc. | Lipid nanoparticle formulation |
WO2018170306A1 (en) | 2017-03-15 | 2018-09-20 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
WO2018167320A1 (en) | 2017-03-17 | 2018-09-20 | Curevac Ag | Rna vaccine and immune checkpoint inhibitors for combined anticancer therapy |
US20210403925A1 (en) * | 2017-03-24 | 2021-12-30 | Curevac Ag | Nucleic acids encoding crispr-associated proteins and uses thereof |
US11739335B2 (en) * | 2017-03-24 | 2023-08-29 | CureVac SE | Nucleic acids encoding CRISPR-associated proteins and uses thereof |
WO2018232120A1 (en) | 2017-06-14 | 2018-12-20 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of agents |
WO2019008001A1 (en) | 2017-07-04 | 2019-01-10 | Curevac Ag | Novel nucleic acid molecules |
EP3424524A2 (en) | 2017-07-04 | 2019-01-09 | CureVac AG | Cancer rna-vaccine |
WO2019036638A1 (en) | 2017-08-18 | 2019-02-21 | Modernatx, Inc. | Methods of preparing modified rna |
WO2019038332A1 (en) | 2017-08-22 | 2019-02-28 | Curevac Ag | Bunyavirales vaccine |
US11602557B2 (en) | 2017-08-22 | 2023-03-14 | Cure Vac SE | Bunyavirales vaccine |
WO2019046809A1 (en) | 2017-08-31 | 2019-03-07 | Modernatx, Inc. | Methods of making lipid nanoparticles |
WO2019077001A1 (en) * | 2017-10-19 | 2019-04-25 | Curevac Ag | Novel artificial nucleic acid molecules |
WO2019092153A1 (en) | 2017-11-08 | 2019-05-16 | Curevac Ag | Rna sequence adaptation |
WO2019115635A1 (en) | 2017-12-13 | 2019-06-20 | Curevac Ag | Flavivirus vaccine |
WO2019193183A2 (en) | 2018-04-05 | 2019-10-10 | Curevac Ag | Novel yellow fever nucleic acid molecules for vaccination |
JP2021519595A (en) * | 2018-04-17 | 2021-08-12 | キュアバック アーゲー | New RSV RNA molecule and vaccination composition |
EP4227319A1 (en) | 2018-04-17 | 2023-08-16 | CureVac SE | Novel rsv rna molecules and compositions for vaccination |
WO2020002525A1 (en) | 2018-06-27 | 2020-01-02 | Curevac Ag | Novel lassa virus rna molecules and compositions for vaccination |
WO2020061367A1 (en) | 2018-09-19 | 2020-03-26 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
WO2020061457A1 (en) | 2018-09-20 | 2020-03-26 | Modernatx, Inc. | Preparation of lipid nanoparticles and methods of administration thereof |
WO2020128031A2 (en) | 2018-12-21 | 2020-06-25 | Curevac Ag | Rna for malaria vaccines |
WO2020160397A1 (en) | 2019-01-31 | 2020-08-06 | Modernatx, Inc. | Methods of preparing lipid nanoparticles |
WO2020160430A1 (en) | 2019-01-31 | 2020-08-06 | Modernatx, Inc. | Vortex mixers and associated methods, systems, and apparatuses thereof |
WO2020161342A1 (en) | 2019-02-08 | 2020-08-13 | Curevac Ag | Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases |
WO2020254535A1 (en) | 2019-06-18 | 2020-12-24 | Curevac Ag | Rotavirus mrna vaccine |
WO2021028439A1 (en) | 2019-08-14 | 2021-02-18 | Curevac Ag | Rna combinations and compositions with decreased immunostimulatory properties |
WO2021123332A1 (en) | 2019-12-20 | 2021-06-24 | Curevac Ag | Lipid nanoparticles for delivery of nucleic acids |
DE202021003575U1 (en) | 2020-02-04 | 2022-01-17 | Curevac Ag | Coronavirus Vaccine |
DE112021000012T5 (en) | 2020-02-04 | 2021-11-18 | Curevac Ag | Coronavirus vaccine |
WO2021156267A1 (en) | 2020-02-04 | 2021-08-12 | Curevac Ag | Coronavirus vaccine |
EP4147717A1 (en) | 2020-02-04 | 2023-03-15 | CureVac SE | Coronavirus vaccine |
DE202021004130U1 (en) | 2020-02-04 | 2022-10-26 | Curevac Ag | Coronavirus Vaccine |
WO2021160758A3 (en) * | 2020-02-11 | 2021-09-23 | Pantherna Therapeutics Gmbh | Recombinant nucleic acid construct and use thereof |
EP3865579A1 (en) * | 2020-02-12 | 2021-08-18 | Pantherna Therapeutics GmbH | Recombinant nucleic acid construct and use thereof |
WO2021204179A1 (en) | 2020-04-09 | 2021-10-14 | Suzhou Abogen Biosciences Co., Ltd. | Nucleic acid vaccines for coronavirus |
WO2021204175A1 (en) | 2020-04-09 | 2021-10-14 | Suzhou Abogen Biosciences Co., Ltd. | Lipid nanoparticle composition |
WO2021239880A1 (en) | 2020-05-29 | 2021-12-02 | Curevac Ag | Nucleic acid based combination vaccines |
WO2022002040A1 (en) | 2020-06-30 | 2022-01-06 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2022023559A1 (en) | 2020-07-31 | 2022-02-03 | Curevac Ag | Nucleic acid encoded antibody mixtures |
WO2022037652A1 (en) | 2020-08-20 | 2022-02-24 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2022043551A2 (en) | 2020-08-31 | 2022-03-03 | Curevac Ag | Multivalent nucleic acid based coronavirus vaccines |
WO2022135993A2 (en) | 2020-12-22 | 2022-06-30 | Curevac Ag | Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration |
US11918643B2 (en) | 2020-12-22 | 2024-03-05 | CureVac SE | RNA vaccine against SARS-CoV-2 variants |
US11872280B2 (en) | 2020-12-22 | 2024-01-16 | CureVac SE | RNA vaccine against SARS-CoV-2 variants |
WO2022137133A1 (en) | 2020-12-22 | 2022-06-30 | Curevac Ag | Rna vaccine against sars-cov-2 variants |
WO2022152141A2 (en) | 2021-01-14 | 2022-07-21 | Suzhou Abogen Biosciences Co., Ltd. | Polymer conjugated lipid compounds and lipid nanoparticle compositions |
WO2022152109A2 (en) | 2021-01-14 | 2022-07-21 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2022162027A2 (en) | 2021-01-27 | 2022-08-04 | Curevac Ag | Method of reducing the immunostimulatory properties of in vitro transcribed rna |
WO2022200575A1 (en) | 2021-03-26 | 2022-09-29 | Glaxosmithkline Biologicals Sa | Immunogenic compositions |
WO2022207862A2 (en) | 2021-03-31 | 2022-10-06 | Curevac Ag | Syringes containing pharmaceutical compositions comprising rna |
WO2022233880A1 (en) | 2021-05-03 | 2022-11-10 | Curevac Ag | Improved nucleic acid sequence for cell type specific expression |
WO2022247755A1 (en) | 2021-05-24 | 2022-12-01 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2022256597A1 (en) | 2021-06-04 | 2022-12-08 | Translate Bio, Inc. | Assay for quantitative assessment of mrna capping efficiency |
WO2023006999A2 (en) | 2021-07-30 | 2023-02-02 | CureVac SE | Mrnas for treatment or prophylaxis of liver diseases |
WO2023025404A1 (en) | 2021-08-24 | 2023-03-02 | BioNTech SE | In vitro transcription technologies |
WO2023031394A1 (en) | 2021-09-03 | 2023-03-09 | CureVac SE | Novel lipid nanoparticles for delivery of nucleic acids |
WO2023031392A2 (en) | 2021-09-03 | 2023-03-09 | CureVac SE | Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine |
WO2023044343A1 (en) | 2021-09-14 | 2023-03-23 | Renagade Therapeutics Management Inc. | Acyclic lipids and methods of use thereof |
WO2023044333A1 (en) | 2021-09-14 | 2023-03-23 | Renagade Therapeutics Management Inc. | Cyclic lipids and methods of use thereof |
WO2023056914A1 (en) | 2021-10-08 | 2023-04-13 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
EP4162950A1 (en) | 2021-10-08 | 2023-04-12 | Suzhou Abogen Biosciences Co., Ltd. | Nucleic acid vaccines for coronavirus |
WO2023056917A1 (en) | 2021-10-08 | 2023-04-13 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2023133525A1 (en) * | 2022-01-07 | 2023-07-13 | Precision Biosciences, Inc. | Optimized polynucleotides for protein expression |
WO2023144193A1 (en) | 2022-01-25 | 2023-08-03 | CureVac SE | Mrnas for treatment of hereditary tyrosinemia type i |
WO2023144330A1 (en) | 2022-01-28 | 2023-08-03 | CureVac SE | Nucleic acid encoded transcription factor inhibitors |
WO2023196931A1 (en) | 2022-04-07 | 2023-10-12 | Renagade Therapeutics Management Inc. | Cyclic lipids and lipid nanoparticles (lnp) for the delivery of nucleic acids or peptides for use in vaccinating against infectious agents |
WO2023227608A1 (en) | 2022-05-25 | 2023-11-30 | Glaxosmithkline Biologicals Sa | Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide |
WO2024037578A1 (en) | 2022-08-18 | 2024-02-22 | Suzhou Abogen Biosciences Co., Ltd. | Composition of lipid nanoparticles |
WO2024068545A1 (en) | 2022-09-26 | 2024-04-04 | Glaxosmithkline Biologicals Sa | Influenza virus vaccines |
Also Published As
Publication number | Publication date |
---|---|
US10080809B2 (en) | 2018-09-25 |
ES2660129T3 (en) | 2018-03-20 |
CN108929880A (en) | 2018-12-04 |
BR112014023898A2 (en) | 2017-07-11 |
AU2013242405A1 (en) | 2014-09-25 |
SG11201405545XA (en) | 2014-11-27 |
CA2866945C (en) | 2021-05-04 |
RU2660565C2 (en) | 2018-07-06 |
JP6301906B2 (en) | 2018-03-28 |
US20150050302A1 (en) | 2015-02-19 |
JP2015517803A (en) | 2015-06-25 |
CN104321432B (en) | 2018-08-10 |
SG10201607966UA (en) | 2016-11-29 |
RU2014142881A (en) | 2016-05-20 |
CA2866945A1 (en) | 2013-10-03 |
AU2013242405B2 (en) | 2018-06-28 |
MX2014011625A (en) | 2014-10-17 |
CN104321432A (en) | 2015-01-28 |
WO2013143700A3 (en) | 2013-12-27 |
KR20140139101A (en) | 2014-12-04 |
MX358706B (en) | 2018-08-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2018264081C1 (en) | Artificial nucleic acid molecules for improved protein or peptide expression | |
AU2013242405B2 (en) | Artificial nucleic acid molecules comprising a 5'TOP UTR | |
EP3317424B1 (en) | Method for analysis of an rna molecule | |
EP2831241B1 (en) | Artificial nucleic acid molecules for improved protein or peptide expression | |
EP2831240B1 (en) | Artificial nucleic acid molecules comprising a 5'top utr | |
EP1974042A1 (en) | Hybrid 3 untranslated regions suitable for efficient protein expression in mammalian cells | |
EP1728859A1 (en) | Sequence capable of accelerating gene expression at moderately low temperature |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13713359 Country of ref document: EP Kind code of ref document: A2 |
|
REEP | Request for entry into the european phase |
Ref document number: 2013713359 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2013713359 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2866945 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2013242405 Country of ref document: AU Date of ref document: 20130327 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14388224 Country of ref document: US |
|
ENP | Entry into the national phase |
Ref document number: 2015502143 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: MX/A/2014/011625 Country of ref document: MX |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112014023898 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 20147030143 Country of ref document: KR Kind code of ref document: A Ref document number: 2014142881 Country of ref document: RU Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 112014023898 Country of ref document: BR Kind code of ref document: A2 Effective date: 20140926 |