CN101203245A - Rna最佳注射配方 - Google Patents
Rna最佳注射配方 Download PDFInfo
- Publication number
- CN101203245A CN101203245A CNA2006800171263A CN200680017126A CN101203245A CN 101203245 A CN101203245 A CN 101203245A CN A2006800171263 A CNA2006800171263 A CN A2006800171263A CN 200680017126 A CN200680017126 A CN 200680017126A CN 101203245 A CN101203245 A CN 101203245A
- Authority
- CN
- China
- Prior art keywords
- rna
- mrna
- injection
- buffer
- injection buffer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002347 injection Methods 0.000 title claims abstract description 218
- 239000007924 injection Substances 0.000 title claims abstract description 218
- 239000000243 solution Substances 0.000 title abstract description 15
- 239000000872 buffer Substances 0.000 claims abstract description 166
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 39
- 238000013519 translation Methods 0.000 claims abstract description 39
- 238000012546 transfer Methods 0.000 claims abstract description 24
- 230000001965 increasing effect Effects 0.000 claims abstract description 16
- 159000000007 calcium salts Chemical class 0.000 claims abstract description 11
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims abstract description 10
- 159000000000 sodium salts Chemical class 0.000 claims abstract description 10
- 238000000338 in vitro Methods 0.000 claims abstract description 9
- 108020004999 messenger RNA Proteins 0.000 claims description 190
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 174
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 73
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 57
- 235000002639 sodium chloride Nutrition 0.000 claims description 53
- 239000011780 sodium chloride Substances 0.000 claims description 37
- 241000699670 Mus sp. Species 0.000 claims description 34
- 230000014509 gene expression Effects 0.000 claims description 30
- 238000002360 preparation method Methods 0.000 claims description 25
- 239000001103 potassium chloride Substances 0.000 claims description 24
- 235000011164 potassium chloride Nutrition 0.000 claims description 24
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 22
- 239000001110 calcium chloride Substances 0.000 claims description 20
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 20
- 239000011734 sodium Substances 0.000 claims description 20
- 230000004048 modification Effects 0.000 claims description 17
- 238000012986 modification Methods 0.000 claims description 17
- 150000003839 salts Chemical class 0.000 claims description 17
- 108091026890 Coding region Proteins 0.000 claims description 13
- 238000001890 transfection Methods 0.000 claims description 12
- 241000124008 Mammalia Species 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 9
- 230000008859 change Effects 0.000 claims description 7
- 201000010099 disease Diseases 0.000 claims description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 7
- 238000004520 electroporation Methods 0.000 claims description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 5
- 108010007568 Protamines Proteins 0.000 claims description 5
- 102000007327 Protamines Human genes 0.000 claims description 5
- 229940048914 protamine Drugs 0.000 claims description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 3
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 claims description 3
- 241000282472 Canis lupus familiaris Species 0.000 claims description 3
- 241000283073 Equus caballus Species 0.000 claims description 3
- 241000282326 Felis catus Species 0.000 claims description 3
- 241000700159 Rattus Species 0.000 claims description 3
- 241000282898 Sus scrofa Species 0.000 claims description 3
- 108020004566 Transfer RNA Proteins 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 claims description 3
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- 108020004459 Small interfering RNA Proteins 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- 150000002016 disaccharides Chemical class 0.000 claims description 2
- 150000002772 monosaccharides Chemical class 0.000 claims description 2
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 1
- 108091028664 Ribonucleotide Proteins 0.000 claims 1
- 239000007983 Tris buffer Substances 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 125000000129 anionic group Chemical group 0.000 claims 1
- 239000002336 ribonucleotide Substances 0.000 claims 1
- 125000002652 ribonucleotide group Chemical group 0.000 claims 1
- 238000001727 in vivo Methods 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 81
- 108020004705 Codon Proteins 0.000 description 50
- 238000004043 dyeing Methods 0.000 description 46
- 229940001447 lactate Drugs 0.000 description 38
- 108090000623 proteins and genes Proteins 0.000 description 37
- 102000004169 proteins and genes Human genes 0.000 description 31
- 150000001768 cations Chemical class 0.000 description 30
- 206010028980 Neoplasm Diseases 0.000 description 27
- 239000000427 antigen Substances 0.000 description 27
- 108091007433 antigens Proteins 0.000 description 27
- 102000036639 antigens Human genes 0.000 description 27
- 235000018102 proteins Nutrition 0.000 description 27
- 108060001084 Luciferase Proteins 0.000 description 25
- 239000005089 Luciferase Substances 0.000 description 25
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 24
- 230000000694 effects Effects 0.000 description 23
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 22
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 description 21
- 239000000203 mixture Substances 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- 239000011575 calcium Substances 0.000 description 20
- 108010005774 beta-Galactosidase Proteins 0.000 description 19
- ZAINTDRBUHCDPZ-UHFFFAOYSA-M Alexa Fluor 546 Chemical group [H+].[Na+].CC1CC(C)(C)NC(C(=C2OC3=C(C4=NC(C)(C)CC(C)C4=CC3=3)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=C2C=3C(C(=C(Cl)C=1Cl)C(O)=O)=C(Cl)C=1SCC(=O)NCCCCCC(=O)ON1C(=O)CCC1=O ZAINTDRBUHCDPZ-UHFFFAOYSA-M 0.000 description 18
- 102100026189 Beta-galactosidase Human genes 0.000 description 17
- 235000011148 calcium chloride Nutrition 0.000 description 17
- 239000002777 nucleoside Substances 0.000 description 16
- 150000003833 nucleoside derivatives Chemical class 0.000 description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 13
- 238000001514 detection method Methods 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 13
- 238000011081 inoculation Methods 0.000 description 13
- 101150066555 lacZ gene Proteins 0.000 description 13
- 210000001519 tissue Anatomy 0.000 description 13
- 241000282414 Homo sapiens Species 0.000 description 12
- 150000001413 amino acids Chemical group 0.000 description 12
- 102000039446 nucleic acids Human genes 0.000 description 12
- 108020004707 nucleic acids Proteins 0.000 description 12
- 150000007523 nucleic acids Chemical class 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 241000700605 Viruses Species 0.000 description 11
- 239000000975 dye Substances 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- -1 cationic organic anion Chemical class 0.000 description 10
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 description 9
- 210000000612 antigen-presenting cell Anatomy 0.000 description 9
- 150000002500 ions Chemical class 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000001540 sodium lactate Substances 0.000 description 9
- 235000011088 sodium lactate Nutrition 0.000 description 9
- 229940005581 sodium lactate Drugs 0.000 description 9
- 201000011510 cancer Diseases 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 238000012545 processing Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 7
- 230000034994 death Effects 0.000 description 7
- 201000001441 melanoma Diseases 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 101900264058 Escherichia coli Beta-galactosidase Proteins 0.000 description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 239000006166 lysate Substances 0.000 description 6
- 229910052700 potassium Inorganic materials 0.000 description 6
- 239000011591 potassium Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 102100024458 Cyclin-dependent kinase inhibitor 2A Human genes 0.000 description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 5
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 5
- 108010075704 HLA-A Antigens Proteins 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 239000008156 Ringer's lactate solution Substances 0.000 description 5
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 5
- 229910052783 alkali metal Inorganic materials 0.000 description 5
- 150000001340 alkali metals Chemical class 0.000 description 5
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 5
- 150000001342 alkaline earth metals Chemical class 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- ZEWYCNBZMPELPF-UHFFFAOYSA-J calcium;potassium;sodium;2-hydroxypropanoic acid;sodium;tetrachloride Chemical compound [Na].[Na+].[Cl-].[Cl-].[Cl-].[Cl-].[K+].[Ca+2].CC(O)C(O)=O ZEWYCNBZMPELPF-UHFFFAOYSA-J 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 210000000172 cytosol Anatomy 0.000 description 5
- 238000011010 flushing procedure Methods 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 239000004310 lactic acid Substances 0.000 description 5
- 235000014655 lactic acid Nutrition 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 150000001450 anions Chemical class 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 229960005069 calcium Drugs 0.000 description 4
- 229910052791 calcium Inorganic materials 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 238000001415 gene therapy Methods 0.000 description 4
- 238000010166 immunofluorescence Methods 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 4
- 230000001960 triggered effect Effects 0.000 description 4
- 238000011725 BALB/c mouse Methods 0.000 description 3
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 3
- 208000032612 Glial tumor Diseases 0.000 description 3
- 206010018338 Glioma Diseases 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010033040 Histones Proteins 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 102100025725 Mothers against decapentaplegic homolog 4 Human genes 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 241000254064 Photinus pyralis Species 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 239000000337 buffer salt Substances 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- 230000003862 health status Effects 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 239000011777 magnesium Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 150000002891 organic anions Chemical class 0.000 description 3
- 230000008521 reorganization Effects 0.000 description 3
- 239000008279 sol Substances 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 240000006409 Acacia auriculiformis Species 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 102000002572 Alpha-Globulins Human genes 0.000 description 2
- 108010068307 Alpha-Globulins Proteins 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 102000009515 Arachidonate 15-Lipoxygenase Human genes 0.000 description 2
- 108010048907 Arachidonate 15-lipoxygenase Proteins 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 2
- 102100036252 Cyclin-dependent kinase 4 Human genes 0.000 description 2
- 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 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 241000710188 Encephalomyocarditis virus Species 0.000 description 2
- 241000709661 Enterovirus Species 0.000 description 2
- 241000991587 Enterovirus C Species 0.000 description 2
- 102400001368 Epidermal growth factor Human genes 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 102100028976 HLA class I histocompatibility antigen, B alpha chain Human genes 0.000 description 2
- 102100028971 HLA class I histocompatibility antigen, C alpha chain Human genes 0.000 description 2
- 108010058607 HLA-B Antigens Proteins 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 2
- 101000986084 Homo sapiens HLA class I histocompatibility antigen, C alpha chain Proteins 0.000 description 2
- 101000835893 Homo sapiens Mothers against decapentaplegic homolog 4 Proteins 0.000 description 2
- 101000733249 Homo sapiens Tumor suppressor ARF Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 2
- 102000005741 Metalloproteases Human genes 0.000 description 2
- 108010006035 Metalloproteases Proteins 0.000 description 2
- 102100025751 Mothers against decapentaplegic homolog 2 Human genes 0.000 description 2
- 241000714177 Murine leukemia virus Species 0.000 description 2
- 102000002233 Myelin-Oligodendrocyte Glycoprotein Human genes 0.000 description 2
- 108010000123 Myelin-Oligodendrocyte Glycoprotein Proteins 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- 240000001307 Myosotis scorpioides Species 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 102000014160 PTEN Phosphohydrolase Human genes 0.000 description 2
- 108010011536 PTEN Phosphohydrolase Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 241000713311 Simian immunodeficiency virus Species 0.000 description 2
- 206010041067 Small cell lung cancer Diseases 0.000 description 2
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 2
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 2
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 2
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 208000009956 adenocarcinoma Diseases 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
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 102000005936 beta-Galactosidase Human genes 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 201000000053 blastoma Diseases 0.000 description 2
- 201000008275 breast carcinoma Diseases 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- 208000030381 cutaneous melanoma Diseases 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 210000005069 ears Anatomy 0.000 description 2
- 239000003792 electrolyte Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 201000008184 embryoma Diseases 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 201000005619 esophageal carcinoma Diseases 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 208000005017 glioblastoma Diseases 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- COHYTHOBJLSHDF-BUHFOSPRSA-N indigo dye Chemical compound N\1C2=CC=CC=C2C(=O)C/1=C1/C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-BUHFOSPRSA-N 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 150000008040 ionic compounds Chemical class 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 2
- 238000002690 local anesthesia Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000005075 mammary gland Anatomy 0.000 description 2
- 201000006417 multiple sclerosis Diseases 0.000 description 2
- 210000000663 muscle cell Anatomy 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 108091027963 non-coding RNA Proteins 0.000 description 2
- 102000042567 non-coding RNA Human genes 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 235000014593 oils and fats Nutrition 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000002161 passivation Methods 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229920000724 poly(L-arginine) polymer Polymers 0.000 description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 201000001514 prostate carcinoma Diseases 0.000 description 2
- 201000010174 renal carcinoma Diseases 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 201000003708 skin melanoma Diseases 0.000 description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 description 2
- 229940063675 spermine Drugs 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UWTATZPHSA-M (R)-lactate Chemical compound C[C@@H](O)C([O-])=O JVTAAEKCZFNVCJ-UWTATZPHSA-M 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- JRYMOPZHXMVHTA-DAGMQNCNSA-N 2-amino-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrrolo[2,3-d]pyrimidin-4-one Chemical compound C1=CC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JRYMOPZHXMVHTA-DAGMQNCNSA-N 0.000 description 1
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- ORWQBKPSGDRPPA-UHFFFAOYSA-N 3-[2-[ethyl(methyl)amino]ethyl]-1h-indol-4-ol Chemical compound C1=CC(O)=C2C(CCN(C)CC)=CNC2=C1 ORWQBKPSGDRPPA-UHFFFAOYSA-N 0.000 description 1
- AXDJCCTWPBKUKL-UHFFFAOYSA-N 4-[(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]aniline;hydron;chloride Chemical compound Cl.C1=CC(=N)C(C)=CC1=C(C=1C=CC(N)=CC=1)C1=CC=C(N)C=C1 AXDJCCTWPBKUKL-UHFFFAOYSA-N 0.000 description 1
- OMJRFWRSVILQLV-UHFFFAOYSA-N 6-amino-5-methyl-1h-pyrimidin-2-one Chemical compound CC1=CNC(=O)N=C1N.CC1=CNC(=O)N=C1N OMJRFWRSVILQLV-UHFFFAOYSA-N 0.000 description 1
- OXEUETBFKVCRNP-UHFFFAOYSA-N 9-ethyl-3-carbazolamine Chemical compound NC1=CC=C2N(CC)C3=CC=CC=C3C2=C1 OXEUETBFKVCRNP-UHFFFAOYSA-N 0.000 description 1
- 108050006685 Apoptosis regulator BAX Proteins 0.000 description 1
- 101100226366 Arabidopsis thaliana EXT3 gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 102100032306 Aurora kinase B Human genes 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 208000028564 B-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 102000036365 BRCA1 Human genes 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 102000052609 BRCA2 Human genes 0.000 description 1
- 108700020462 BRCA2 Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101150008921 Brca2 gene Proteins 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 1
- 108010083123 CDX2 Transcription Factor Proteins 0.000 description 1
- 102000006277 CDX2 Transcription Factor Human genes 0.000 description 1
- QCMYYKRYFNMIEC-UHFFFAOYSA-N COP(O)=O Chemical class COP(O)=O QCMYYKRYFNMIEC-UHFFFAOYSA-N 0.000 description 1
- 101100297347 Caenorhabditis elegans pgl-3 gene Proteins 0.000 description 1
- 102100032220 Calcium and integrin-binding family member 2 Human genes 0.000 description 1
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 102100026548 Caspase-8 Human genes 0.000 description 1
- 108090000538 Caspase-8 Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 241000710777 Classical swine fever virus Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 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 1
- 108010009392 Cyclin-Dependent Kinase Inhibitor p16 Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 102100034157 DNA mismatch repair protein Msh2 Human genes 0.000 description 1
- 102100021147 DNA mismatch repair protein Msh6 Human genes 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- CZGGKXNYNPJFAX-UHFFFAOYSA-N Dimethyldithiophosphate Chemical compound COP(S)(=S)OC CZGGKXNYNPJFAX-UHFFFAOYSA-N 0.000 description 1
- 101100168884 Drosophila melanogaster alpha-Cat gene Proteins 0.000 description 1
- 102100023274 Dual specificity mitogen-activated protein kinase kinase 4 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102100029055 Exostosin-1 Human genes 0.000 description 1
- 102100029074 Exostosin-2 Human genes 0.000 description 1
- 102100027280 Fanconi anemia group A protein Human genes 0.000 description 1
- 229910015475 FeF 2 Inorganic materials 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010008488 Glycylglycine Proteins 0.000 description 1
- 102100028970 HLA class I histocompatibility antigen, alpha chain E Human genes 0.000 description 1
- 102100040505 HLA class II histocompatibility antigen, DR alpha chain Human genes 0.000 description 1
- 102100040485 HLA class II histocompatibility antigen, DRB1 beta chain Human genes 0.000 description 1
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 description 1
- 102220391613 HLA-B*2702 Human genes 0.000 description 1
- 108010046732 HLA-DR4 Antigen Proteins 0.000 description 1
- 108010039343 HLA-DRB1 Chains Proteins 0.000 description 1
- 102100027685 Hemoglobin subunit alpha Human genes 0.000 description 1
- 108091005902 Hemoglobin subunit alpha Proteins 0.000 description 1
- 108091027305 Heteroduplex Proteins 0.000 description 1
- 101000980814 Homo sapiens CAMPATH-1 antigen Proteins 0.000 description 1
- 101000943456 Homo sapiens Calcium and integrin-binding family member 2 Proteins 0.000 description 1
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 1
- 101000911952 Homo sapiens Cyclin-dependent kinase 7 Proteins 0.000 description 1
- 101000944365 Homo sapiens Cyclin-dependent kinase inhibitor 1C Proteins 0.000 description 1
- 101001134036 Homo sapiens DNA mismatch repair protein Msh2 Proteins 0.000 description 1
- 101000968658 Homo sapiens DNA mismatch repair protein Msh6 Proteins 0.000 description 1
- 101001115395 Homo sapiens Dual specificity mitogen-activated protein kinase kinase 4 Proteins 0.000 description 1
- 101000918311 Homo sapiens Exostosin-1 Proteins 0.000 description 1
- 101000918275 Homo sapiens Exostosin-2 Proteins 0.000 description 1
- 101000914673 Homo sapiens Fanconi anemia group A protein Proteins 0.000 description 1
- 101000986085 Homo sapiens HLA class I histocompatibility antigen, alpha chain E Proteins 0.000 description 1
- 101000968009 Homo sapiens HLA class II histocompatibility antigen, DR alpha chain Proteins 0.000 description 1
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 1
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 description 1
- 101000738901 Homo sapiens PMS1 protein homolog 1 Proteins 0.000 description 1
- 101000695187 Homo sapiens Protein patched homolog 1 Proteins 0.000 description 1
- 101000579425 Homo sapiens Proto-oncogene tyrosine-protein kinase receptor Ret Proteins 0.000 description 1
- 101000932478 Homo sapiens Receptor-type tyrosine-protein kinase FLT3 Proteins 0.000 description 1
- 101000795659 Homo sapiens Tuberin Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 235000000177 Indigofera tinctoria Nutrition 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 108010010995 MART-1 Antigen Proteins 0.000 description 1
- 229910015837 MSH2 Inorganic materials 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101150076359 Mhc gene Proteins 0.000 description 1
- 108010074346 Mismatch Repair Endonuclease PMS2 Proteins 0.000 description 1
- 102100037480 Mismatch repair endonuclease PMS2 Human genes 0.000 description 1
- 101710143123 Mothers against decapentaplegic homolog 2 Proteins 0.000 description 1
- 101710143112 Mothers against decapentaplegic homolog 4 Proteins 0.000 description 1
- 102100023123 Mucin-16 Human genes 0.000 description 1
- 101000753280 Mus musculus Angiopoietin-1 receptor Proteins 0.000 description 1
- 102000013609 MutL Protein Homolog 1 Human genes 0.000 description 1
- 108010026664 MutL Protein Homolog 1 Proteins 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 102100021010 Nucleolin Human genes 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000382923 Oxya chinensis Species 0.000 description 1
- 102100037482 PMS1 protein homolog 1 Human genes 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102100028680 Protein patched homolog 1 Human genes 0.000 description 1
- 102100028286 Proto-oncogene tyrosine-protein kinase receptor Ret Human genes 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 101100390989 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FMS1 gene Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102100026715 Serine/threonine-protein kinase STK11 Human genes 0.000 description 1
- 101710181599 Serine/threonine-protein kinase STK11 Proteins 0.000 description 1
- 108700032504 Smad2 Proteins 0.000 description 1
- 101150102611 Smad2 gene Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 102000007000 Tenascin Human genes 0.000 description 1
- 108010008125 Tenascin Proteins 0.000 description 1
- 241000053227 Themus Species 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102100031638 Tuberin Human genes 0.000 description 1
- 102100039094 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 244000225942 Viola tricolor Species 0.000 description 1
- 235000004031 Viola x wittrockiana Nutrition 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 241000269368 Xenopus laevis Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229910001413 alkali metal ion Inorganic materials 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000001449 anionic compounds Chemical class 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003172 anti-dna Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000014102 antigen processing and presentation of exogenous peptide antigen via MHC class I Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 108010087236 cobra venom endonuclease Proteins 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 231100000284 endotoxic Toxicity 0.000 description 1
- 230000002346 endotoxic effect Effects 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- SEACYXSIPDVVMV-UHFFFAOYSA-L eosin Y Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 SEACYXSIPDVVMV-UHFFFAOYSA-L 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940043257 glycylglycine Drugs 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000024278 histolysis Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- YAGKRVSRTSUGEY-UHFFFAOYSA-Q hydron;iron(3+);hexacyanide Chemical compound [H+].[H+].[H+].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] YAGKRVSRTSUGEY-UHFFFAOYSA-Q 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229940097275 indigo Drugs 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910001412 inorganic anion Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 208000003243 intestinal obstruction Diseases 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 150000002505 iron Chemical class 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 229940116871 l-lactate Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000003893 lactate salts Chemical group 0.000 description 1
- 229940099584 lactobionate Drugs 0.000 description 1
- JYTUSYBCFIZPBE-AMTLMPIISA-M lactobionate Chemical compound [O-]C(=O)[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O JYTUSYBCFIZPBE-AMTLMPIISA-M 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229910003002 lithium salt Inorganic materials 0.000 description 1
- 159000000002 lithium salts Chemical class 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004880 lymph fluid Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 229940049920 malate Drugs 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-L malate(2-) Chemical compound [O-]C(=O)C(O)CC([O-])=O BJEPYKJPYRNKOW-UHFFFAOYSA-L 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- RSMUVYRMZCOLBH-UHFFFAOYSA-N metsulfuron methyl Chemical compound COC(=O)C1=CC=CC=C1S(=O)(=O)NC(=O)NC1=NC(C)=NC(OC)=N1 RSMUVYRMZCOLBH-UHFFFAOYSA-N 0.000 description 1
- 238000012737 microarray-based gene expression Methods 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 208000034420 multiple type III exostoses Diseases 0.000 description 1
- 238000012243 multiplex automated genomic engineering Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- JTSLALYXYSRPGW-UHFFFAOYSA-N n-[5-(4-cyanophenyl)-1h-pyrrolo[2,3-b]pyridin-3-yl]pyridine-3-carboxamide Chemical compound C=1C=CN=CC=1C(=O)NC(C1=C2)=CNC1=NC=C2C1=CC=C(C#N)C=C1 JTSLALYXYSRPGW-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229940023146 nucleic acid vaccine Drugs 0.000 description 1
- 108010044762 nucleolin Proteins 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000007500 overflow downdraw method Methods 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000000276 potassium ferrocyanide Substances 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000003825 pressing Methods 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
- 238000003672 processing method Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000012925 reference material Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940086735 succinate Drugs 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 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 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 229960004854 viral vaccine Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Images
Classifications
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- 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
- A61K48/0008—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- 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
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Inorganic Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Pulmonology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明涉及用RNA和包含钠盐、钙盐和可选的钾盐和可选的乳酸盐的水性注射缓冲液制备RNA注射液,来增加寄助物中的RNA转移和/或RNA翻译。本发明还涉及一种RNA注射液和用于增加体内和体外RNA的RNA转移和/或RNA翻译的方法。
Description
技术领域
本发明涉及在制备用于增加寄助物内RNA转移到寄助物和/或RNA翻译的RNA注射液时,RNA和包含钠盐、钙盐、可选的钾盐和可选的乳酸盐的水性注射缓冲液的使用。
背景技术
在许多疾病的治疗和预防中,例如基因治疗和遗传接种等分子医学方法,起着重要的作用。这些方法的基础是将核酸引入到病人的细胞或组织中,然后处理引入的核酸所编码的信息,也就是说,翻译成所需的多肽或蛋白质。DNA和RNA都可考虑作为被引入的核酸。
在20世纪90年代早期,注射入裸露的质粒DNA的遗传接种方法首先在大鼠上得到证实。然而,临床I/II期研究显示,在人类身上,此技术不能达到在对老鼠的研究中所引起的期望1。许多基于DNA的遗传接种和引入DNA到细胞中的方法(包括磷酸钙转染法、聚戊二烯转染法、原生质体融合法、电穿孔法、显微注射法、脂质体法等,使用DNA病毒作为DNA载体)已经被研究出来。
15年前,Wolff等研究显示了向大鼠中以质粒DNA(pDNA)或mRNA的形式注入裸露的遗传信息可引起蛋白质表达2。对这些结果的跟踪研究显示裸露的pDNA可用于接种3-5。然而,直到20世纪90年代末期,用mRNA来接种才略为引起人们的注意,当时被证实将mRNA转移到树突状细胞(dendriticcells)可触发免疫反应6。然而,直接注射裸露的mRNA来接种仍然不被重视,并且仅仅在三个不同的研究小组的四篇论文中给予讨论7-10。这种情况的一个重要原因是mRNA的由于其被核酶迅速降解所导致的不稳定性以及与此有关的mRNA作为体内的遗传工具的有限效果。然而,同时,现有技术中已经有许多方法用来稳定mRNA,例如,EP-A-1083232、WO 99/14346、US 5 580 859和US 6 214 804。
RNA作为用于遗传载体的核酸具有比DNA更多的优势,包括:
i)引入到细胞中的RNA并不结合到基因组中(而DNA在一定程度上结合到基因组中,并且因此插入到寄主细胞(host cell)的基因组的完整基因中,因此此基因可能变异并且可导致遗传信息的全部丢失或误传)。
ii)RNA的有效转录不需要例如启动子(promoters)等的病毒序列(而引入到细胞中的DNA的表达需要强的启动子(例如,病毒CMV启动子))。启动子结合到寄主细胞的基因组中可导致基因表达调控中不希望的改变。
iii)引入的RNA的降解发生在有限的时间内(几个小时)11,12,因此有可能实现瞬时基因表达,其在需要的治疗期过后可停止(而这在引入到基因组中的DNA的情况下是不可能的)。
iv)RNA不会在病人身上引起致病的抗-RNA抗体的诱导(而抗-DNA抗体的诱导据知可引起不希望的免疫反应)。
v)RNA广泛可用——任何所需的感兴趣的蛋白的RNA可在短时间内制备出来以用于接种,甚至是基于个体病人的。
总之,仍然应注意到,mRNA代表了所有有机体中的编码遗传信息的过渡副本,作为合成蛋白质的模型,并且,不像DNA,其代表了制备合适的用于体内外源性遗传信息转移的遗传媒介(vector)的所有必要先决条件。
所描述的将核酸转移到寄助物(尤其在哺乳动物中)的独特的合适程序是注射。为此类注射用的DNA一般在水、NaCl或PBS注射缓冲液中稀释,而RNA一般只在注射缓冲液中稀释。作为RNA注射缓冲液使用的标准缓冲液有例如磷酸盐缓冲液,尤其是PBS和HEPES缓冲盐溶液(HBS)。在转移mRNA的情况下,这样的RNA注射液最好在注射前进行短时间的加热来去除mRNA的二级结构。使用这样的标准缓冲液用于RNA注射液的的缺点是皮下RNA的转移是很低效率的。另一个缺点是转移的RNA的翻译率非常低。另一个缺点是,在这样的标准缓冲液中,RNA常常形成二级结构(例如,所谓的发夹结构(hairpin structure)),其可大大降低RNA被吸收到细胞溶质中的效率。
发明内容
因此,本发明的目的是提供一种系统,通过该系统,一方面皮下RNA到寄助物中的转移得到改善,另一方面转移的RNA的翻译率得到提高。
此目标在权利要求中加以特征化的本发明的各实施例中得以实现。
本发明的一个实施例提供在制备用于增加RNA转移到寄助物和/或寄助物内RNA翻译的RNA注射液时RNA和水性注射缓冲液的用途,所述水性注射缓冲液包含钠盐,优选为至少50mM的钠盐;钙盐,优选为至少0.01mM的钙盐;和可选的钾盐,优选为至少3mM的钾盐。本发明的另一方面还提供一种如此制备的注射液。所述注射液是由注射缓冲液和溶解在注射缓冲液中的RNA得到的。
根据优选实施例,包含在注射缓冲液中的钠盐、钙盐和可选的钾盐是以卤化物的形式存在,例如,氯化物、碘化物或溴化物,或以它们的氢氧化物、碳酸盐、碳酸氢盐或硫酸盐的形式存在。此处提到的例子是:对于钠盐有NaCl,NaI,NaBr,Na2CO3,NaHCO3,Na2SO4;对于可选的钾盐有KCl,KI,KBr,K2CO3,KHCO3,K2SO4;对于钙盐有CaCl2,CaI2,CaBr2,CaCO3,CaSO4,Ca(OH)2。上面所提到的阳离子的有机阴离子也可包含在注射缓冲液中。
根据本发明RNA和注射缓冲液的的用途的特别优选实施例,根据本发明的注射缓冲液包含氯化钠(NaCl),氯化钙(CaCl2)和可选的氯化钾(KCl),除了氯化物之外还可以出现其他的阴离子。这些盐在注射缓冲液中的典型浓度是至少50mM的氯化钠(NaCl),至少3mM的氯化钾(KCl)和至少0.01mM的氯化钙(CaCl2)。
根据本发明的注射缓冲液可以是高渗的或等渗的或低渗的注射缓冲液。结合本发明,注射缓冲液是高渗的或等渗的或低渗的,与不同的参考媒质有关,就是说,本发明的注射缓冲液相比于各个参考媒质具有较高的、相等的或较低的盐含量,上述的盐的优选浓度是不会由于渗透作用或其它浓度效果对细胞造成损害的那些浓度。此处的参考媒质是,例如,在“体内”处理过程中产生的液体,例如,血液、淋巴液、细胞溶质液或其它在体内出现的液体,或现有的在“体外”(“in vitro”)处理过程中使用的液体或缓冲液。这些液体和缓冲液为所属领域技术人员所知悉。
注射缓冲液可包含有另外的成分,例如,糖类(单糖、二糖、三糖或多糖),尤其是葡萄糖或甘露醇。然而,在优选实施例中,用于本发明的用途所采用的注射缓冲液中不会出现糖类。本发明的缓冲液中最好也不包含任何不带电荷的成分,例如,糖类。本发明的缓冲液一般仅包含金属阳离子,尤其是来自碱金属或碱土金属的阳离子,以及阴离子,尤其是上面所述的阴离子。
本发明的注射缓冲液的优选pH值是1到8.5,优选的是3到5,更优选的是5.5到7.5,尤其最佳的是5.5到6.5。该注射缓冲液还可选地包含有缓冲系统,将注射缓冲液固定到缓冲后的pH值。这样的系统可以是,例如,磷酸缓冲系统、HEPES或Na2HPO4/NaH2PO4。然而,很特别的优选是根据本发明使用的注射缓冲液不含有上面所述的任何缓冲系统或者根本就没有缓冲系统。
如前所述,根据本发明使用的注射缓冲液包含钠盐、钙盐和可选的钾盐,在该注射缓冲液中钾和纳一般以一价阳离子(Na+,K+)的形式存在,钙以二价阳离子(Ca2+)的形式存在。根据本发明的优选实施例,除了根据本发明的包含在或可选地包含在注射缓冲液中的一价和二价阳离子之外还可以有二价阳离子,尤其是碱土金属族的阳离子,例如,镁(Mg2+),或也包括铁(Fe2+);和一价阳离子,尤其是碱金属离子,例如,锂(Li+)。这些一价阳离子优选以它们的盐的形式存在,例如,以卤盐的形式,例如,氯化物、碘化物或溴化物,或以它们的氢氧化物、碳酸盐、碳酸氢盐或硫化物的形式存在。例如,在此可列举的例子有:对于锂盐有LiCl,LiI,LiBr,Li2CO3,LiHCO3,Li2SO4;对于镁盐有MgCl2,MgI2,MgBr2,MgCO3,MgSO4和Mg(OH)2;对于铁盐有FeCl2,FeBr2,FeI2,FeF2,Fe2O3,FeCO3,FeSO4,Fe(OH)2。也包括上述的二价和/或一价阳离子的结合。因此,根据本发明的注射缓冲液可仅包含二价阳离子,或仅有一价阳离子,或同时包含二价和一价阳离子。根据本发明的注射缓冲液还可仅包括一种类型的二价离子或一价离子,尤其优选的是,例如,仅包含Ca2+或钙盐,例如,CaCl2。
根据本发明,在制备注射液时,在注射液中使用碱金属或碱土金属的不同于Ca2+和Na1+的二价或一价阳离子或不同的二价阳离子和阴离子来全部替代或部分替代Ca2+或Na1+时,在注射缓冲液中,也可以考虑上面所提到的Ca2+(作为二价离子)和Na1+(作为一价离子)的摩尔浓度(也就是说,一般浓度是至少50mM的Na+,至少0.01mM的Ca2+,可选的至少3mM的K+)。虽然根据本发明,如上提及的在注射缓冲液中的Ca2+和Na1+可完全用不同的二价或一价阳离子来替代,例如,被不同的二价阳离子的组合(替代Ca2+)和/或不同的一价阳离子的组合(替代Na1+)来替代(特别地,用碱金属的不同一价阳离子的组合或碱土金属的不同二价阳离子的组合),优选的是,部分地替代Ca2+或Na1+),也就是说,在具有Ca2+或Na1+的注射缓冲液中加入各自总摩尔浓度的至少20%、较好的是至少40%、更好的是至少60%、最好是至少80%的一价或二价阳离子。然而,根据本发明的特别优选的注射缓冲液只包含Ca2+作为二价阳离子和Na1+作为一价阳离子,也就是说,Ca2+占了二价阳离子的总摩尔浓度的100%,Na1+占了一价阳离子的总摩尔浓度的100%。
注射缓冲液的制备最好在室温(25℃)和大气压下进行。制备可根据现有技术中的任何想要的处理过程来执行。优选地,其内所包含的离子或盐在水溶液中稀释,所依据的浓度比是根据特定条件(RNA注射液将要注射到的寄助物,尤其是哺乳动物,寄助物的健康状态、年龄等,溶解度和各成分的干扰、反应温度、反应时间等)来选择的。
包含在水性注射缓冲液中的钠、钙和氯离子和可选的钾离子和可选的乳酸盐(参见下面的实施例)各成分的浓度特别依赖于它们在水中的溶解度、各成分彼此之间的干扰以及在制备该注射液或制备RNA注射液时的反应温度和反应压力。
根据本发明使用的注射缓冲液是基于水性溶液的,也就是,包含水和根据本发明的注射溶液中使用的盐、和可选的乳酸盐。在这样的水性溶液中,上述盐的一价或二价阳离子可以可选地是微溶的或甚至不溶的。盐的溶解度可用溶度积计算得到。精确地确定溶度和溶度积的方法为所述领域的技术人员所熟知。此水性溶液可包含高至30mol%的盐,较好是25mol%,再好是20mol%,更好是15mol%,再更好是10mol%,再更好是5mol%,最好是2mol%的不溶或微溶的盐。在本发明的范围内,溶度积小于10-4的盐认为是微溶的。溶度积大于10-4的盐认为是可溶的。
盐或离子或离子化合物在水中的溶解性是依赖于它的晶格能和水合能,考虑到发生的熵效应的话。术语“溶度积”也可更精确地指当盐或离子或离子化合物溶解在水里时建立的平衡。溶度积一般定义为在电解液饱和溶液中离子的浓度之积。例如,碱金属(例如,Na+,K+)在水中的溶解度比碱土金属(例如,Ca2+)的更大,就是说,碱金属有更大的溶度积。也就是说,根据本发明包含在注射缓冲液的水性溶液中的钾盐和钠盐比钙盐更容易溶解。因此,在确定这些离子的浓度时,需要考虑钾盐、钠盐和钙盐之间的干扰等。
根据本发明的应用的一个优选实施例,注射缓冲液中包含50mM到800mM、较优为60mM到500mM、更优为70mM到250mM、尤其较优为60mM到110mM的氯化钠(NaCl),0.01mM到100mM、较优为0.5mM到80mM、更优为1.5mM到40mM的氯化钙(CaCl2),以及可选地3mM到500mM、优选为4mM到300mM、更优地为5mM to 200mM的氯化钾(KCl)。
除了上述的无机阴离子例如卤素离子、硫酸根或碳酸根外,也可包括有机阴离子。这些阴离子可由琥珀酸盐、乳糖醛酸盐、乳酸盐、苹果酸盐、maleonate等形成,也可是这些物质的组合。根据本发明使用的注射缓冲液最好还包括乳酸盐,尤其最好在含有有机阴离子的注射缓冲液中,只包含有乳酸盐作为有机阴离子。本发明的范围内的乳酸盐可以是任何形式的乳酸盐,例如L-乳酸盐和D-乳酸盐。结合本发明,乳酸钠和/或乳酸钙一般是以乳酸盐的形式存在,尤其是在注射缓冲液中仅有Na+作为一价阳离子和Ca2+作为二价阳离子时。
本发明应用的一种优选形式中,本发明的注射缓冲液优选包含15mM到500mM的乳酸盐,其中以包含15mM到200mM的乳酸盐为较佳,最好包含15mM到100mM的乳酸盐。
本发明已发现,具有上述成分且可选地含有或不含有乳酸盐(下文中,“RL注射缓冲液”指不含乳酸盐的缓冲液,而“含乳酸的RL注射缓冲液”指含乳酸盐的缓冲液)的注射缓冲液用于RNA注射液(也就是,包含RNA且适于用来注射RNA的注射液)时,相对于现有技术中使用的注射缓冲液,明显地增加了RNA到/在寄助物(哺乳类动物)细胞/组织中的转移和翻译。
含有上述氯化钠((NaCl)、氯化钙(CaCl2)、乳酸盐(特别是乳酸钠)和可选的氯化钾(KCl)成分的溶液又称为“林格氏液”(Ringer′s solution)或“林格氏乳酸盐”(Ringer′s lactate)。林格氏乳酸盐是全结晶电解质溶液,用于容积替代和作为载体溶液,例如,用于兼容药剂。例如,在体液和电解质丢失(由于呕吐、腹泻、肠梗阻或烧伤)的情况下,林格氏乳酸盐可用作初级容积替代剂,尤其用于婴儿和儿童,并且也用来维持外周和/或中心静脉通路的开放。然而,本发明的用林格氏乳酸盐来作为RNA注射液中的注射缓冲液,在现有技术中未曾有记载。
本发明范围内的RNA是指任何形式的RNA,例如,mRNA、tRNA、rRNA、siRNA、单链或双链RNA、异源双链核酸分子RNA等。所使用的RNA可用来编码任何感兴趣的蛋白质。本发明使用的RNA优选是裸露RNA,其中以mRNA较佳,最好是裸露mRNA。
本发明所述的裸露RNA,特别是裸露mRNA,应被理解为不复杂的RNA,例如,带有聚阳离子分子的RNA。裸露RNA可以单链形式存在,但也可以双链形式存在,也就是说,以二级结构存在,例如,所谓的“发夹结构”。这样的双链形式尤其在裸露RNA中发生,特别是当分子中出现互补核苷酸时,在裸露mRNA中发生。
然而,本发明所述的RNA,尤其是mRNA,也可以复杂形式存在。本发明中,由于RNA尤其是mRNA的复杂化/浓缩,通过将RNA与(多个)阳离子聚合物、多肽或蛋白质联合或绑定,RNA到有机体的需要治疗的细胞中或组织中有效转移将得到改善。这样的RNA(mRNA)最好与至少一种阳离子或聚阳离子剂进行复合或浓缩。所述阳离子或据聚阳离子剂优选地为从以下一组中选出的试剂:鱼精蛋白(protamine)、聚左旋赖氨酸(poly-L-lysine)、聚左旋精氨酸(poly-L-arginine)、核仁素(nucleolin)、精胺(spermine)、和组蛋白(histone)、或组蛋白或鱼精蛋白的衍生物。较佳的选择是采用鱼精蛋白来作为聚阳离子、核酸绑定的蛋白。稳定RNA的程序在EP-A-1083232中有描述,其相关的揭示完整地结合在本发明中。
本发明的RNA可进一步修饰。这些改进是为了增加RNA的稳定性。优选地,RNA有一种或多种(自然发生的或非自然发生的)修饰,尤其是化学修饰,例如,利于增加RNA在有机体中的半衰期,使mRNA比没有经过修饰的mRNA在细胞溶质中的翻译效率更高。优选地,根据本发明,经过修饰的mRNA与没有经过修饰的mRNA在细胞溶质中的翻译效率相比,可提高至少10%,较佳的是提高至少20%,更佳的是提高至少40%,再更佳的可提高至少50%,再更佳的可提高至少60%,再更佳的可提高至少75%,再更佳的可提高至少85%,最高可提高至少100%。
例如,经修饰后的mRNA的编码区的G/C含量可比相应的野生型的mRNA的编码区的G/C含量更多,优选地,经修饰后的mRNA的编码氨基酸序列相对于野生型的mRNA的编码氨基酸序列仍未改变。这一修饰基于如下事实:mRNA的序列区对于mRNA的有效翻译来说是重要的。各种核苷的成分和序列是非常重要的。特别是,具有较高的G(鸟苷)/C(胞核嘧啶)含量的序列比具有较高的A(腺苷)/U(尿嘧啶)含量的序列更稳定。因此,在保持翻译的氨基酸序列不变的同时,改变密码子,使之相对于野生型的mRNA有更多的G/C核苷,是有利的。由于几个密码子编码同一氨基酸(所谓的“遗传密码的兼并”)的事实,确定对稳定性有利的密码子是可能的,尤其具有最大G/C含量时。结果,注射缓冲液中的RNA的G/C含量优选根据G/C含量的最大含量而提高至少30%,较佳是提高至少50%,更佳是提高至少70%,最好是提高至少80%(即,为了最大化G/C含量,在编码区修饰了所有的可能的三联密码而不改变编码的氨基酸序列之后的G/C含量),并且,最好就是最大G/C含量,该最大G/C含量由G/C含量得到最大化而不未改变编码的氨基酸序列的序列给出。
根据将由修饰后的mRNA编码的氨基酸序列,相比较于野生型序列,有各种修饰mRNA的可能。对于由仅含有G或C核苷的密码子编码氨基酸的情况,不需要修饰密码子。这样的例子是Pro(脯氨酸)(CCC或CCG)、Arg(精氨酸)(CGC或CGG)、Ala(丙氨酸)(GCC或GCG)和Gly(甘氨酸)(GGC或GGG)的密码子。
另一万面,包含A和/或U核苷的密码子可用编码同样的氨基酸但不含A和/或U的密码子来代替。下面是一些例子:
编码Pro的密码子可从CCU或CCA变成CCC或CCG;
编码Arg的密码子可从CGU或CGA或AGA或AGG变成CGC或CGG;
编码Ala的密码子可从GCU或GCA变成GCC或GCG;
编码Gly的密码子可从GGU或GGA变成GGC或GGG。
在某些情况下,虽然不可能从密码子中去除A和U核苷,但可用具有较少的A和/或U核苷的密码子来减少A和U的含量。下面是一些例子:
-编码Phe(苯丙氨酸)的密码子可从UUU变成UUC;
-编码Leu(亮氨酸)的密码子可从UUA、UUG、CUU或CUA变成CUC或CUG;
-编码Ser(丝氨酸)的密码子可从UCU或UCA或AGU变成UCC、UCG或AGC;
-编码Tyr(络氨酸)的密码子可从UAU变成UAC;
-编码Cys(胱氨酸)的密码子可从UGU变成UGC;
-编码His(组氨酸)的密码子可从CAU变成CAC;
-编码Gln(谷氨酸)的密码子可从CAA变成CAG;
-编码Ile(异亮氨酸)的密码子可从AUU或AUA变成AUC;
-编码Thr(苏氨酸)的密码子可从ACU或ACA变成ACC或ACG;
-编码Asn(酪氨酸)的密码子可从AAU变成AAC;
-编码Lys(赖氨酸)的密码子可从AAA变成AAG;
-编码Val(缬氨酸)的密码子可从GUU或GUA变成GUC或GUG;
-编码Asp(门冬氨酸)的密码子可从GAU变成GAC;
-编码Glu(谷氨酸)的密码子可从GAA变成GAG,
-终止密码子UAA可变成UAG或UGA。
上面所列出的替代可单独使用或以所有可能的组合来使用,以使修饰后的mRNA的G/C含量比野生型的mRNA(原始序列)的G/C含量更高。上述的替代可能的各种组合,例如,可优选使用如下:
用ACC(或ACG)替代原始序列(野生型的mRNA)中编码Thr的所有密码子,并且用UCC(或UCG或AGC)替代原始序列(野生型的mRNA)中编码Ser的所有密码子;
用AUC替代原始序列中编码Ile的所有密码子,并且用AAG替代原始序列中编码Lys的所有密码子,并且用UAC替代原始序列中编码Tyr的所有密码子。
用GUC(或GUG)替代原始序列中编码Val的所有密码子,并且用GAG替代原始序列中编码Glu的所有密码子,并且用GCC(或GCG)替代原始序列中编码Ala的所有密码子,并且用CGC(或CGG)替代原始序列中编码Arg的所有密码子;
用GUC(或GUG)替代原始序列中编码Val的所有密码子,并且用GAG替代原始序列中编码Glu的所有密码子,并且用GCC(或GCG)替代原始序列中编码Ala的所有密码子,并且用GGC(或GGG)替代原始序列中编码Gly的所有密码子,并且用AAC替代原始序列中编码Asn的所有密码子;
用GUC(或GUG)替代原始序列中编码Val的所有密码子,并且用UUC替代原始序列中编码Phe的所有密码子,并且用UGC替代原始序列中编码Cys的所有密码子,并且用CUG(或CUC)替代原始序列中编码Leu的所有密码子,并且用CAG替代原始序列中编码Gln的所有密码子,并且用CCC(或CCG)替代原始序列中编码Pro的所有密码子;
等等。
编码蛋白质的修饰后的mRNA的编码区G/C含量改变的情况下,该改变与编码蛋白质的野生型mRNA的编码区G/C含量相比,为增加至少7%,较佳是增加至少15%,更佳是增加至少20%,最佳是增加30%。在此关系中尤其优选的是相对于野生型的序列,将修饰后的mRNA尤其编码蛋白质的区域内的G/C含量增加到最大的范围。
进一步优选是增加修饰后的mRNA的核糖体结合点的编码区内的A/U含量,使之比相应的野生型mRNA的核糖体结合点的编码区内的A/U含量更高。这一修饰增加了核糖体结合到mRNA的效率。核糖体有效地结合到核糖体结合点(Kozak序列:GCCGCCACCAUGG,启动子的AUG形式)将影响mRNA的有效翻译。这一增加在于在结合点的编码区引入了至少一个额外的A/U单位,一般至少3个,也就是说,从AUG启动子的A开始的-20到+20。
优选的修饰与mRNA有关,其中mRNA的编码区和/或修饰后的mRNA的5′-和/或3′-未翻译区已经相对于不含任何扰动序列元素的野生型mRNA而改变,修饰后的mRNA的编码氨基酸序列相对于野生型的mRNA最好没变。人们知道,扰动序列元素(DSE)在真核mRNA的序列中出现,扰动序列元素信号蛋白与其结合并调节体内mRNA的酶降解。因此,本发明为了进一步稳定修饰后的mRNA,与野生型mRNA的对应区域相比,可选地可在编码蛋白质的区域内进行一种或多种这样的改变,从而就没有或几乎没有扰动序列元素出现在其中。根据本发明,通过这样的改变,可消除mRNA中出现在未翻译区域(3′-和/或5′-UTR)的DSE。
例如,这样的扰动序列是含丰富的AU的序列(“AURES”),其可在许多不稳定的mRNA的3′-UTR部分出现(Caput et at.,Proc.Natl.Acad.Sci.USA1986,83:1670 to 1674),也可以是被核酸内切酶识别的序列模体(motif)(例如,Binder et al.,EMBO J.1994,13:1969 to 1980)。
同样优选地,经修饰的mRNA具有5′-帽(5′-cap)的稳定结构,本发明使用的帽结构的例子是m7G(5′)ppp,5′(A,G(5′)ppp(5′)A和G(5′)ppp(5′)G.
同样优选地,经修饰的mRNA具有聚(A)尾部(ploy(A)tail),较佳的是具有至少25个核苷,再较佳的是具有至少50个核苷,更较佳的是具有至少70个核苷,再较佳的是具有至少100个核苷,最好具有至少200个核苷。
同样优选地,修饰了的mRNA具有至少一个IRES和/或至少一个5′-和/或3′-稳定序列。根据本发明,一个或多个所谓的IRES(“内部核糖体进入位”″internal ribosome entry side″)可引入到修饰了的mRNA当中。因此,IRES可作为唯一的核糖体结合点,但它也可用来提供mRNA,通过核糖体(″multicistronic mRNA″,多顺反子mRNA)来编码将彼此单独翻译的多个蛋白质、肽或多肽。根据本发明的IRES序列的例子来自于小核糖核酸病毒(例如,FMDV)、瘟疫病毒(CFFV)、脊髓灰质炎病毒(PV)、脑心肌炎病毒(ECMV)、口蹄疫病毒(FMDV)、丙肝病毒(HCV)、猪瘟病毒(CSFV)、鼠白血病病毒(MLV)、猴免疫缺陷病毒(SIV)、蟋蟀麻痺病毒(CrPV)。
优选地,修饰后的mRNA具有至少一个5′-和/或3′-稳定序列。这些在5′-和/或3′未翻译区的稳定序列可使细胞溶质中的mRNA的半衰期增长。这些稳定序列与病毒、细菌、真核生物中自然存在的序列具有100%的同源性,但也可具有部分或完全的合成性质。作为本发明的稳定序列的一个例子是人类(Homo sapiens)或非洲爪蟾(Xenopus laevis)的球蛋白基因的未翻译序列(UTR)。稳定序列的另一个例子具有如下通式(C/U)CCANxCCC(U/A)PyxUC(C/U)CC,其包含在用于编码α-球蛋白(α-globin)、(I)-胶原((I)-collagen)、15-脂氧酶(15-lipoxygenase)或酪氨酸羟化酶(tyrosine-hydroxylase)的很稳定的mRNA的3′-UTR中(参见Holciket al.,Proc.Natl.Acad.Sci.USA 1997,94:2410 to 2414)。当然,对于本领域技术人员来说,这样的稳定序列可单独使用或彼此结合使用,以及与其他的稳定序列组合起来使用。
在本发明的优选实施例中,修饰了的mRNA包含自然存在的核苷的至少一个相似物。这个/这些相似物可进一步稳定修饰后的mRNA,这是基于如下事实:细胞中存在的RNA降解酶优先识别出自然存在的核苷来作为底物。通过引入核苷类似物到RNA中,从而RNA的降解变得更困难了,然而,这些类似物的引入,特别是引入到mRNA的编码区内,对翻译效率有或正或负的影响。在此举出本发明可用的核苷类似物的例子(但不限于这些)有氨基磷酸酯(phosphoramidates)、硫代磷酸酯类(phosphorothioates)、肽核苷(peptidenucleotides)、甲基磷酸酯(methyl phosphonates)、7-deazaguanosine、5-甲基胞嘧啶(5-methylcytosine)和肌苷(inosine)。例如,根据专利号为US 4,373,071、US 4,401,796、US 4,415,732、US 4,458,066、US 4,500,707、US 4,668,777、US4,973,679、US 5,047,524、US 5,132,418、US 5,153,319、US 5,262,530和US5,700,642的美国专利的相关介绍,所属领域的技术人员熟悉这些类似物的制备。这些类似物可存在于修饰后的mRNA的未翻译区和翻译区。
在本发明的较佳实施例中,修饰后的mRNA还包含编码信号肽的序列。编码信号肽的序列优选地长度是30到300个碱基,编码10到100个氨基酸。较佳的,编码信号肽的序列的长度是45到180个碱基,编码15到60个氨基酸序列。通过示例的方式,下表1中的序列可用来修饰本发明使用的RNA。表1中也包括有优选1到20个、较佳1到10个、最好是1到5个碱基的序列替代A、T、G、C,与表1中列出的一个序列相比较。
信号序列的名称 | 序列(肽和核苷酸序列) |
HLA-B*07022 | MLVMAPRTVLLLLSAALALTETWAGRNA序列(富含GC)AUG CUG GUG AUG GCC CCG CGG ACC GUC CUC CUGCUG CUG AGC GCG GCC CUG GCC CUG ACG GAG ACCUGG GCC GGC |
HLA-A*3202 | MAVMAPRTLLLLLLGALALTQTWAGRNA序列(富含GC)AUG GCC GUG AUG GCG CCG CGG ACC CUG CUC CUGCUG CUG CUG GGC GCC CUG GCC CUC ACG CAG ACCUGG GCC GGG |
HLA-A*01011 | MAVMAPRTLLLLLSGALALTQTWAGAUG GCC GUG AUG GCG CCG CGG ACC CUG CUC CUGCUG CUG AGC GGC GCC CUG GCC CUG ACG CAG ACCUGG GCC GGG |
HLA-A*0102 | MAVMAPRTLLLLLSGALALTQTWAGAUG GCC GUG AUG GCG CCG CGG ACC CUG CUC CUGCUG CUG AGC GGC GCC CUG GCC CUG ACG CAG ACCUGG GCC GGG |
HLA-A*0201 | MAVMAPRTLVLLLSGALALTQTWAGAUG GCC GUG AUG GCG CCG CGG ACC CUG GUC CUCCUG CUG AGC GGC GCC CUG GCC CUG ACG CAG ACCUGG GCC GGG |
HLA-A*0301 | MAVMAPRTLLLLLSGALALTQTWAGAUG GCC GUG AUG GCG CCG CGG ACC CUG CUC CUGCUG CUG AGC GGC GCC CUG GCC CUG ACG CAG ACCUGG GCC GGG |
HLA-A*1101 | MAVMAPRTLLLLLSGALALTQTWAGAUG GCC GUG AUG GCG CCG CGG ACC CUG CUC CUGCUG CUG AGC GGC GCC CUG GCC CUG ACG CAG ACCUGG GCC GGG |
HLA-B*070201 | MLVMAPRTVLLLLSAALALTETWAGAUG CUG GUG AUG GCC CCG CGG ACC GUC CUC CUGCUG CUG AGC GCG GCC CUG GCC CUG ACG GAG ACCUGG GCC GGC |
HLA-B*2702 | MRVTAPRTLLLLLWGAVALTETWAGAUG CGG GUG ACC GCC CCG CGC ACG CUG CUC CUGCUG CUG UGG GGC GCG GUC GCC CUG ACC GAG ACCUGG GCC GGG |
HLA-Cw*010201 | MRVMAPRTLILLLSGALALTETWACSAUG CGG GUG AUG GCC CCG CGC ACC CUG AUC CUCCUG CUG AGC GGC GCG CUG GCC CUG ACG GAG ACCUGG GCC UGC UCG |
HLA-Cw*02021 | MRVMAPRTLLLLLSGALALTETWACSAUG CGG GUG AUG GCC CCG CGC ACC CUG CUC CUGCUG CUG AGC GGC GCG CUG GCC CUG ACG GAG ACCUGG GCC UGC UCG |
HLA-E*0101 | MVDGTLLLLSSEALALTQTWAGSHSAUG GUG GAC GGC ACC CUG CUC CUG CUG AGC UCGGAG GCC CUG GCG CUG ACG CAG ACC UGG GCC GGGAGC CAC AGC |
HLA-DRB1 | MVCLKLPGGSCMTALTVTLMVLSSPLALAAUG GUG UGC CUG AAG CUC CCG GGC GGG AGC UGCAUG ACC GCC CUG ACG GUC ACC CUG AUG GUG CUGUCG AGC CCC CUG GCG CUG GCC |
HLA-DRA1 | MAISGVPVLGFFIIAVLMSAQESWAAUG GCC AUC AGC GGC GUG CCG GUC CUG GGG UUCUUC AUC AUC GCG GUG CUC AUG UCG GCC CAG GAGAGC UGG GCC |
HLA-DR4 | MVCLKFPGGSCMAALTVTLMVLSSPLALAAUG GUG UGC CUG AAG UUC CCG GGC GGG AGC UGCAUG GCC GCG CUC ACC GUC ACG CUG AUG GUG CUGUCG AGC CCC CUG GCC CUG GCC |
髓磷脂少突胶质细胞糖蛋白 | MACLWSFSWPSCFLSLLLLLLLQLSCSYAAUG GCC UGC CUG UGG AGC UUC UCG UGG CCG AGCUGC UUC CUC AGC CUG CUG CUG CUG CUG CUC CUGCAG CUG AGC UGC AGC UAC GCG |
表1:信号肽序列的示例:由第一类MHC或第二类MHC基因的第一外显子编码的氨基酸序列和髓磷脂少突胶质细胞糖蛋白
所属领域的技术人员熟知实现上述的修饰的各种处理方法。例如,对于根据本发明修饰了的mRNA中密码子的替换,在相对短的编码区的情况下,可以使用标准技术用化学地方法合成整个mRNA。然而,在借助现有的面向目标的诱变的技术制备经修饰的mRNA时,碱基的替代、增加或消除最好利用DNA模板(matrix)来引入(参见例如Maniatis et al.,Molecular Cloning:ALaboratory Manual,Cold Spring Harbor Laboratory Press,3rd ed.,Cold SpringHarbor,NY,2001)。在此过程中,相应的DNA分子在体外被转录以便制备mRNA。此DNA模板有合适的启动子,例如,用于体外转录的T7或SP6启动子,接着是要制备的mRNA所需的核苷酸序列和体外转录的终止信号。形成要制备的RNA模板的DNA分子可通过发酵繁殖和随后分离为细菌中的质粒复制品的一部分来制备。所以,根据所属领域的技术人员已知的分子生物学的方法,使用在分裂点具有短的单链过渡的短合成DNA寡核苷酸,或者使用通过化学合成制备的基因,可将所需的核苷序列克隆到合适的质粒内(参见前面提到的Maniatis等所著的文献)。然后,用限制性内切酶消化的方法将在质粒中的以一个副本形式存在的或多个副本形式存在的DNA分子从质粒中切除。
上述的RNA的修饰,尤其是mRNA的修饰,可在本发明的范围内单独或相互组合起来进行。类似地,一个或多个修饰可与上述的RNA的复杂化相结合,尤其是mRNA。
本发明的目标是增加寄助物中的RNA转移和/或RNA翻译。本发明范围内所指的寄助物可以理解为RNA可转移入其细胞或组织并随后在其中进行翻译的任何生物体。本发明范围内所指的寄助物特别是哺乳动物例如小鼠、大鼠、猪、牛、马、狗、猫、猿,尤其是人。
根据本发明,荧光素酶编码的RNA,尤其是mRNA,稀释在本发明的RL注射缓冲液(含或不含乳酸盐)中,比稀释在标准的传统的用来稀释RNA的缓冲液例如HBS或PBS中具有明显更好的翻译率(参见图1)。更进一步,注射的mRNA的转移和翻译效率很大程度上取决于钙离子的存在与否。在相应的RL注射缓冲液(含或不含乳酸盐)中含有/不含有钙离子的对比测试中,不含钙会大大地降低RNA的转移率至与标准缓冲液PBS和HBS的转移率相当的水平(参见图2)。
因此发现,首先,本发明的RL注射缓冲液(含或不含乳酸盐)显著地增加RNA转移;其次,用含高达100mM的钙浓度的RL注射缓冲液(含或不含乳酸盐)是改进RNA的转移率的另一个因素。
本发明的注射缓冲液最好与RNA注射液中的RNA相结合使用。因此本发明进一步提供了一种包含RNA的RNA注射液和一种包含至少50mM氯化钠(NaCl)、至少0.01mM氯化钙(CaCl2)和可选的至少3mM氯化钾(KCl)的注射缓冲液,用于增加细胞中的RNA转移和/或RNA翻译。本发明优选的RNA注射液中,注射缓冲液包含有至少50mM到800mM的氯化钠,较佳地含氯化钠60mM到500mM,更佳地含氯化钠70mM到250mM,尤其更佳的是含氯化钠60mM到110mM;包含有至少0.01mM到100nM的氯化钙(CaCl2),较佳地至少含氯化钙0.5mM到80mM,更佳地含氯化钙至少1.5mM到40mM;和可选的至少3mM到500mM的氯化钾(KCl),较佳地含氯化钾至少4mM到300mM,更佳地含氯化钾至少5mM到200mM。
本发明的RNA注射液的注射缓冲液优选地进一步包含乳酸盐。本发明的RNA注射液的注射缓冲液优选地包含至少15mM的乳酸盐。本发明的一种优选RNA注射液中,注射缓冲液较佳地包含15mM到500mM的乳酸盐,更佳地包含15mM到200mM的乳酸盐,最好含15mM到100mM的乳酸盐。
RNA注射液可根据现有技术中的任何方法来制备。优选地,RNA稀释在RL注射缓冲液或含乳酸盐的RNA注射缓冲液中。类似地,RNA可以干RNA的形式(例如,冷干的)使用,并且可加入RNA注射缓冲液或含乳酸盐的RL注射液,可选地通过提高温度、搅拌、超声等来加速溶解。浓度比可根据特定条件来选择(RNA注射液要注射入的寄助物,尤其是哺乳动物以及寄助物的健康状态、年龄等)。
本发明的RNA注射液中的RNA优选是裸露RNA,较佳是mRNA,最好是如上所定义的裸露mRNA。
如上所述,本发明的RNA注射液尤其可用来增加到/在寄助物内的RNA转移和RNA翻译。
因此,本发明还提供上述的RNA注射液用于增加到/在寄助物内的RNA转移和RNA翻译的用途。
RL注射缓冲液(含或不含乳酸)内RNA的剂量(尤其是指临床应用的量和持续时间)也已经进行了研究。研究表明,随着mRNA的量在小鼠中增加到0.1μg(100μl的注射体积中),在人体中增加到3mg(150μl注射体积中),莹光素酶的表达也增加。mRNA的翻译瞬时发生并且随后被调节,因此,为了使外来分子(蛋白质)能够持续、一致的表达,依据各种因素例如要表达的外来分子和倾向的活动、接受注射的有机体和其(健康)状态,大约每3天要重复注射一次,甚至两天重复注射一次或每天重复注射。RNA的量(同样取决于上述的各种因素)可以是100μl的注射体积中包含0.01μg到1000μg,较佳地包含1μg到800μg,更佳地包含2μg到200μg,再更佳地包含5μg到100μg,再更佳地包含10μg到90μg,最好包含20μg到80μg。在100μl注射体积中,RNA的量特别优选的是60μg。
因此,本发明提出的RNA和RL注射缓冲液或含乳酸盐的RL注射缓冲液以及RNA注射液的用途,可用于例如癌症或肿瘤疾病的治疗和/或预防,或用于治疗和/或预防癌症或肿瘤疾病的药剂的制备,该癌症或肿瘤疾病指的是例如黑素瘤,比如,恶性黑素瘤、皮肤黑素瘤,癌,比如大肠癌、肺癌,比如小细胞肺癌、腺癌、前列腺癌、食管癌、乳腺癌、肾癌、肉瘤、骨髓瘤、白血病,特别是AML(急性髓细胞白血病,acute myeloid leukaemia)、神经胶质瘤、淋巴瘤和胚细胞瘤、敏感症、自体免疫疾病,例如,多发性硬化症、病毒和/或细菌感染。
例如,本发明包括RNA和RL注射缓冲液或含乳酸盐的RL注射缓冲盐的用途,还包括RNA注射液的用途,用于基因治疗和接种疫苗,例如,用于抗病毒或肿瘤接种,用于预防上述的疾病。
本发明所述的“基因治疗”是指引入功能基因到患病的细胞中恢复机体或细胞的失去了的功能,或通过相应的遗传信息抑制受损的功能。例如,对于肿瘤抑制基因,例如,丢失p53基因或其只有少量表达时,可以以其mRNA的形式引入细胞内并插入到DNA中,并且最初表达不足的蛋白就能够重新生成到生理上需要的量。本发明所述的肿瘤抑制基因的例子是p53 TP53,RB1,APC,WT1,NF1,NF2,VHL,BRCA1,BRCA2,DCC,MEN1,MEN2,PTCH,p57/KIP2,MSH2,MLH1,FMS1,FMS2,MET,p16/INK4a/CDKN2,CDK4,RET,EXT1,EXT2,EXT3,PTEN/MMAC1,ATM,BLM,XPB,XPD,XPA,XPG,FACC,FACA,SMAD4/DPC4,p14Art(p19Art),DPC4,E-CAD,LKB1/STK1,TSC2,PMS1,PMS2,MSH6,TGF-β type II R,BAX,α-CAT,MADR2/SMAD2,CDX2,MKK4,PP2R1B,MCC,等等。
本发明所述的接种是指以RNA的形式,尤其是以mRNA的形式,将遗传信息引入到有机体中,特别是引入到有机体的一个/几个细胞或组织中。如此给药的mRNA在有机体中翻译成目标分子(例如,肽、多肽、蛋白质),也就是说,由mRNA编码的目标分子被表达并触发免疫反应。人们知道,抗原递呈细胞(APC)在触发免疫反应时起到专门的关键作用,因为它们是如此的唯一的细胞类型:当激活后,所有的启动抗原特异性免疫细胞的增殖所需的所有信号被触发。本发明所述的接种可使用编码抗原的RNA,特别是mRNA来进行,所述的抗原在肿瘤免疫接种时是肿瘤抗原,在接种对抗外来病原体时是外来抗原。本发明所述的肿瘤抗原是T-细胞定义的肿瘤抗原,例如,“癌/睾丸”(″cancer/testis″antigens)抗原,例如,MAGE、RAGE、NY-ESO-1,分化抗原(differentiation antigens),例如,MART-1/Melan-A、酪氨酸酶(tyrosinase)、gp100、PSA、CD20,变异基因的抗原决定基(antigenic epitopes of mutatedgenes),例如,CDK4,caspase-8,或癌胚抗原(oncofetal antigen),例如,CEA、AF。其它的肿瘤抗原有,例如,存在于T-细胞和B-细胞淋巴瘤中的肿瘤抗原CD5和CAMPATH-1(CDw52),存在于非霍奇金B-细胞淋巴瘤(non-Hodgkin’sB-cell lymphomas)中的CD20,肿瘤抗原CEA(carcinoembryogenic antigen),粘液素,存在于实心肿瘤特别是上皮性肿瘤(乳腺、肠和肺)中的CA-125和FAP-a,TN蛋白(tenascin),和还存在于成胶质细胞瘤(glioblastoma)中的金属蛋白酶(metalloproteinases)。进一步的肿瘤抗原是,例如,肿瘤抗原EGF(表皮生长因子),存在于肺、乳腺、头部和颈部以及T-细胞和B-细胞肿瘤中的p185HER2和IL-2受体,肿瘤抗原SV40等。
RNA,特别是mRNA,也可编码多个这样的抗原。因此,黑素瘤、癌、AML或神经胶质瘤可有效地得到控制,因为特定肿瘤的不同抗原的组合具有极其广泛的活动范围。本发明的RNA特别是mRNA可进一步编码免疫原性蛋白。这样的免疫原性蛋白可调节免疫反应的再激活。这样的再激活基于以下的发现:几乎每个有机体都有对某些外来分子(如蛋白质,尤其是病毒蛋白、抗原)的所谓的“记忆免疫反应”(memory immune responses)。这意味着,有机体在较早时已经被这样的外来分子感染过,并且,对此外来分子(例如,病毒蛋白)的免疫反应已经通过此次感染被触发,此免疫反应就保存在“记忆”中,也就是说被存储起来。当有机体再次被同样的外来分子感染时,免疫反应就被再次激活。根据本发明,这样的免疫反应的再激活可通过接种包含有至少一个免疫原性蛋白的编码区的RNA特别是mRNA来实现。优选的是编码一个或多个抗原并编码一个或多个免疫原性蛋白的RNA,尤其是mRNA。
本发明所述的免疫原性蛋白优选地是病毒的结构蛋白,特别是基质蛋白、壳蛋白和脂膜的表面蛋白。这样的病毒蛋白的其它例子是腺病毒蛋白、鼻病毒(rhinoviruses)蛋白、冠病毒(corona viruses)蛋白、逆转录酶病毒(retroviruses)蛋白,特别优选的是乙肝表面抗原(hepatitis B surface antigen)(下面以“HBS抗原”代表)和流感基质蛋白,特别是流感基质M1蛋白。
本发明进一步涉及RNA和上述的RL注射缓冲液或含乳酸盐的RL注射缓冲液、或上述的RNA注射液在“体外”处理时增加RNA转移和/或RNA翻译的用途,例如,用于基因表达分析或用于体外筛选,例如,通过HTS(高吞吐量筛选)。
本发明进一步提供一种方法,用于增加寄助物中的RNA转移和/或RNA翻译,例如,用于治疗和/预防癌或肿瘤疾病,例如,黑素瘤,如良性黑素瘤、皮肤黑素瘤,癌如大肠癌、肺癌如小细胞肺癌、腺癌、前列腺癌、食管癌、乳腺癌、肾癌、肉瘤、骨髓瘤、白血病,特别是AML(急性髓细胞白血病,acutemyeloid leukaemia)、神经胶质瘤、淋巴瘤和胚细胞瘤、敏感症、自体免疫疾病,例如,多发性硬化症、病毒和/或细菌感染,以及用于基因治疗和/或接种,可选的用于抗病毒疫苗接种,用于上述疾病的预防,所述方法包括下述步骤:
a.)制备本发明的RNA注射液;
b.)将步骤a得到的RNA注射液给药到寄助物中。
步骤a中RNA注射液的制备可按前述进行,也就是说,根据现有技术的任何方法进行,优选地,通过将RNA稀释在RL注射缓冲液或含乳酸盐的RL注射缓冲盐中来实现。这里所述的浓度比率也依赖上述的条件来选择(例如,RNA注射液要注射到的寄助物,特别是哺乳动物,以及寄助物的健康状态、年龄等)。RNA注射液可用例如注射器(例如,Sub-Q,Becton Dickinson,Heidelberg,Germany)以任何合适的方式给药,例如,皮内给药、上皮内给药、皮下给药、静脉内给药、动脉内给药、腹腔给药、腹膜内给药、结点内给药(例如,给药到淋巴结中)等。
本发明所述的寄助物最好是从下列选出的哺乳动物:小鼠、大鼠、猪、牛、马、狗、猫、猿,特别是人。
根据本发明制备的注射液还可用于使用RNA特别是mRNA进行的细胞的体外转染。此体外转染可适用于实验室使用或作为生物体外基因治疗的一部分,也就是说,将细胞从病人身上移除,包含在本发明的注射液中的RNA的生物体外转染,然后重新移植到病人身上。转染可借助电穿孔的方法进行,可选地还可借助电压脉冲来实现,其场强不超过2到10kVcm-1,并且脉冲持续时间是10到200μs,电流密度至少有2Acm-2。然而,如果不是用来进行转染的话,也可用1到100ms的脉冲。如果本发明的注射溶液是用于实验室目的,所有的实验室细胞系都能采用此方法用RNA转染。对于生物体外基因治疗来说,许多细胞类型可以用来转染,尤其是初级人类血液细胞、多向潜能祖细胞和成纤维细胞、神经细胞、上皮细胞或肌细胞,以上给出的是示例,而不是为了限制。
本申请中引用的所有参考文献均完整地包括在本申请中。
下列的图和例子是为了进一步解释本发明,而不是对本发明进行限制。
附图说明
图1至图5所示的试验中,每种情况所示的包含有编码萤火虫(Photinuspyralis)荧光素酶的mRNA(图4中100μl PBS中的pDNA)的100μl缓冲液(缓冲液的成分在下面的材料1.注射缓冲液中给出),被皮内注射到BALB/c小鼠13的耳翼。分析整个小鼠的耳的荧光素酶的活动。这以百万荧光素酶分子表示。检测范围在图中用标有数字的粗线表示。图上的每个点展示了单只耳朵上的荧光素酶表达。图上的短横杠表示各组的平均值。针对各组给出了p值,明显不同于其平均值(根据Mann-Whitney检验)。在图1、2和5所示的实验中,注射后15小时,耳朵被切除。数据显示每组有至少3个独立的实验。
图1比较了mRNA的不同注射缓冲液:磷酸缓冲盐(PBS)和HEPES-缓冲盐(HBS)和含乳酸的RL注射缓冲液(RL)。lacZ mRNA用作负控制。本发明显示,稀释在含乳酸的RL注射缓冲液中的mRNA具有比稀释在HBS或PBS中的mRNA明显更高的荧光素酶表达(p<0.001)(图1A)。
图2展示了RL注射缓冲液(含乳酸盐和不含乳酸盐,以及含钙或钾和不含钙或钾)中不含钙(-CaCl)、钾(-KCl)、乳酸钠(-NaLa)时对mRNA的吸收效率的影响。PBS和HBS与RL注射缓冲液或含乳酸盐的注射缓冲液(含或不含钙)之间的主要区别是不含乳酸盐和钙(在HBS或PBS中)。因此,研究一方面使用完全RL注射缓冲液(含乳酸盐的RL注射缓冲液),另一方面使用不含钙或不含钾或不含乳酸盐的RL注射缓冲液配方,来比较编码荧光素酶的mRNA的转移和翻译。这些研究显示,不含乳酸盐使荧光素酶的表达可与使用完全RL注射缓冲液(含乳酸盐的RL注射缓冲液)时的表达相当。但是,RL注射缓冲液或含乳酸盐的RL注射缓冲液中不含钙时,显著地降低RNA的转移效率(p=0.004)至与PBS和HBS相当的水平。
图3和图4展示了直接体内mRNA翻译的动力学。进行了RNA在本发明的含乳酸盐的RL中和DNA在PBS标准缓冲液中的平行动力学实验并进行比较。mRNA(在含乳酸盐的RL注射缓冲液中)(图3)或pDNA(在PBS中)(图4)在注射10天之后的翻译被记录下来并展示在图中。在两个测试(RNA和DNA)过程中,在活的小鼠上的荧光素酶活性被记录下来。具有代表性的耳朵的结果在图中示出。在注射(编码荧光素酶)溶在含乳酸盐的RL注射缓冲液中的mRNA之后检测到的荧光素酶的表达很早就达到了其最大值(17个小时),并且9天之后就再也检测不到(图3)。比较起来,在PBS中注射(编码荧光素酶)pDNA可导致较晚的蛋白质表达,注射3天后才达到其最大值并且持续到超过9天(图4)。这些结果再次确认了本发明的RL注射缓冲液的效率,而且确认了RNA远比DNA更适合用作寄助物特别是哺乳动物中瞬时基因表达的载体。RNA一方面很快地表达,另一方面瞬时表达,这意味着目标基因表达可被较早地触发并持续有限的时间,相应地以更有目标和分化的方式。这些研究证实了成功的RNA转移的增加以及随后的有效翻译。
图5展示了不同量的mRNA对荧光素酶表达的影响。这些实验是为了更精确的确定特别是在临床应用中使用的RNA在含乳酸盐的RL注射缓冲液中的剂量,结合考虑量和持续时间。为此目的,增加的RNA量被注射到多个小鼠体内。随着mRNA的量增加到5μg(100μl注射体积),检测到荧光素酶表达的增加。超过5μg的剂量不能进一步增加表达。在相应的人体的实验中(图中没有示出),使用了120μg的mRNA的剂量,当增加到200μg时,导致表达增加。人体使用的200μg的mRNA,与小鼠的5μg剂量相比,可由注射部位的大小来获得,即人体大约有小鼠的40倍大小。在解释了背景和研究可能导致的后果和获得同意之后,人体实验是在健康的志愿者身上进行的。
关于不同时间的剂量,应该注意到,mRNA的翻译是瞬时发生的(如图3所示,在12小时后达到最大值并且在9天之后便检测不到)并且因此被调节。因此,为了持续、均一地翻译外来分子(蛋白质),要大约每天、每两天或每3天重复注射(取决于一些因素,如外来分子或mRNA要注射入的有机体)是合适的。
图6再次显示了CaCl2对荧光素酶活性的影响。为此目的,制备了一系列的CaCl2荧光素酶溶解缓冲液(最终浓度在图中示出),并且相同量的重组荧光素酶蛋白被加到所有的样本中(最终浓度大约是4.7pM)。混合物的光发射用光度计测量(加入ATP和荧光素后)。CaCl2浓度对荧光素酶活性的影响根据下式计算出:
%相对荧光素酶活性(RLA)=(具有确定的CaCl2浓度的样本的RLA-纯溶解缓冲液的RLA)/(不含CaCl2的样本的RLA-纯溶解缓冲液的RLA)×100%
较高浓度的Ca2+离子(大约2mM以上)的出现不能增加荧光素酶的活性。
图7再次展示了CaCl2浓度对mRNA在体内转移的影响。使用了各种浓度的含乳酸盐的RL注射缓冲液,以制备具有相同量的用于编码萤火虫荧光素酶的mRNA(20μg)的RNA注射液(100μl),但具有不同的同渗容摩(osmol.)。这些RNA注射液被注射到BALB/c小鼠的耳缘。15小时后,处死小鼠,制备耳的溶解产物。计算出的每只耳产生的荧光素酶分子总量、各组的平均值(带数字的横杠)、每组的大小(n)和测试的检测范围(带数字的粗线)在图中示出。结果发现,体内的mRNA的有效转移和接着的有效翻译需要的最小浓度是170mOsm。
图8A-E展示了表达体内补充的mRNA的细胞的特征。20μg用于编码Escherichia coli(大肠杆菌)β-半乳糖苷酶(β-galactosidase)的mRNA,稀释到包含100μl RL含有乳酸盐的注射缓冲液的RNA注射液总量中,被注射到小鼠体内,14小时后,处死小鼠,切除耳朵,制备冷冻切片。图8A和8C至8E用颜色示出其特征。
更进一步,研究了注射缓冲液(含或不含乳酸盐)中的RNA的直接表达。为此目的,定义了吸收并翻译RL注射缓冲液(含或不含乳酸盐)中转移的外源性RNA的细胞类型(也参见例5,图8A-E和类似的,图11、12、16和17)。之后,在本发明的基于mRNA的免疫接种的范围内,分析了如何用转移到免疫系统的确定的目标细胞中的RL注射缓冲液(含或不含乳酸盐)内的外源性RNA来触发免疫反应。
图8A展示的实验中,每个第五样本切片用含X-gal溶液染色。在连续的20μm厚的冰冻切片中检测到多达10个β-半乳糖苷酶阳性细胞。表达区域,也就是说,β-半乳糖苷酶阳性细胞(箭头所示),覆盖了耳的纵向和径向的方向1至2毫米,并且位于耳肌肉的表皮层和软骨层之间的较窄的层上。
本发明也研究了APC是否用直接吸收和自体翻译转移的mRNA或吸收另外的细胞转移的RNA的翻译产物(所谓的“交叉递呈(cross presentation)”)来检测外来抗原。由于细胞的局部化、它们的形状和它们的MHC第二类基因型,可能得出结论:在注射部位吸收和表达外来的裸露的mRNA的主要是肌肉细胞和/或成纤维细胞(图8A)。结果对应于上述的被其他细胞翻译的抗原的“交叉递呈”。此程序可同样用来解释抗核酸疫苗编码蛋白的抗体的形成。根据本发明,首次可能确定相应的免疫反应的触发通过所谓的“交叉触发”产生,在其中,肌肉细胞或真皮细胞(成纤维细胞)吸收并表达转移的RNA,并且APC是被这些细胞激活的。
图8B的柱状图展示了连续的切片中β-半乳糖苷酶阳性细胞的数目。每个柱代表一个切片。
图8C到8E的实验中,每个第五样本切片被染色,即用于MHC第二类表达(用Alexa 546免疫荧光染色检测出,绿色的)和β-半乳糖苷酶表达(用洋红-gal(magenta-gal)染色检测出,蓝紫色的)。左边一列的图显示了洋红-gal染色单独与蓝紫色的β-半乳糖苷酶阳性细胞=(深)暗区。中间和右边的图中,洋红-gal染色(在中间和右边图中所示的感兴趣的区域)和单独的MHC第二类染色(桔色=中间图中的亮区)(绿色=右边图中的亮区)的重叠。如图所示的,绝大多数的β-半乳糖苷酶阳性细胞清楚地表现出是MHC第二类阴性的。
图9A-B展示了在小鼠和人体身上裸露mRNA的体内转移。制备编码荧光素酶的mRNA并将其溶解在含有RL注射缓冲液的RL注射液中。检测范围在图中用带数字的粗线表示。
图9A所示的实验中,总量100μl、含有20μg mRNA的注射液注射到小鼠的耳部肌肉中。注射后14小时,处死小鼠,耳部细胞被溶解,并且研究溶解物的荧光素酶的表达。计算每只耳朵的荧光素酶分子数量(重组的荧光素酶用作标准)。每组的数据来自至少3个独立的实验。
图9B所示的实验中,总量200μl、含相同mRNA(120μg)的注射液注射到人的皮肤中(到志愿者的腿部)。16小时后,在局部麻醉下,取下直径为2mm的活组织进行检查,也就是,从注射部位的中间(“mRNA”)和离注射点一定距离(“假的(mock)”)。荧光素酶的活性只能在注射点的中间检测到。图中示出了两个独立实验中的一个。这些结果显示,裸露的mRNA直接转移到人的皮肤中。因此,本发明允许在人体进行有效定向的基于mRNA的疫苗接种。
图10A-D展示了注射的含乳酸盐的RL注射缓冲盐中的mRNA的完整性和翻译能力。其完整性用甲醛-琼脂糖凝胶电泳来检测(1.2% w/v)。为此目的,1μg的编码萤火虫荧光素蛋白酶(luc,1.9kB,图10A)或编码大肠杆菌β-半乳糖苷酶(lacZ,3.5kB,图10C)的mRNA被分离出来。没有检测到在各自的注射缓冲液(原料溶液)中稀释之前(注射之前)和在各自的注射缓冲液稀释之后的mRNA的完整性有何不同。相比而言,当从注射器中收集RNA注射液的残液进行检测时,可以检测到mRNA的看得见的、完全的降解(在注射之后)。由于注射器与小鼠或人体组织的接触,这些残液显然被核糖核酸酶所污染。
注射的mRNA的翻译能力用含10μg的mRNA的BHK21细胞的电穿孔来检测。用来控制10μg不相关的mRNA或非mRNA(假的)。这些细胞随后被溶解且用光度计研究它们的荧光素酶活性(图10B),或用X-gal染色并且用光学显微镜研究它们的荧光素酶活性(图10D)。
图11展示了在细胞水平的mRNA转移的识别。该图展示了小鼠的图片。图中,外侧(背侧)对观察者可见。含乳酸的RL注射缓冲液中的mRNA被注射到小鼠的耳部肌肉。制备出耳朵的连续横切面(1、2、3、4)。这些切面收集在各种装置中(1、2、3、4),在空气中干燥并存储在-20℃直到进行各种染色操作。
图12A-C展示了在细胞水平的mRNA转移。含乳酸盐的RL注射缓冲液中的5μg编码大肠杆菌β-半乳糖苷酶的mRNA被注射到小鼠耳朵部位。注射后15小时,耳朵被植入到TissueTek O.C.T介质中,并制备60μm厚的冰冻切片。切片用X-gal染色一整夜。图12A展示了mRNA转移阴性耳朵的冰冻切片。没有lacZ阳性细胞是可检测出的。图12B展示了概貌。图12C展示了mRNA转移阳性耳朵的细节视图。lacZ阳性细胞呈深蓝色,用箭头标示出。
图13展示了Alexa Fluor 546信号与洋红-gal阳性细胞的颜色的相容性。为了确定是否可能在洋红-gal阳性细胞中检测到Alexa Fluor 546,用eGFPmRNA(eGFP=enhanced green fluorescence protein,增强型绿色荧光蛋白)或lacZ mRNA的组合来转染BHK细胞。下列的被转染细胞的组合被分析:
-仅用eGFP mRNA或lacZ mRNA进行单独的转染
-如上的被单独转染的细胞(eGFP/lacZ)的混合物
-被双重转染的细胞(eGFP+lacZ)
该细胞先用带Alexa Fluor 546检测的抗-eGFP的抗体染色,然后用洋红-gal染色。经洋红-gal软色的阳性细胞(表达lacZ)通过广角光学显微镜检测到(顶行),经Alexa Fluor 546染色的阳性细胞(表达eGFP)通过荧光显微镜检测出(中间行)。为了得到细胞之间的相对位置的准确结果,将以上两种检测的结果重叠(底行),尽管此图中的Alexa Fluor 546信号覆盖了光学显微镜的图象。不能排除提供给APC的mRNA的直接吸收和自体翻译有发生并足够引起免疫反应。在某些APC中,轻微的或不完全的不能检测到的翻译可能已经发生,并且(在不完全翻译的情况下)可能影响外来抗原的处理和递呈。
图14A-B展示了冰冻切片的第二类MHC染色的特征。总量为100μl的含乳酸盐RL注射液中的20μg编码大肠杆菌β-半乳糖苷酶的mRNA被注射到体内。注射后14小时,处死小鼠,切除耳朵,制备冰冻切片。该冰冻切片首先用抗第二类MHC的抗体染色(图14A),或用对应的同型对照抗体(isotypecontrol antibody)软色(图14B),并通过用Alexa 546进行的免疫荧光染色检验法进行检测。然后,该冰冻切片用洋红-gal染色(用于β-半乳糖苷酶表达)。图中显示出了洋红-gal染色(左列),第二类MHC染色(中间列,lacZ阳性细胞的位置用轮廓线表示出来),和两种染色的重叠(右列,lacZ阳性细胞用轮廓线表示出来,第二类MHC阳性细胞在图中用亮区表示)。
图15展示了X-gal染色和AEC染色阳性细胞的相容性。为了确定X-gal沉积是否与AEC阳性细胞相容,BHK细胞与eGFP mRNA和lacZ mRNA共同转染。细胞用AEC(红色:阳性细胞,表达eGFP)和X-gal溶液(蓝绿色:阳性细胞,表达lacZ)或用AEC和X-gal的组合来进行抗-eGFP免疫染色。染色后的细胞用广角显微镜来分析。双阳性细胞呈黑色(黑色箭头)。当各次染色足够强时,很难单独地区分染色后的阳性细胞(绿色和红色箭头),因而倾向于呈黑色。
图16A-B展示了第二类MHC阳性细胞和mRNA转移阳性细胞的协同定位(co-localisation)。含20μg编码β-半乳糖苷酶的mRNA的100μl含乳酸盐RL注射液被注射到体内。注射后14小时,处死小鼠,切除耳朵,制备冰冻切片。该冰冻切片首先用抗第二类MHC的抗体(图16A+B)或用对应的同型对照抗体(图16C)(用Alexa 546染色法检测)进行染色,然后用X-gal进行染色(用于β-半乳糖苷酶表达)。对mRNA转移为阳性的细胞呈现出蓝绿色,对第二类MHC为阳性的细胞呈红色,双重阳性的细胞呈现出黑色。mRNA转移阳性细胞用箭头标示出来,与第二类MHC表达无关。
图17展示了mRNA转移和耳部肌肉的形态。含20μg编码β-半乳糖苷酶的mRNA的100μl含乳酸盐RL注射液被注射到体内。注射后14小时,处死小鼠,切除耳朵,制备冰冻切片。该冰冻切片首先用X-gal(用于β-半乳糖苷酶表达)染色,然后用苏木精和伊红(四溴荧光素)染色。对于mRNA转移为阳性的细胞用箭头标示出来,并靠近软组织层。
具体实施方式
材料
1、注射缓冲液
使用了下述的缓冲液:
-2x磷酸缓冲盐(PBS)
(PBS:274mM氯化钠,5.4mM氯化钾,20mM磷酸一氢钠,4mM磷酸二氢钾,温度20.8℃,pH7.3)
-2x HEPES缓冲盐(HBS)
(HBS:300mM氯化钠,20mM Hepes,温度20.8℃,pH7.4)
-1x RL注射缓冲液(不含乳酸盐)
(如果没有指出其它成分和浓度的话,含82.2mM氯化钠,4.3mM氯化钾,1.44mM氯化钙)
-1x RL注射缓冲液(含乳酸盐)
(如果没有指出其它成分和浓度的话,含102.7mM氯化钠,5.4mM氯化钾,1.8mM氯化钙,20mM乳酸钠)
-1x RL注射缓冲液(含乳酸盐、不含氯化钠)
(如果没有指出其它成分和浓度的话,含4.3mM氯化钾,1.44mM氯化钙,22.4mM乳酸钠)
-1x RL注射缓冲液(含乳酸盐不含氯化钾)
(如果没有指出其它成分和浓度的话,含82.2mM氯化钠,1.44mM氯化钙,22.4mM乳酸钠)
-1x RL注射缓冲液(含乳酸盐不含氯化钙)
(如果没有指出其它成分和浓度的话,含82.2mM氯化钠,4.3mM氯化钾,22.4mM乳酸钠)
当使用2x PBS和2x HBS时,所有的成分溶解在水中,调节pH。焦碳酸二乙脂(DEPC,Sigma,Schnelldorf,Germany)加至浓度为0.1%(v/v)。缓冲液在37℃下放置1小时以上。然后将缓冲液高压灭菌。
含乳酸盐的1x RL注射缓冲液用四种不同盐(氯化钠、氯化钾、氯化钙、乳酸钠)的20x储备溶液来制备。类似地,1x RL注射缓冲液用三种不同盐(氯化钠、氯化钾、氯化钙)的20x储备溶液来制备。在进一步的实施例中,略去氯化钠或氯化钾或氯化钙而不补偿被降低的同渗容摩。这些含乳酸盐不含氯化钠、氯化钾或氯化钙的的RL注射缓冲液,也用20x储备溶液来制备。除了乳酸钠的外消旋酸盐(Fluka,Schnelldorf,Germany)外,每种成分都经DEPC和高压灭菌处理,如对2x PBS和2x HBS所描述的那样。
所有的缓冲液和缓冲成分都通过将1x缓冲液中的1μg mRNA在37℃下培养2小时,来检测核糖核酸酶的活性。用甲醛-琼脂糖凝胶电泳的方法来分析mRNA时,使用没有观察到降解的缓冲液。
2、小鼠
所有的动物实验都根据实验室和国家指导方针来进行。使用的年龄为8周到15周的雌性BALB/c小鼠来自于德国Sulzfeld州的查尔斯河(CharlesRiver,Sulzfeld,Germany)。
在皮内注射之前,小鼠被麻醉并且耳部肌肉用异丙醇进行了处理。为了分析mRNA的吸收和翻译,在特定的时间后处死小鼠,切除耳朵,用刀片剃去麻烦的毛发。
3、人
人体实验在健康的男性志愿者身上进行,向他们介绍了研究的背景和可能的结果,并征得他们的同意。
例1:制备核酸
mRNA
“加帽”的mRNA通过利用T7 RNA聚合酶(T7-Opti mRNA kits,CureVac,Tübingen,Germany)进行体外“径流转录(run-off transcription)”来制备。
此mRNA的编码序列(从Acc.U02445克隆出的大肠杆菌β-半乳糖苷酶[lacZ]或从Acc.U47295克隆出的萤火虫荧光蛋白酶[luc])在其3’端用α球蛋白未翻译区和人工的聚A尾部(n=70)侧面相接。小鼠实验中,mRNA用苯酚/氯仿/异戊基醇提取并用氯化锂沉淀。然后将mRNA重新悬浮在水中,用分光光度计在260nm测量产量。最终,mRNA用醋酸铵沉淀并以无菌的方式重新悬浮在水中。
pDNA
不含内毒素的pCMV-荧光素酶DNA用去内毒索的质粒提取试剂盒(EndoFree Plasmid Maxi Kit)(Qiagen,Hilden,Germany)来制备。pDNA用醋酸铵沉积并最后以无菌方式重新悬浮在水中。pCMV-荧光素酶质粒通过将来自pGL3(Acc.U47295)的Xba I-(用Klenow片段钝化)Hind III片段插入到pCMV-HB-S(Acc.A44171)的Nsi I-(用Klenow片段钝化)Hind III-消化质粒的方式来修饰。pDNA的报道基因受控于CMV启动子。
储备溶液
储备溶液通过将mRNA或DNA稀释在无菌水中来制得并用分光光度计(以260、280和320nm)来确定浓度和纯度。
质量控制
对于所有的核酸样本,其浓度用分光光度计确定,并且其完整性用甲醛-琼脂凝胶电泳(mRNA)或限制性消化和TBE-琼脂凝胶电泳(DNA)的方法来检验(图10)。除了完整性之外,所有核酸样本的翻译能力用电穿孔BHK21细胞来分析。为此目的,200μl的含10μg核酸的PBS中的1至3百万个细胞在0.4cm试管中以300V和150μF进行电穿孔。电穿孔后8到24小时后,用适当的方法(X-gal染色或荧光检测)分析经转染的细胞的蛋白质表达(图10)。对于体内实验,只有在BHK21细胞中呈现了蛋白质表达并在凝胶电泳中有合适的完整性的核酸样本才被注射。
例2:RNA注射溶液的制备
对于HBS和PBS来说,mRNA在1x浓度的缓冲液中稀释。对于含或不含乳酸盐的RL注射缓冲液和其各种变化(不含Ca2+,K+,Na+中的一个)(成分和浓度参考材料1.注射缓冲液),mRNA稀释在0.8x浓度的缓冲液中。除非特别提到,否则含20μg mRNA的100μl注射缓冲液用于每只鼠耳。为了去除mRNA中的二级结构,RNA注射缓冲液在80℃加热5分钟。然后,将该溶液放在冰上5分钟。最后,将RNA注射液吸到Sub-Q(Becton Dickinson,Heidelberg,Germany)注射器中。每次注射用不同的注射器。质粒DNA(pDNA)稀释在1x浓度的PBS中。
例3:体外检测荧光素酶的活性
为了体外检测荧光素酶的活性,制备了组织溶解产物。为此目的,该组织用杵和研钵在液氮下粉碎,残留的小块用800μl溶解缓冲液(25mM Tris HCl,2mM EDTA,10%(w/v)甘油,1%(w/v)氚核X-100加上新加的2mM DTT和1mM PMSF)均匀化。在掌上离心机上离心(10分钟,13000转/分,4℃)后得到匀浆上清液。110μl此溶解产物的等分试样在-80℃储存。
为了测量荧光素酶的活性,等分试样在冰上解冻,50μl溶解产物的光发射用光度计(LB 9507,Berthold,Bad Wildbad,Germany)测量15秒。测量之前,光度计自动加入300μl缓冲液A(25mM氨基乙酰氨基乙酸,15mM硫酸镁,5mM新加入的ATP,pH 7.8)和缓冲液B(250μM水中的荧光素)到溶解产物中。
为了标准化,在所有的测量中使用了一系列的重组荧光蛋白稀释液(QuantiLum,Promega,Madison,USA)。用此标准方法,计算出每次单独测量的荧光素酶分子的量。对于每份溶解产物,荧光素酶的活性在不同的日期两次测量,计算出荧光素酶的平均活性。在所有具有高于检测极限的荧光素酶活性的溶解产物,荧光素酶分子的量的变化系数(n=4)低于10%。此检测极限(在图中用带数字的粗线表示)用溶解缓冲液的测量值加上这些值的3倍标准偏差的平均值来计算(n=80)。
例4:体内生物发光检测
为了检测活的动物体上注射的荧光素酶,在注射入核酸后一段特定时间后,麻醉小鼠。小鼠被分成3个不同的组:在第I组小鼠中,100μl RL注射缓冲液被注射到左耳,并且含在100μl RL注射缓冲液中的20μg编码荧光素酶的mRNA被注射到左耳。在第II组小鼠中,含20μg编码荧光素酶的mRNA的100μl RL注射缓冲液被注射到左耳和右耳。在第III组中,100μl RL注射缓冲液被注射到右耳,含在100μl RL注射缓冲液中的20μg编码荧光素酶的mRNA被注射到左耳。然后小鼠被i.p.注射含20mg/ml荧光素(Synchem,Kassel,Germany)的200μl PBS(过滤除菌的)。荧光素注射后5分钟,收集20分钟内小鼠的光发射。为此目的,小鼠被放到预先加热至37℃的黑盒子中(第I组在左侧,第II组在中间,第III组在右侧)。盒子装有Aequoria全景成像照相机(Hamamatsu,Japan)。光发射以假彩色图象显示,与小鼠在正常光线下的灰度图象相重叠。类似地,还用含20μg编码荧光素酶的mRNA的含乳酸盐RL注射缓冲液、不含氯化钠而含乳酸盐的RL注射缓冲液和不含氯化钙而含乳酸盐的RL注射缓冲液进行相同的实验。
例5:β-半乳糖苷酶活性和组织结构
刮干净了的鼠耳被切除,植入在含Tissue-TekO.C.TTM化合物(Sakura,Zoeterwuode,Netherlands)的介质中并储存在-80℃。从这些小块中切下的20个连续的20μm厚的横向冰冻切片被分成五组放置在SuperFrost+标本固定器(Langenbrinck,Emmendingen,Germany)上(图11),一组内两个切片之间的垂直距离大约是100μm。然后切片在空气中干燥并储存在-20℃,直到它们被染色。为了对转移了的mRNA(编码大肠杆菌β-半乳糖苷酶)已被吸收并已被翻译的区域进行第一次筛选,第一组切片用X-gal染色。为此目的,标本固定器暴露在室温中,用ImmEdgeTM笔(Vektor,Burlingame,USA)勾出轮廓。然后,切片用含2%甲醛的PBS来固定15分钟。然后用PBS将标本固定器洗3次,2分钟,接着在含X-gal染色溶液(1mg/ml刚加入的X-gal,5mM氰铁酸钾,5mM氰亚铁酸钾,1mM氯化镁,15mM氯化钠,60mM磷酸氢二钠,40mM磷酸二氢钠)的潮湿箱中37℃下染色一整夜。冲洗标本固定器2次、2分钟来终止染色,并用Hydro-Matrix(Micro-Tech-Lab,Graz,Austria,用水稀释了二次)介质来处理它们。
为了得到有关组织形态的信息,对于另一组切片,X-gal染色与苏木精-伊红(HE)染色相结合进行。为此目的,在X-gal染色之后,切片在PBS中冲洗3次,每次2分钟,并再在双蒸水里面洗5分钟,然后,用Mayershaemalaun(Merck,Darmstadt,Germany)进行二次染色。该染色处理在活水下进行10分钟,然后,在水中用0.1%伊红Y(Sigma,Schnelldorf,Germany)进行10分钟的对比染色。然后在双蒸水中快速地冲洗,终止染色,然后用递增浓度的酒精脱水(2分钟80%乙醇,2分钟95%乙醇,2分钟100%乙醇,5分钟100%二甲苯)。最后,干燥了的切片用Roti-Histokitt(Roth,Karlsruhe,Germany)介质处理。
为了确定用mRNA转染的目标细胞是否是抗原递呈细胞,对MHC第II类分子(用APC表达)和mRNA转移(与β-半乳糖苷酶的表达有关)进行双染色。对MHC第II类分子进行免疫组织化学和免疫荧光检测。对于两种方法,在所有的3个步骤之间,切片都用PBS冲洗3次,每次2分钟。对于免疫组织化学检测程序,切片用PBS内1%(w/v)甲醛(Fluka)来固定。然后用纯的丙酮浸泡30秒去除油脂。之后,立即在室温下用含4%山羊血(Vektor LaboratoriesInc.,Burlingame,CA)和50μg/ml抗生素蛋白的PBS进行阻断(blocking)。剩余的生物素结合位点用50μg/ml生物素(AppliChem,Darmstadt,Germany)阻断,同时用单克隆抗体2G9(Becton Dickinson,Heidelberg,Germany)或合适的同型对照抗体(大鼠IgG 2a,R35-95,Becton Dickinson,Heidelberg,Germany)染色MHC第II类分子,每种情况下都稀释成1μg/ml(均在PBS中)。之后,在室温下用生物素化的山羊/抗大鼠IgG(3μg/ml)媒介和2%小鼠血清(CCPro,Neustadt,Germany)在PBS中将切片培养30分钟。然后加入ABC复合体(PBS中的1∶100的反应物A和B)(Vektor Laboratories Inc.,Burlingame,CA),在室温下维持30分钟。用刚制备的已通过0.45μm过滤器过滤的3-氨-9-乙基咔吧唑(3-amino-9-ethylcarbazole,AEC,Sigma)基质溶液(0.5mg/ml AEC,0.015%过氧化氢,50mM醋酸钠,pH5.5)来检验MHC第II类分子染色的完成。底物反应通过用水冲洗5分钟两次并用PBS冲洗5分钟3次来终止。如上所述那样,接着进行X-gal染色。
免疫荧光检测也使用了类似的染色方法。在丙酮处理步骤之后,切片在室温下在阻断缓冲液(PBS中1%的牛血清白蛋白)中阻断50分钟。然后将切片用在阻断缓冲液中稀释至1μg/ml的原发抗体(2G9或同型对照抗体)培养40分钟。然后,在室温下用Alexa Fluor 546山羊/抗大鼠IgG(1∶400,分子探针,Leiden,Netherlands)在阻断缓冲液中培养40分钟。最后,进行洋红色-gal染色。为此目的,染液中的X-gal用0.1mg/ml的洋红色-gal(Peqlab,Erlangen,Germany)来代替。
切片用带有Axiocam HRc照相机和Axiovision 4.0软件的Zeiss(Oberkochen,Germany)Axioplan 2显微镜来进行分析,并线性调节照片的颜色和对比度。
例6:人体的RNA转移和翻译
此实验在健康的男性志愿者身上进行。注射部位被刮去毛发,消毒并用RnaseZap(Ambion,Austin,USA)溶液进行了处理。然后,在一组中注射0.8xRL注射缓冲液中的120μg mRNA,总量为200μl。在其它组中,用下列RL注射缓冲液代替:
-不含氯化钠含乳酸盐的RL注射缓冲液,
-不含氯化钾含乳酸盐的RL注射缓冲液,
-不含氯化钙含乳酸盐的RL注射缓冲液。
注射后15小时,局部麻醉状态下取直径4mm的活组织(模压取出)。活组织在液氮中迅速冷冻,并如上描述的那样进行制备(例3)。研碎了的活组织重悬在600μl消化缓冲液中。
统计评估
两个不同组的平均值采用“非参数曼-惠特尼秩和检验(non-parametricMann-Whitney rank sum test)”来进行比较。p值<0.05被认为是显著的差异,并且在图中示出。
例7:对各种染色处理的评价
为了识别出在体内吸收并表达mRNA的细胞类型,使用了组织学处理,使得可以将mRNA的检测与细胞类型特异性染色相结合。
因为mRNA的转移不能用荧光探针检测出,因而执行了一种处理,在其内,编码大肠杆菌β-半乳糖苷酶的mRNA与各种靛青染料(X-gal或洋红-gal)相结合使用。
1、β-半乳糖苷酶检测系统的特别特征
为了确保靛青染色处理中所有的细胞都在单层中呈现,制备了小鼠耳的薄切片。考虑到小鼠耳部肌肉的形态结构(大约0.5mm至1mm厚的薄层)和只可用冰冻切片的事实(β-半乳糖苷酶在制备石蜡切片所需的某些步骤中被热失活),以及需要尽可能多的高质量的切片的需求,制备这些切片事实上很困难。尽管如此,也可能制备出几组厚度为20μm的高质量的切片。两种不同的染料用来检测β-半乳糖苷酶的活性。X-gal(阳性细胞被染成蓝绿色)的结果比用洋红-gal(阳性细胞被染成紫罗兰色)具有更好的对比度。然而,同时,非特定的背景染色,例如,由于毛囊引起的染色,在X-gal染色中很易见到。尽管如此,清楚地区分mRNA的转移也是可能的。对于洋红-gal染色来说,看不到非特定的背景染色。
2、靛青和免疫组合染色的要求
将mRNA转移染色(靛青染料)和特定细胞特定的标记物染色(特定抗体)相结合,需要对固定剂和组合染色(靛青染色包括在37℃下培育14小时)的顺序进行一些适应性修饰。先用丙酮固定再用抗体染色,可得到最好的抗体染色结果(对第II类MHC分子)。相反,首先用甲醛和戊二醛的混合物固定在进行靛青染色,可得到β-半乳糖苷酶活性的最佳结果。考虑到环境的各种变化,采用了如下步骤:用甲醛固定但不用戊二醛,然后进行抗体染色。不用戊二醛是因为虽然它对靛青染色有轻微的影响,但它会大大地增加组织的自身荧光。用甲醛来固定是由于下列原因:
1、比用丙酮可更好地保护组织的形态;
2、灵敏强烈的靛青染色需要用甲醛固定;并且
3、用甲醛固定时抗-MHC第II类抗体染色的质量仍然是可接受的。
然后在纯丙酮中进行短暂的培养来去除油脂和脂肪。使用水溶性的介质时,这可得到更好的质量(很少或没有气泡)。最后,当首先进行此染色时,可得到高质量的抗体染色。染色顺序(甲醛固定)只对靛青染色的质量有轻微的影响。
3、在靛青和免疫组合染色中染料的相容性
两种不同染色的组合不仅需要两种方法中各种步骤的相容性,也需要用于检测的探针的相容性。原则上,免疫染色可使用沉淀染料(酶探针)或荧光染料(标记的探针)。为了将靛青染料和沉淀染料组合起来,采用X-gal和AEC。双阳性细胞在此染色中呈现出黑色(图15 8)。很难区分高强度染色了的各个阳性细胞。因此,最好将洋红-gal与荧光染料Alexa Fluor 546组合。用洋红-gal来代替X-gal是基于两个原因:
1、洋红-gal染色的强度更弱(甚至当染料加到饱和量时),
2、洋红-gal阳性细胞的颜色与荧光染料Alexa Fluor 546发射的波长对应得更好。
两个因素使Alexa Fluor 546荧光信号的淬熄(quenching)降到最低程度。此染料组合实际上允许两个信号的检测(图13),至少当靛青染色不是太强时(这便是切片中mRNA转移阳性细胞的情况)。
参考文献
1.Ulmer J.B.,Curr.Opin.Drug Discov.Devel.,2001,4:192-197
2.J.A.Wolff et al.,Science247,1465-1468(1990).
3.H.L.Robinson,L.A.Hunt,R.G.Webster,Vaccine 11,957-960(1993).
4.J.B.Ulmer et al.,Science 259,1745-1749(1993).
5.J.A.Wolff et al.,Science247,1465-1468(1990).
6.D.Boczkowski,S.K.Nair,D.Snyder,E.Gilboa,J.Exp.Med.184,465-472(1996).
7.J.P.Carralot et al.,Cell Mol.Life Sci.(2004).
8.R.M.Conry et al.,Cancer Res.55,1397-1400(1995).
9.R.D.Granstein,W.Ding,H.Ozawa,J Invest Dermatol 114,632-636(2000).
10.I.Hoerr,R.Obst,H.G.Rammensee,G.Jung,Eur.J Immunol.30,1-7(2000).
11.J.Donnelly,K.Berry,J.B.Ulmer,Int J Parasitol.33,457-467(2003).
12.D.M.Klinman et al.,Springer Semin.Immunopathol.19,245-256(1997).
13.P.Forg,P.von Hoegen,W.Dalemans,V.Schirrmacher,Gene Ther.5,789-797(1998).
Claims (21)
1.一种RNA和包含钠盐、钙盐和可选的钾盐的水性注射缓冲液在制备用于增加RNA转移到寄助物和/或寄助物内RNA翻译的RNA注射液中的用途。
2.根据权利要求1所述的用途,其特征在于,所述注射缓冲液包含具有从下组中选出的阴离子的盐:氯化物、碘化物、溴化物、氢氧化物、碳酸盐、碳酸氢盐或硫酸盐,特别地所述注射缓冲液包含NaCl、CaCl2和可选的KCl作为盐。
3.根据权利要求2所述的用途,其特征在于,所述注射缓冲液包含至少50mM氯化钠(NaCl)、至少0.01mM氯化钙(CaCl2)、和可选的至少3mM的氯化钾。
4.根据权利要求1至3所述的用途,其特征在于,所述注射缓冲液包含50mM到800mM、较优为60mM到500mM、更优为70mM到250mM、尤其较优为60mM到110mM的氯化钠(NaCl),0.01mM到100mM、较优为0.5mM到80mM、更优为1.5mM到40mM的氯化钙(CaCl2),以及可选地3mM到500mM、优选为4mM到300mM、更优地为5mM to 200mM的氯化钾(KCl)。
5.根据权利要求1至4中任意一项所述的用途,其特征在于,所述注射缓冲液进一步包含乳酸盐。
6.根据权利要求1至5中任意一项所述的用途,其特征在于,所述注射缓冲液不含缓冲系统,例如HEPES、tris/HCl、Na(K)2HPO4/Na(K)H2PO4等。
7.根据权利要求1至6中任意一项所述的用途,其特征在于,所述注射缓冲液不含单糖、二糖或多糖。
8.根据权利要求1至7中任意一项所述的用途,其特征在于,所述注射缓冲液含至少50mM的氯化钠(NaCl)、至少0.1mM的氯化钙(CaCl2)、至少15mM的乳酸盐和可选的至少3mM的氯化钾(KCl)。
9.根据权利要求8所述的用途,其特征在于,所述注射缓冲液包含15mM到500mM的乳酸盐,更佳地包含15mM到200mM的乳酸盐,最好含15mM到100mM的乳酸盐。
10.根据权利要求1至9中任意一项所述的用途,其特征在于,所述RNA是rRNA、tRNA、siRNA或最好是mRNA。
11.根据权利要求1至10中任意一项所述的用途,其特征在于,所述RNA是裸露RNA,特别是裸露的mRNA;或者是与聚阳离子结合的RNA特别是mRNA。
12.根据权利要求11所述的用途,其特征在于,所述RNA特别是mRNA与鱼精蛋白相结合。
13.根据要求10至12中任意一项所述的用途,其特征在于,mRNA具有至少一种自然或非自然发生的修饰,所述修饰从以下方式中选择:增加mRNA的编码区内的G/C含量而不改变编码氨基酸序列;引入至少一种核糖核苷酸相似物,消除WT序列中的不稳定序列;或增加核糖体结合位点的编码区内的A/U含量。
14.一种根据要求1至13所定义的包含RNA和注射缓冲液的RNA注射液,用于增加RNA转移到寄助物和/或寄助物内的RNA表达。
15.根据权利要求14所述的RNA注射液,其特征在于,所述缓冲液进一步包含乳酸盐。
16.根据权利要求15所述的RNA注射液,其特征在于,所述注射缓冲液包含至少15mM的乳酸盐,较佳的含15mM至100mM的乳酸盐。
17.根据权利要求14至16所述的RNA注射液,其特征在于,所述RNA是tRNA、rRNA或优选为mRNA。
18.根据权利要求14至17所述的RNA注射液,其特征在于,所述RNA是裸露的RNA,特别是裸露的mRNA。
19.一种增加RNA转移到寄助物和/或寄助物内RNA翻译的方法,其特征在于,所述方法包括下列步骤:
a.根据权利要求14到18中任意一项制备RNA注射液;
b.将步骤a得到的RNA注射液给药到寄助物中。
20.根据权利要求19所述的方法,其特征在于,所述寄助物为从以下选择的哺乳动物:小鼠、大鼠、猪、牛、马、狗、猫、猿,特别是人。
21.一种用于体外细胞转染的方法,其特征在于,根据权利要求14到18中任一项所述的注射缓冲液注射到细胞中,特别是实验室细胞系或疾病细胞中,优选借助于电穿孔。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102005023170A DE102005023170A1 (de) | 2005-05-19 | 2005-05-19 | Optimierte Formulierung für mRNA |
DE102005023170.5 | 2005-05-19 | ||
PCT/EP2006/004784 WO2006122828A2 (de) | 2005-05-19 | 2006-05-19 | Optimierte injektionsformulierung für mrna |
Publications (2)
Publication Number | Publication Date |
---|---|
CN101203245A true CN101203245A (zh) | 2008-06-18 |
CN101203245B CN101203245B (zh) | 2012-11-07 |
Family
ID=37116756
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2006800171263A Active CN101203245B (zh) | 2005-05-19 | 2006-05-19 | Rna最佳注射配方 |
Country Status (9)
Country | Link |
---|---|
US (3) | US20080267873A1 (zh) |
EP (3) | EP3583953B1 (zh) |
JP (1) | JP5295760B2 (zh) |
CN (1) | CN101203245B (zh) |
AU (1) | AU2006249093B2 (zh) |
DE (1) | DE102005023170A1 (zh) |
ES (1) | ES2604538T5 (zh) |
RU (1) | RU2418593C2 (zh) |
WO (1) | WO2006122828A2 (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101961343A (zh) * | 2010-09-15 | 2011-02-02 | 河南辅仁怀庆堂制药有限公司 | 注射用核糖核酸及其生产方法 |
CN102174513A (zh) * | 2011-02-17 | 2011-09-07 | 杨俊海 | 一种哺乳动物核糖核酸小分子复合物及其应用 |
CN102099041B (zh) * | 2008-07-18 | 2013-07-31 | Ogx科技公司 | 反义制剂 |
Families Citing this family (159)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102005023170A1 (de) | 2005-05-19 | 2006-11-23 | Curevac Gmbh | Optimierte Formulierung für mRNA |
LT2578685T (lt) | 2005-08-23 | 2019-06-10 | The Trustees Of The University Of Pennsylvania | Rnr, apimančios modifikuotus nukleozidus ir jų panaudojimo būdai |
AU2007280690C1 (en) | 2006-07-31 | 2012-08-23 | Curevac Gmbh | Nucleic acid of formula (I): GIXmGn, or (II): CIXmCn, in particular as an immune-stimulating agent/adjuvant |
DE102007001370A1 (de) | 2007-01-09 | 2008-07-10 | Curevac Gmbh | RNA-kodierte Antikörper |
WO2009030254A1 (en) | 2007-09-04 | 2009-03-12 | Curevac Gmbh | Complexes of rna and cationic peptides for transfection and for immunostimulation |
WO2009046738A1 (en) | 2007-10-09 | 2009-04-16 | Curevac Gmbh | Composition for treating lung cancer, particularly of non-small lung cancers (nsclc) |
WO2009046739A1 (en) | 2007-10-09 | 2009-04-16 | Curevac Gmbh | Composition for treating prostate cancer (pca) |
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 |
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 |
EP2548958B1 (en) * | 2010-03-15 | 2017-11-22 | Yamaguchi University | Agent for improving gene transfer efficiency to mammalian cells |
CA2801523C (en) | 2010-07-30 | 2021-08-03 | Curevac Gmbh | Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation |
EP3578205A1 (en) | 2010-08-06 | 2019-12-11 | ModernaTX, Inc. | A pharmaceutical formulation comprising engineered nucleic acids and medical use thereof |
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 |
HUE058896T2 (hu) | 2010-10-01 | 2022-09-28 | Modernatx Inc | N1-metil-pszeudo-uracilt tartalmazó ribonukleinsavak és azok felhasználásai |
AU2011358150B2 (en) * | 2010-12-16 | 2016-11-03 | Sprna Gmbh | Pharmaceutical composition consisting of RNA having alkali metal as counter ion and formulated with dications |
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 |
JP2014511687A (ja) | 2011-03-31 | 2014-05-19 | モデルナ セラピューティクス インコーポレイテッド | 工学操作された核酸の送達および製剤 |
US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
RU2648950C2 (ru) | 2011-10-03 | 2018-04-02 | Модерна Терапьютикс, Инк. | Модифицированные нуклеозиды, нуклеотиды и нуклеиновые кислоты и их применение |
EP2791160B1 (en) | 2011-12-16 | 2022-03-02 | ModernaTX, Inc. | Modified mrna compositions |
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 |
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 |
KR102186497B1 (ko) | 2012-03-27 | 2020-12-04 | 큐어백 아게 | 인공 핵산 분자 |
AU2013242404B2 (en) | 2012-03-27 | 2018-08-30 | CureVac SE | Artificial nucleic acid molecules for improved protein or peptide expression |
BR112014023898A2 (pt) | 2012-03-27 | 2017-07-11 | Curevac Gmbh | moléculas de ácido nucleico artificiais compreendendo 5''utr top |
US9572897B2 (en) | 2012-04-02 | 2017-02-21 | Modernatx, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
AU2013243951A1 (en) | 2012-04-02 | 2014-10-30 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of secreted proteins |
US9254311B2 (en) | 2012-04-02 | 2016-02-09 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins |
US9283287B2 (en) | 2012-04-02 | 2016-03-15 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of nuclear proteins |
WO2013151666A2 (en) | 2012-04-02 | 2013-10-10 | modeRNA Therapeutics | Modified polynucleotides for the production of biologics and proteins associated with human disease |
US9512456B2 (en) | 2012-08-14 | 2016-12-06 | Modernatx, Inc. | Enzymes and polymerases for the synthesis of RNA |
EP2922554B1 (en) | 2012-11-26 | 2022-02-23 | ModernaTX, Inc. | Terminally modified rna |
US9974845B2 (en) | 2013-02-22 | 2018-05-22 | Curevac Ag | Combination of vaccination and inhibition of the PD-1 pathway |
EP3292873B1 (en) | 2013-02-22 | 2019-05-01 | CureVac AG | Combination of vaccination and inhibition of the pd-1 pathway |
US20160032316A1 (en) | 2013-03-14 | 2016-02-04 | The Trustees Of The University Of Pennsylvania | Purification and Purity Assessment of RNA Molecules Synthesized with Modified Nucleosides |
US10258698B2 (en) | 2013-03-14 | 2019-04-16 | Modernatx, 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 |
JP6625521B2 (ja) | 2013-05-15 | 2020-01-08 | リボカイン,エルエルシー | 環状rnaの細胞内翻訳 |
SG11201510746WA (en) | 2013-08-21 | 2016-03-30 | Curevac Ag | Respiratory syncytial virus (rsv) vaccine |
SG11201510747RA (en) | 2013-08-21 | 2016-03-30 | Curevac Ag | Method for increasing expression of rna-encoded proteins |
CA2915730A1 (en) | 2013-08-21 | 2015-02-26 | Karl-Josef Kallen | A combination rsv/influenza a vaccine |
CN105517569A (zh) | 2013-08-21 | 2016-04-20 | 库瑞瓦格股份公司 | 狂犬病疫苗 |
WO2015034928A1 (en) | 2013-09-03 | 2015-03-12 | Moderna Therapeutics, Inc. | Chimeric polynucleotides |
WO2015048744A2 (en) | 2013-09-30 | 2015-04-02 | Moderna Therapeutics, Inc. | Polynucleotides encoding immune modulating polypeptides |
EA201690675A1 (ru) | 2013-10-03 | 2016-08-31 | Модерна Терапьютикс, Инк. | Полинуклеотиды, кодирующие рецептор липопротеинов низкой плотности |
CA2925021A1 (en) | 2013-11-01 | 2015-05-07 | Curevac Ag | Modified rna with decreased immunostimulatory properties |
WO2015101416A1 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Methods for rna analysis |
BR112016014462A2 (pt) | 2013-12-30 | 2017-10-24 | Curevac Ag | moléculas de ácido nucleico artificiais |
US11254951B2 (en) | 2014-12-30 | 2022-02-22 | Curevac Ag | Artificial nucleic acid molecules |
CA2927254C (en) | 2013-12-30 | 2023-10-24 | Curevac Ag | Artificial nucleic acid molecules |
ES2754239T3 (es) | 2014-03-12 | 2020-04-16 | Curevac Ag | Combinación de vacunación y agonistas de OX40 |
CA2936286A1 (en) | 2014-04-01 | 2015-10-08 | Curevac Ag | Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant |
PT3134131T (pt) | 2014-04-23 | 2022-03-24 | Modernatx Inc | Vacinas de ácidos nucleicos |
JP6748579B2 (ja) | 2014-06-10 | 2020-09-02 | キュアバック リアル エステート ゲゼルシャフト ミット ベシュレンクテル ハフツング | Rna生成を強化する方法及び手段 |
US10626400B2 (en) | 2014-07-04 | 2020-04-21 | Biontech Ag | Stabilised formulations of RNA |
US20170204152A1 (en) | 2014-07-16 | 2017-07-20 | Moderna Therapeutics, Inc. | Chimeric polynucleotides |
EP3169334B1 (en) * | 2014-07-16 | 2021-04-28 | Ethris GmbH | Rna for use in the treatment of ligament or tendon lesions |
WO2016068228A1 (ja) * | 2014-10-29 | 2016-05-06 | 株式会社高研 | 薬剤徐放担体及び薬剤徐放方法 |
EP4241784A3 (en) | 2014-12-12 | 2023-11-15 | CureVac SE | Artificial nucleic acid molecules for improved protein expression |
BR112017014090A2 (pt) * | 2014-12-29 | 2018-03-06 | Bonac Corporation | composição, e, métodos para produção de uma composição e estabilização de uma molécula de ácido nucleico. |
WO2016165825A1 (en) | 2015-04-13 | 2016-10-20 | Curevac Ag | Method for producing rna compositions |
EP3283125B1 (en) | 2015-04-17 | 2021-12-29 | CureVac Real Estate GmbH | Lyophilization of rna |
MX2017013321A (es) | 2015-04-22 | 2018-07-06 | Curevac Ag | Composicion que contiene arn para tratamiento de enfermedades tumorales. |
EP3289101B1 (en) | 2015-04-30 | 2021-06-23 | CureVac AG | Immobilized poly(n)polymerase |
EP3294885B1 (en) | 2015-05-08 | 2020-07-01 | CureVac Real Estate GmbH | Method for producing rna |
US11559570B2 (en) | 2015-05-15 | 2023-01-24 | CureVac SE | Prime-boost regimens involving administration of at least one mRNA construct |
US10517827B2 (en) | 2015-05-20 | 2019-12-31 | Curevac Ag | Dry powder composition comprising long-chain RNA |
CN107530448A (zh) | 2015-05-20 | 2018-01-02 | 库瑞瓦格股份公司 | 包含长链rna的干粉组合物 |
WO2016193226A1 (en) | 2015-05-29 | 2016-12-08 | Curevac Ag | Method for adding cap structures to rna using immobilized enzymes |
EP3744843A1 (en) | 2015-05-29 | 2020-12-02 | CureVac Real Estate GmbH | A method for producing and purifying rna, comprising at least one step of tangential flow filtration |
US10501768B2 (en) | 2015-07-13 | 2019-12-10 | Curevac Ag | Method of producing RNA from circular DNA and corresponding template DNA |
LT3350157T (lt) | 2015-09-17 | 2022-02-25 | Modernatx, Inc. | Junginiai ir kompozicijos terapinei medžiagai teikti intraceliuliniu būdu |
JP6990176B2 (ja) | 2015-10-05 | 2022-02-03 | モデルナティエックス インコーポレイテッド | メッセンジャーリボ核酸薬物の治療投与のための方法 |
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 |
WO2017066789A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Mrna cap analogs with modified sugar |
WO2017066791A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Sugar substituted mrna cap analogs |
CA3001014A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Mrna cap analogs and methods of mrna capping |
EP3362461B1 (en) | 2015-10-16 | 2022-03-16 | Modernatx, Inc. | Mrna cap analogs with modified phosphate linkage |
WO2017066782A1 (en) | 2015-10-16 | 2017-04-20 | Modernatx, Inc. | Hydrophobic mrna cap analogs |
EP3373965A1 (en) | 2015-11-09 | 2018-09-19 | CureVac AG | Rotavirus vaccines |
AU2016375021B2 (en) | 2015-12-22 | 2022-02-03 | CureVac SE | Method for producing RNA molecule compositions |
AU2016377681B2 (en) | 2015-12-22 | 2021-05-13 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of agents |
US11248223B2 (en) | 2015-12-23 | 2022-02-15 | Curevac Ag | Method of RNA in vitro transcription using a buffer containing a dicarboxylic acid or tricarboxylic acid or a salt thereof |
SG11201806340YA (en) | 2016-02-17 | 2018-09-27 | Curevac Ag | Zika virus vaccine |
EP3423595A1 (en) | 2016-03-03 | 2019-01-09 | CureVac AG | Rna analysis by total hydrolysis |
US11446398B2 (en) | 2016-04-11 | 2022-09-20 | Obsidian Therapeutics, Inc. | Regulated biocircuit systems |
US11596699B2 (en) | 2016-04-29 | 2023-03-07 | CureVac SE | RNA encoding an antibody |
EP3452493A1 (en) | 2016-05-04 | 2019-03-13 | CureVac AG | Nucleic acid molecules and uses thereof |
WO2017191274A2 (en) | 2016-05-04 | 2017-11-09 | Curevac Ag | Rna encoding a therapeutic protein |
KR20190029576A (ko) | 2016-06-09 | 2019-03-20 | 큐어백 아게 | 핵산 카고용 하이브리드 담체 |
JP2019525901A (ja) | 2016-06-14 | 2019-09-12 | モデルナティエックス インコーポレイテッドModernaTX,Inc. | 脂質ナノ粒子の安定化製剤 |
WO2018089540A1 (en) | 2016-11-08 | 2018-05-17 | Modernatx, Inc. | Stabilized formulations of lipid nanoparticles |
WO2018096179A1 (en) | 2016-11-28 | 2018-05-31 | Curevac Ag | Method for purifying rna |
CN110177544A (zh) | 2016-11-29 | 2019-08-27 | 普尔泰克健康有限公司 | 用于递送治疗剂的外泌体 |
WO2018104540A1 (en) | 2016-12-08 | 2018-06-14 | Curevac Ag | Rnas for wound healing |
CN110582304A (zh) | 2016-12-08 | 2019-12-17 | 库尔维科公司 | 用于治疗或预防肝脏疾病的rna |
EP3558355A2 (en) | 2016-12-23 | 2019-10-30 | CureVac AG | Henipavirus vaccine |
WO2018115527A2 (en) | 2016-12-23 | 2018-06-28 | Curevac Ag | Mers coronavirus vaccine |
EP3558354A1 (en) | 2016-12-23 | 2019-10-30 | CureVac AG | Lassa virus vaccine |
PE20200735A1 (es) | 2017-02-28 | 2020-07-23 | Sanofi Sa | Arn terapeutico |
RS63953B1 (sr) | 2017-03-15 | 2023-02-28 | Modernatx Inc | Jedinjenje i kompozicije za intracelularnu isporuku terapeutskih sredstava |
CA3055653A1 (en) | 2017-03-15 | 2018-09-20 | Modernatx, Inc. | Lipid nanoparticle formulation |
EP3601576A1 (en) | 2017-03-24 | 2020-02-05 | CureVac AG | Nucleic acids encoding crispr-associated proteins and uses thereof |
WO2018191657A1 (en) | 2017-04-13 | 2018-10-18 | Acuitas Therapeutics, Inc. | Lipids for delivery of active agents |
US20210198200A1 (en) | 2017-06-14 | 2021-07-01 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of agents |
CN111328287A (zh) | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | 新型核酸分子 |
US20200362382A1 (en) | 2017-08-18 | 2020-11-19 | Modernatx, Inc. | Methods of preparing modified rna |
EP3673069A1 (en) | 2017-08-22 | 2020-07-01 | CureVac AG | Bunyavirales vaccine |
AU2018326799A1 (en) | 2017-08-31 | 2020-02-27 | Modernatx, Inc. | Methods of making lipid nanoparticles |
WO2019092153A1 (en) | 2017-11-08 | 2019-05-16 | Curevac Ag | Rna sequence adaptation |
EP3723796A1 (en) | 2017-12-13 | 2020-10-21 | CureVac AG | Flavivirus vaccine |
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 |
EP3773702A2 (en) | 2018-04-05 | 2021-02-17 | CureVac AG | Novel yellow fever nucleic acid molecules for vaccination |
CN112292395A (zh) | 2018-04-17 | 2021-01-29 | 库瑞瓦格股份公司 | 用于疫苗接种的新型rsv rna分子和组合物 |
WO2019241315A1 (en) | 2018-06-12 | 2019-12-19 | Obsidian Therapeutics, Inc. | Pde5 derived regulatory constructs and methods of use in immunotherapy |
EP3813874A1 (en) | 2018-06-27 | 2021-05-05 | CureVac AG | Novel lassa virus rna molecules and compositions for vaccination |
JP7410135B2 (ja) | 2018-09-19 | 2024-01-09 | モデルナティエックス インコーポレイテッド | 治療薬の細胞内送達のための化合物及び組成物 |
US20210378980A1 (en) | 2018-09-20 | 2021-12-09 | Modernatx, Inc. | Preparation of lipid nanoparticles and methods of administration thereof |
US20210386788A1 (en) | 2018-10-24 | 2021-12-16 | Obsidian Therapeutics, Inc. | Er tunable protein regulation |
AU2019410737A1 (en) | 2018-12-21 | 2021-06-10 | CureVac SE | RNA for malaria vaccines |
WO2020160430A1 (en) | 2019-01-31 | 2020-08-06 | Modernatx, Inc. | Vortex mixers and associated methods, systems, and apparatuses thereof |
CN113939282A (zh) | 2019-01-31 | 2022-01-14 | 摩登纳特斯有限公司 | 制备脂质纳米颗粒的方法 |
WO2020163654A1 (en) * | 2019-02-06 | 2020-08-13 | Paul Leo Mcgrane | Biologically modified vascular grafts for improved bypass surgery outcomes |
WO2020161342A1 (en) | 2019-02-08 | 2020-08-13 | Curevac Ag | Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases |
EP3986452A1 (en) | 2019-06-18 | 2022-04-27 | CureVac AG | Rotavirus mrna vaccine |
US11576966B2 (en) | 2020-02-04 | 2023-02-14 | CureVac SE | Coronavirus vaccine |
US11241493B2 (en) | 2020-02-04 | 2022-02-08 | Curevac Ag | Coronavirus vaccine |
BR112022014627A2 (pt) | 2020-02-04 | 2022-09-27 | Curevac Ag | Vacina contra coronavírus |
WO2021204179A1 (en) | 2020-04-09 | 2021-10-14 | Suzhou Abogen Biosciences Co., Ltd. | Nucleic acid vaccines for coronavirus |
CA3178455A1 (en) | 2020-04-09 | 2021-10-14 | Suzhou Abogen Biosciences Co., Ltd. | Lipid nanoparticle composition |
EP3993828A1 (en) | 2020-05-29 | 2022-05-11 | 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 |
US20230272052A1 (en) | 2020-07-31 | 2023-08-31 | CureVac SE | Nucleic acid encoded antibody mixtures |
JP2023537887A (ja) | 2020-08-20 | 2023-09-06 | スージョウ・アボジェン・バイオサイエンシズ・カンパニー・リミテッド | 脂質化合物及び脂質ナノ粒子組成物 |
CA3205569A1 (en) | 2020-12-22 | 2022-06-30 | 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 |
WO2022152109A2 (en) | 2021-01-14 | 2022-07-21 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
WO2022152141A2 (en) | 2021-01-14 | 2022-07-21 | Suzhou Abogen Biosciences Co., Ltd. | Polymer conjugated 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 |
EP4334446A1 (en) | 2021-05-03 | 2024-03-13 | CureVac SE | Improved nucleic acid sequence for cell type specific expression |
KR20240013087A (ko) | 2021-05-24 | 2024-01-30 | 쑤저우 아보젠 바이오사이언시스 컴퍼니 리미티드 | 지질 화합물 및 지질 나노입자 조성물 |
WO2023044343A1 (en) | 2021-09-14 | 2023-03-23 | Renagade Therapeutics Management Inc. | Acyclic lipids and methods of use thereof |
CA3232386A1 (en) | 2021-09-14 | 2023-03-23 | Renagade Therapeutics Management Inc. | Cyclic lipids and methods of use thereof |
CN116064598B (zh) | 2021-10-08 | 2024-03-12 | 苏州艾博生物科技有限公司 | 冠状病毒的核酸疫苗 |
AU2022358824A1 (en) | 2021-10-08 | 2024-04-11 | Suzhou Abogen Biosciences Co., Ltd. | Lipid compounds and lipid nanoparticle compositions |
AR127312A1 (es) | 2021-10-08 | 2024-01-10 | Suzhou Abogen Biosciences Co Ltd | Compuestos lipídicos ycomposiciones de nanopartículas lipídicas |
WO2023064612A2 (en) | 2021-10-15 | 2023-04-20 | BioNTech SE | Pharmaceutical compositions for delivery of viral antigens and related methods |
WO2023116804A1 (zh) | 2021-12-23 | 2023-06-29 | 苏州艾博生物科技有限公司 | 脂质化合物和脂质纳米颗粒组合物 |
WO2023122752A1 (en) | 2021-12-23 | 2023-06-29 | Renagade Therapeutics Management Inc. | Constrained lipids and methods of use thereof |
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 |
WO2024037578A1 (en) | 2022-08-18 | 2024-02-22 | Suzhou Abogen Biosciences Co., Ltd. | Composition of lipid nanoparticles |
DE202023106198U1 (de) | 2022-10-28 | 2024-03-21 | CureVac SE | Impfstoff auf Nukleinsäurebasis |
Family Cites Families (40)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5132418A (en) | 1980-02-29 | 1992-07-21 | University Patents, Inc. | Process for preparing polynucleotides |
US4500707A (en) | 1980-02-29 | 1985-02-19 | University Patents, Inc. | Nucleosides useful in the preparation of polynucleotides |
US4458066A (en) | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
US4973679A (en) | 1981-03-27 | 1990-11-27 | University Patents, Inc. | Process for oligonucleo tide synthesis using phosphormidite intermediates |
US4415732A (en) | 1981-03-27 | 1983-11-15 | University Patents, Inc. | Phosphoramidite compounds and processes |
US4668777A (en) | 1981-03-27 | 1987-05-26 | University Patents, Inc. | Phosphoramidite nucleoside compounds |
US4373071A (en) | 1981-04-30 | 1983-02-08 | City Of Hope Research Institute | Solid-phase synthesis of polynucleotides |
US4401796A (en) | 1981-04-30 | 1983-08-30 | City Of Hope Research Institute | Solid-phase synthesis of polynucleotides |
US5153319A (en) | 1986-03-31 | 1992-10-06 | University Patents, Inc. | Process for preparing polynucleotides |
US5047524A (en) | 1988-12-21 | 1991-09-10 | Applied Biosystems, Inc. | Automated system for polynucleotide synthesis and purification |
US5262530A (en) | 1988-12-21 | 1993-11-16 | Applied Biosystems, Inc. | Automated system for polynucleotide synthesis and purification |
US5703055A (en) | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
US6214804B1 (en) | 1989-03-21 | 2001-04-10 | Vical Incorporated | Induction of a protective immune response in a mammal by injecting a DNA sequence |
US5700642A (en) | 1995-05-22 | 1997-12-23 | Sri International | Oligonucleotide sizing using immobilized cleavable primers |
US5766903A (en) | 1995-08-23 | 1998-06-16 | University Technology Corporation | Circular RNA and uses thereof |
US6627616B2 (en) * | 1995-12-13 | 2003-09-30 | Mirus Corporation | Intravascular delivery of non-viral nucleic acid |
US20020165183A1 (en) * | 1999-11-29 | 2002-11-07 | Hans Herweijer | Methods for genetic immunization |
US6379966B2 (en) * | 1999-02-26 | 2002-04-30 | Mirus Corporation | Intravascular delivery of non-viral nucleic acid |
US20030143204A1 (en) * | 2001-07-27 | 2003-07-31 | Lewis David L. | Inhibition of RNA function by delivery of inhibitors to animal cells |
US20030083272A1 (en) | 1997-09-19 | 2003-05-01 | Lahive & Cockfield, Llp | Sense mrna therapy |
AU750106B2 (en) * | 1997-10-07 | 2002-07-11 | University Of Maryland Biotechnology Institute | Method for introducing and expressing RNA in animal cells |
US6383811B2 (en) * | 1997-12-30 | 2002-05-07 | Mirus Corporation | Polyampholytes for delivering polyions to a cell |
US20040106567A1 (en) * | 1999-09-07 | 2004-06-03 | Hagstrom James E. | Intravascular delivery of non-viral nucleic acid |
ATE289630T1 (de) | 1999-09-09 | 2005-03-15 | Curevac Gmbh | Transfer von mrnas unter verwendung von polykationischen verbindungen |
US20020132788A1 (en) * | 2000-11-06 | 2002-09-19 | David Lewis | Inhibition of gene expression by delivery of small interfering RNA to post-embryonic animal cells in vivo |
WO2002086134A2 (de) * | 2001-04-23 | 2002-10-31 | Amaxa Gmbh | Pufferlössung für die elektroporation und verfahren umfassend die verwendung derselben |
EP1800697B1 (de) * | 2001-06-05 | 2010-04-14 | CureVac GmbH | Stabilisierte mRNA mit erhöhtem G/C-Gehalt für die Gentherapie |
DE10162480A1 (de) * | 2001-12-19 | 2003-08-07 | Ingmar Hoerr | Die Applikation von mRNA für den Einsatz als Therapeutikum gegen Tumorerkrankungen |
DE10229872A1 (de) | 2002-07-03 | 2004-01-29 | Curevac Gmbh | Immunstimulation durch chemisch modifizierte RNA |
WO2004063331A2 (en) * | 2003-01-03 | 2004-07-29 | Gencia Corporation | SiRNA MEDIATED POST-TRANSRIPTIONAL GENE SILENCING OF GENES INVOLVED IN ALOPECIA |
US20040167090A1 (en) * | 2003-02-21 | 2004-08-26 | Monahan Sean D. | Covalent modification of RNA for in vitro and in vivo delivery |
CA2520406A1 (en) * | 2003-03-27 | 2004-10-14 | Emory University | Cxcr4 antagonists and methods of their use |
BRPI0418384A (pt) * | 2003-05-30 | 2007-06-26 | Arc Pharmaceuticals Inc | composições farmacêuticas e métodos referentes à inibição de adesões fibrosas utilizando diversos agentes |
EP1636385A4 (en) * | 2003-06-24 | 2010-06-02 | Mirus Bio Corp | INHIBITION OF GENE FUNCTION BY IN VIVO DISTRIBUTION OF GENE EXPRESSION INHIBITORS BASED ON POLYNUCLEOTIDES IN MAMMALIAN CELLS |
DE10335833A1 (de) * | 2003-08-05 | 2005-03-03 | Curevac Gmbh | Transfektion von Blutzellen mit mRNA zur Immunstimulation und Gentherapie |
DE102004035227A1 (de) | 2004-07-21 | 2006-02-16 | Curevac Gmbh | mRNA-Gemisch zur Vakzinierung gegen Tumorerkrankungen |
DE102004042546A1 (de) * | 2004-09-02 | 2006-03-09 | Curevac Gmbh | Kombinationstherapie zur Immunstimulation |
WO2006037038A1 (en) | 2004-09-27 | 2006-04-06 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Optimized vaccines to provide protection against ebola and other viruses |
DE102005023170A1 (de) | 2005-05-19 | 2006-11-23 | Curevac Gmbh | Optimierte Formulierung für mRNA |
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 |
-
2005
- 2005-05-19 DE DE102005023170A patent/DE102005023170A1/de not_active Withdrawn
-
2006
- 2006-05-19 EP EP19176589.0A patent/EP3583953B1/de active Active
- 2006-05-19 EP EP06742996.9A patent/EP1881847B8/de active Active
- 2006-05-19 JP JP2008511654A patent/JP5295760B2/ja active Active
- 2006-05-19 CN CN2006800171263A patent/CN101203245B/zh active Active
- 2006-05-19 RU RU2007146610/15A patent/RU2418593C2/ru active
- 2006-05-19 AU AU2006249093A patent/AU2006249093B2/en active Active
- 2006-05-19 WO PCT/EP2006/004784 patent/WO2006122828A2/de active Application Filing
- 2006-05-19 US US11/914,945 patent/US20080267873A1/en not_active Abandoned
- 2006-05-19 EP EP16001381.9A patent/EP3153179B1/de active Active
- 2006-05-19 ES ES06742996T patent/ES2604538T5/es active Active
-
2017
- 2017-12-07 US US15/835,403 patent/US20180214523A1/en not_active Abandoned
-
2021
- 2021-03-12 US US17/200,693 patent/US20210308238A1/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102099041B (zh) * | 2008-07-18 | 2013-07-31 | Ogx科技公司 | 反义制剂 |
CN101961343A (zh) * | 2010-09-15 | 2011-02-02 | 河南辅仁怀庆堂制药有限公司 | 注射用核糖核酸及其生产方法 |
CN102174513A (zh) * | 2011-02-17 | 2011-09-07 | 杨俊海 | 一种哺乳动物核糖核酸小分子复合物及其应用 |
CN102174513B (zh) * | 2011-02-17 | 2013-09-18 | 杨俊海 | 一种哺乳动物核糖核酸小分子复合物及其应用 |
Also Published As
Publication number | Publication date |
---|---|
EP1881847B2 (de) | 2022-12-07 |
ES2604538T5 (es) | 2023-01-30 |
US20180214523A1 (en) | 2018-08-02 |
EP1881847B1 (de) | 2016-09-07 |
EP1881847B8 (de) | 2023-01-11 |
WO2006122828A2 (de) | 2006-11-23 |
CN101203245B (zh) | 2012-11-07 |
EP3583953A1 (de) | 2019-12-25 |
US20210308238A1 (en) | 2021-10-07 |
EP3583953B1 (de) | 2023-06-28 |
ES2604538T3 (es) | 2017-03-07 |
WO2006122828A3 (de) | 2007-05-10 |
US20080267873A1 (en) | 2008-10-30 |
AU2006249093B2 (en) | 2013-09-19 |
JP5295760B2 (ja) | 2013-09-18 |
RU2418593C2 (ru) | 2011-05-20 |
RU2007146610A (ru) | 2009-06-27 |
DE102005023170A1 (de) | 2006-11-23 |
AU2006249093A1 (en) | 2006-11-23 |
EP1881847A2 (de) | 2008-01-30 |
EP3153179A1 (de) | 2017-04-12 |
EP3153179B1 (de) | 2019-06-19 |
JP2008540601A (ja) | 2008-11-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101203245B (zh) | Rna最佳注射配方 | |
BR112020024292A2 (pt) | rna circular para translação em células eucarióticas | |
Sitbon et al. | Hemolytic anemia and erythroleukemia, two distinct pathogenic effects of Friend MuLV: mapping of the effects to different regions of the viral genome | |
ES2967788T3 (es) | Expresión especifica de célula de Arnmond | |
KR20160079901A (ko) | 분열 촉진 인자 활성화 단백질 키나제 의존성 재조합 우두 바이러스(md-rvv) 및 이의 용도 | |
KR20070111542A (ko) | 암 치료유전자의 종양-특이적, 발현 고효율을 위한 신규한hTMC 프로모터 및 벡터 | |
WO2014031693A1 (en) | Compositions containing protein polymers and vaccinia virus, and methods of use thereof | |
PT1509253E (pt) | Cd154 quimérico novo | |
Bardina et al. | Mengovirus-induced rearrangement of the nuclear pore complex: hijacking cellular phosphorylation machinery | |
CN105209617B (zh) | 微囊泡及其制造方法 | |
Liu et al. | PIM1-minicircle as a therapeutic treatment for myocardial infarction | |
Sun et al. | Distribution characteristics of DNA vaccine encoded with glycoprotein C from Anatid herpesvirus 1 with chitosan and liposome as deliver carrier in ducks | |
ES2425321T3 (es) | Promotores que muestran especificidad por células endoteliales y métodos de utilización de los mismos | |
US9498499B2 (en) | Imaging-aided gene therapy using mesenchymal stem cells as target-delivery vehicle | |
CN111868234A (zh) | 工程化免疫细胞作为疾病的诊断探针 | |
CN109337928A (zh) | 通过过表达腺相关病毒受体提高基因治疗效率的方法 | |
CN101652148A (zh) | 通过分子成像利用具有反式剪接核酶的腺病毒诊断疾病的方法 | |
CA3150053A1 (en) | Genetically modified enterovirus vectors | |
US9879278B2 (en) | Non-viral episomal suicide construct | |
US20240002855A1 (en) | Nucleic Acid Molecule Binding to YB-1 Protein | |
WO2024029527A1 (ja) | 長波長の光照射で機能する光スイッチタンパク質 | |
CN105968211A (zh) | 一种重组抗病毒蛋白及其制备方法和应用 | |
Jandick et al. | Creation and characterization of a recombinant mammalian orthoreovirus expressing σ1 fusion proteins encoding human epidermal growth factor receptor 2 peptides | |
WO2024137718A1 (en) | Compositions, systems and methods for manipulating area postrema (ap) neurons based on gfral sensing | |
Lin | In vivo imaging of engraftment and enrichment of lentiviral transduced hematopoietic bone marrow cells under MGMT-P140K mediated selection |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C56 | Change in the name or address of the patentee | ||
CP03 | Change of name, title or address |
Address after: Tubingen Patentee after: CUREVAC GMBH Address before: Fort Baden, Germany Patentee before: Curevac GmbH |